1: Mol Biol Cell 2002 Mar;13(3):854-65
Endoplasmic reticulum dynamics, inheritance, and cytoskeletal interactions in
budding yeast.
Fehrenbacher KL, Davis D, Wu M, Boldogh I, Pon LA.
Department of Anatomy and Cell Biology, Columbia University, College of
Physicians and Surgeons, New York, New York 10032.
The endoplasmic reticulum (ER) in Saccharomyces cerevisiae consists of a
reticulum underlying the plasma membrane (cortical ER) and ER associated with
the nuclear envelope (nuclear ER). We used a Sec63p-green fluorescent protein
fusion protein to study motility events associated with inheritance of cortical
ER and nuclear ER in living yeast cells. During M phase before nuclear
migration, we observed thick, apparently rigid tubular extensions emanating from
the nuclear ER that elongate, undergo sweeping motions along the cell cortex,
and shorten. Two findings support a role for microtubules in this process.
First, extension of tubular structures from the nuclear ER is inhibited by
destabilization of microtubules. Second, astral microtubules, structures that
undergo similar patterns of extension, cortical surveillance and retraction,
colocalize with nuclear ER extensions. During S and G(2) phases of the cell
cycle, we observed anchorage of the cortical ER at the site of bud emergence and
apical bud growth. Thin tubules of the ER that extend from the anchored cortical
ER display undulating, apparently random movement and move into the bud as it
grows. Finally, we found that cortical ER morphology is sensitive to a
filamentous actin-destabilizing drug, latrunculin-A, and to mutations in the
actin-encoding ACT1 gene. Our observations support 1) different mechanisms and
cytoskeletal mediators for the inheritance of nuclear and cortical ER elements
and 2) a mechanism for cortical ER inheritance that is cytoskeleton dependent
but relies on anchorage, not directed movement.
PMID: 11907267 [PubMed - in process]
2: Genetics 2002 Mar;160(3):923-34
The Novel Adaptor Protein, Mti1p, and Vrp1p, a Homolog of Wiskott-Aldrich
Syndrome Protein-Interacting Protein (WIP), May Antagonistically Regulate Type I
Myosins in Saccharomyces cerevisiae.
Mochida J, Yamamoto T, Fujimura-Kamada K, Tanaka K.
Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido
University Graduate School of Medicine, Sapporo, Hokkaido, 060-0815, Japan.
Type I myosins in yeast, Myo3p and Myo5p (Myo3/5p), are involved in the
reorganization of the actin cytoskeleton. The SH3 domain of Myo5p regulates the
polymerization of actin through interactions with both Las17p, a homolog of
mammalian Wiskott-Aldrich syndrome protein (WASP), and Vrp1p, a homolog of
WASP-interacting protein (WIP). Vrp1p is required for both the localization of
Myo5p to cortical patch-like structures and the ATP-independent interaction
between the Myo5p tail region and actin filaments. We have identified and
characterized a new adaptor protein, Mti1p (Myosin tail region-interacting
protein), which interacts with the SH3 domains of Myo3/5p. Mti1p
co-immunoprecipitated with Myo5p and Mti1p-GFP co-localized with cortical actin
patches. A null mutation of MTI1 exhibited synthetic lethal phenotypes with
mutations in SAC6 and SLA2, which encode actin-bundling and cortical
actin-binding proteins, respectively. Although the mti1 null mutation alone did
not display any obvious phenotype, it suppressed vrp1 mutation phenotypes,
including temperature-sensitive growth, abnormally large cell morphology,
defects in endocytosis and salt-sensitive growth. These results suggest that
Mti1p and Vrp1p antagonistically regulate type I myosin functions.
PMID: 11901111 [PubMed - in process]
3: J Mol Biol 2002 Mar 8;316(5):1071-81
Mechanistic Implications for Escherichia coli Cofactor-dependent
Phosphoglycerate Mutase Based on the High-resolution Crystal Structure of a
Vanadate Complex.
Bond CS, White MF, Hunter WN.
Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust
Biocentre, University of Dundee, Dundee, DD1 5EH, UK
The structure of Escherichia coli cofactor-dependent phosphoglycerate mutase
(dPGM), complexed with the potent inhibitor vanadate, has been determined to a
resolution of 1.30A (R-factor 0.159; R-free 0.213). The inhibitor is present in
the active site, principally as divanadate, but with evidence of additional
vanadate moieties at either end, and representing a different binding mode to
that observed in the structural homologue prostatic acid phosphatase. The
analysis reveals the enzyme-ligand interactions involved in inhibition of the
mutase activity by vanadate and identifies a water molecule, observed in the
native E.coli dPGM structure which, once activated by vanadate, may
dephosphorylate the active protein. Rather than reflecting the active
conformation previously observed for E.coli dPGM, the inhibited protein's
conformation resembles that of the inactive dephosphorylated Saccharomyces
cerevisiae dPGM. The provision of a high-resolution structure of both active and
inactive forms of dPGM from a single organism, in conjunction with computational
modelling of substrate molecules in the active site provides insight into the
binding of substrates and the specific interactions necessary for three
different activities, mutase, synthase and phosphatase, within a single active
site. The sequence similarity of E.coli and human dPGMs allows us to correlate
stucture with clinical pathology. Copyright 2002 Elsevier Science Ltd.
PMID: 11884145 [PubMed - in process]
4: J Mol Biol 2002 Mar 1;316(4):955-68
Implications for the Ubiquitination Reaction of the Anaphase-promoting Complex
from the Crystal Structure of the Doc1/Apc10 Subunit.
Au SW, Leng X, Harper JW, Barford D.
Section of Structural Biology, Chester Beatty Laboratories, Institute of Cancer
Research, 237 Fulham Road, London, SW3 6JB, UK
The anaphase-promoting complex (APC) is a multi-subunit E3 protein ubiquitin
ligase that is responsible for the metaphase to anaphase transition and the exit
from mitosis. One of the subunits of the APC that is required for its
ubiquitination activity is Doc1/Apc10, a protein composed of a Doc1 homology
domain that has been identified in a number of diverse putative E3 ubiquitin
ligases. Here, we present the crystal structure of Saccharomyces cerevisiae
Doc1/Apc10 at 2.2A resolution. The Doc1 homology domain forms a beta-sandwich
structure that is related in architecture to the galactose-binding domain of
galactose oxidase, the coagulation factor C2 domain and a domain of XRCC1.
Residues that are invariant amongst Doc1/Apc10 sequences, including a
temperature-sensitive mitotic arrest mutant, map to a beta-sheet region of the
molecule, whose counterpart in galactose oxidase, the coagulation factor C2
domains and XRCC1, mediate bio-molecular interactions. This finding suggests the
identification of the functionally important and conserved region of Doc1/Apc10
and, since invariant residues of Doc1/Apc10 colocalise with conserved residues
of other Doc1 homology domains, we propose that the Doc1 homology domains
perform common ubiquitination functions in the APC and other E3 ubiquitin
ligases. Copyright 2002 Elsevier Science Ltd.
PMID: 11884135 [PubMed - in process]
5: Proc Natl Acad Sci U S A 2002 Mar 5;99(5):2684-9
Interactions among the protein and RNA subunits of Saccharomyces cerevisiae
nuclear RNase P.
Houser-Scott F, Xiao S, Millikin CE, Zengel JM, Lindahl L, Engelke DR.
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI
48109-0606; and Department of Biological Sciences, University of
Maryland--Baltimore County, Baltimore, MD 21250.
Ribonuclease P (RNase P) is a ubiquitous endoribonuclease that cleaves precursor
tRNAs to generate mature 5prime prime or minute termini. Although RNase P from
all kingdoms of life have been found to have essential RNA subunits, the number
and size of the protein subunits ranges from one small protein in bacteria to at
least nine proteins of up to 100 kDa. In Saccharomyces cerevisiae nuclear RNase
P, the enzyme is composed of ten subunits: a single RNA and nine essential
proteins. The spatial organization of these components within the enzyme is not
yet understood. In this study we examine the likely binary protein--protein and
protein--RNA subunit interactions by using directed two- and three-hybrid tests
in yeast. Only two protein subunits, Pop1p and Pop4p, specifically bind the RNA
subunit. Pop4p also interacted with seven of the other eight protein subunits.
The remaining protein subunits all showed one or more specific protein--protein
interactions with the other integral protein subunits. Of particular interest
was the behavior of Rpr2p, the only protein subunit found in RNase P but not in
the closely related enzyme, RNase MRP. Rpr2p interacts strongly with itself as
well as with Pop4p. Similar interactions with self and Pop4p were also detected
for Snm1p, the only unique protein subunit so far identified in RNase MRP. This
observation is consistent with Snm1p and Rpr2p serving analogous functions in
the two enzymes. This study provides a low-resolution map of the multisubunit
architecture of the ribonucleoprotein enzyme, nuclear RNase P from S.
cerevisiae.
PMID: 11880623 [PubMed - in process]
6: Mol Cell Biol 2002 Mar;22(6):1615-25
Transcription activator interactions with multiple SWI/SNF subunits.
Neely KE, Hassan AH, Brown CE, Howe L, Workman JL.
Department of Biochemistry and Molecular Biology, Howard Hughes Medical
Institute, The Pennsylvania State University, University Park, PA 16802, USA.
We have previously shown that the yeast SWI/SNF complex stimulates in vitro
transcription from chromatin templates in an ATP-dependent manner. SWI/SNF
function in this regard requires the presence of an activator with which it can
interact directly, linking activator recruitment of SWI/SNF to transcriptional
stimulation. In this study, we determine the SWI/SNF subunits that mediate its
interaction with activators. Using a photo-cross-linking label transfer
strategy, we show that the Snf5, Swi1, and Swi2/Snf2 subunits are contacted by
the yeast acidic activators, Gcn4 and Hap4, in the context of the intact native
SWI/SNF complex. In addition, we show that the same three subunits can interact
individually with acidic activation domains, indicating that each subunit
contributes to binding activators. Furthermore, mutations that reduce the
activation potential of these activators also diminish its interaction with each
of these SWI/SNF subunits. Thus, three distinct subunits of the SWI/SNF complex
contribute to its interactions with activation domains.
PMID: 11865042 [PubMed - indexed for MEDLINE]
7: J Mol Biol 2001 Nov 30;314(3):563-75
Solution structures of two FHA1-phosphothreonine peptide complexes provide
insight into the structural basis of the ligand specificity of FHA1 from yeast
Rad53.
Yuan C, Yongkiettrakul S, Byeon IJ, Zhou S, Tsai MD.
Department of Chemistry, The Ohio State University, Columbus OH 43210, USA.
Rad53, a yeast checkpoint protein involved in regulating the repair of DNA
damage, contains two forkhead-associated domains, FHA1 and FHA2. Previous
combinatorial library screening has shown that FHA1 strongly selects peptides
containing a pTXXD motif. Subsequent location of this motif within the sequence
of Rad9, the target protein, coupled with spectroscopic analysis has led to
identification of a tight binding sequence that is likely the binding site of
FHA1: (188)SLEV(pT)EADATFVQ(200). We present solution structures of FHA1 in
complex with this pT-peptide and with another Rad9-derived pT-peptide that has
ca 30-fold lower affinity, (148)KKMTFQ(pT)PTDPLE(160). Both complexes showed
intermolecular NOEs predominantly between three peptide residues (pT, +1, and +2
residues) and five FHA1 residues (S82, R83, S85, T106, and N107). Furthermore,
the following interactions were implicated on the basis of chemical shift
perturbations and structural analysis: the phosphate group of the pT residue
with the side-chain amide group of N86 and the guanidino group of R70, and the
carboxylate group of Asp (at the +3 position) with the guanidino group of R83.
The generated structures revealed a similar binding mode adopted by these two
peptides, suggesting that pT and the +3 residue Asp are the major contributors
to binding affinity and specificity, while +1 and +2 residues could provide
additional fine-tuning. It was also shown that FHA1 does not bind to the
corresponding pS-peptides or a related pY-peptide. We suggest that
differentiation between pT and pS-peptides by FHA1 can be attributed to
hydrophobic interactions between the methyl group of the pT residue and the
aliphatic protons of R83, S85, and T106 from FHA1. Copyright 2001 Academic
Press.
PMID: 11846567 [PubMed - indexed for MEDLINE]
8: Nucleic Acids Res 2002 Feb 15;30(4):1029-37
Functional and physical interactions between components of the Prp19p-associated
complex.
Chen CH, Yu WC, Tsao TY, Wang LY, Chen HR, Lin JY, Tsai WY, Cheng SC.
Institute of Molecular Biology, Academia Sinica, Nankang, Taiwan, Republic of
China.
The Prp19p-associated complex is essential for the yeast pre-mRNA splicing
reaction. The complex consists of at least eight protein components, but is not
tightly associated with spliceosomal snRNAs. By a combination of genetic and
biochemical methods we previously identified four components of this complex,
Ntc25p, Ntc85p, Ntc30p and Ntc20p, all of them being novel splicing factors. We
have now identified three other components of the complex, Ntc90p, Ntc77p and
Ntc31p. These three proteins were also associated with the spliceosome during
the splicing reaction in the same manner as Prp19p, concurrently with or
immediately after dissociation of U4 snRNA. Two-hybrid analysis revealed that
none of these proteins interacted with Prp19p or Ntc25p, but all interacted with
Ntc85p. An interaction network between the identified components of the
Prp19p-associated complex is demonstrated. Biochemical analysis revealed that
Ntc90p, Ntc31p, Ntc30p and Ntc20p form a subcomplex, which, through interacting
with Ntc85p and Ntc77p, can associate with Prp19p and Ntc25p to form the
Prp19p-associated complex. Genetic analysis suggests that Ntc31p, Ntc30p and
Ntc20p may play roles in modulating the function of Ntc90p.
PMID: 11842115 [PubMed - indexed for MEDLINE]
9: Biochemistry 2002 Feb 19;41(7):2409-20
Novel interactions of Saccharomyces cerevisiae type 1 protein phosphatase
identified by single-step affinity purification and mass spectrometry.
Walsh EP, Lamont DJ, Beattie KA, Stark MJ.
School of Life Sciences Biocentre, University of Dundee, Dundee DD1 5EH, UK.
The catalytic subunit of Saccharomyces cerevisiae type 1 protein phosphatase
(PP1(C)) is encoded by the essential gene GLC7 and is involved in regulating
diverse cellular processes. To identify potential regulatory or targeting
subunits of yeast PP1(C), we tagged Glc7p at its amino terminus with protein A
and affinity-purified Glc7p protein complexes from yeast. The purified proteins
were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and identified by peptide mass fingerprint analysis using
matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. To
confirm the accuracy of our identifications, peptides from some of the proteins
were also sequenced using high-performance liquid chromatography (HPLC) coupled
to tandem mass spectrometry. Only four of the Glc7p-associated proteins that we
identified (Mhp1p, Bni4p, Ref2p, and Sds22p) have previously been shown to
interact with Glc7p, and multiple components of the CPF (cleavage and
polyadenylation factor) complex involved in messenger RNA 3'-end processing were
present as major components in the Glc7p-associated protein fraction. To confirm
the interaction of Glc7p with this complex, we used the same approach to purify
and characterize the components of the yeast CPF complex using protein A-tagged
Pta1p. Six known components of the yeast (CPF) complex, together with Glc7p,
were identified among the Pta1p-associated polypeptides using peptide mass
fingerprint analysis. Thus Glc7p is a novel component of the CPF complex and may
therefore be involved regulating mRNA 3'-end processing.
PMID: 11841235 [PubMed - in process]
10: Biotechnol Prog 2002 Jan-Feb;18(1):116-23
Recovery of recombinant cutinase using detergent foam.
Fernandes S, Mattiasson B, Hatti-Kaul R.
Department of Biotechnology, Center for Chemistry & Chemical Engineering, Lund
University, P.O. Box 124, S-221 00 Lund, Sweden.
Foam generated by vigorous stirring of a nonionic detergent, Triton X-114, was
used for the recovery of recombinant cutinase expressed by Saccharomyces
cerevisiae. The enzyme with a hydrophobic fusion tag, (Trp-Pro)(4), was
recovered with a higher yield as compared to the wild-type cutinase, indicating
the involvement of hydrophobic interactions in protein isolation with the foam.
The influence of various factors including volume, dilution, pH, different
additives, and cell concentration in the medium on enzyme recovery was
investigated. Interaction of the enzyme with detergent was monitored using
fluorescence spectroscopy. No significant changes in protein conformation after
the isolation procedure were observed using circular dichroism.
PMID: 11822909 [PubMed - in process]
11: Proc Natl Acad Sci U S A 2002 Feb 5;99(3):1253-8
Probing protein conformational changes in living cells by using designer binding
proteins: application to the estrogen receptor.
Koide A, Abbatiello S, Rothgery L, Koide S.
Department of Biochemistry and Biophysics, University of Rochester School of
Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
A challenge in understanding the mechanism of protein function in biology is to
establish the correlation between functional form in the intracellular
environment and high-resolution structures obtained with in vitro techniques.
Here we present a strategy to probe conformational changes of proteins inside
cells. Our method involves: (i) engineering binding proteins to different
conformations of a target protein, and (ii) using them to sense changes in the
surface property of the target in cells. We probed ligand-induced conformational
changes of the estrogen receptor alpha (ER alpha) ligand-binding domain (LBD).
By using yeast two-hybrid techniques, we first performed combinatorial library
screening of "monobodies" (small antibody mimics using the scaffold of a
fibronectin type III domain) for clones that bind to ER alpha and then
characterized their interactions with ER alpha in the nucleus, the native
environment of ER alpha, in the presence of various ligands. A library using a
highly flexible loop yielded monobodies that specifically recognize a particular
ligand complex of ER alpha, and the pattern of monobody specificity was
consistent with the structural differences found in known crystal structures of
ER alpha-LBD. A more restrained loop library yielded clones that bind both
agonist- and antagonist-bound ER alpha. Furthermore, we found that a deletion of
the ER alpha F domain that is C-terminally adjacent to the LBD increased the
crossreactivity of monobodies to the apo-ER alpha-LBD, suggesting a dynamic
nature of the ER alpha-LBD conformation and a role of the F domain in
restraining the LBD in an inactive conformation.
PMID: 11818562 [PubMed - indexed for MEDLINE]
12: J Mol Biol 2002 Jan 25;315(4):809-18
Protein-protein interactions of hCsl4p with other human exosome subunits.
Raijmakers R, Noordman YE, van Venrooij WJ, Pruijn GJ.
Department of Biochemistry, University of Nijmegen, Nijmegen, The Netherlands.
The exosome is a complex of 3'-->5' exoribonucleases, which functions in a
variety of cellular processes, all requiring the processing or degradation of
RNA. We demonstrate that the two human proteins hCsl4p and hRrp42p, which have
been identified on the basis of their sequence homology with Saccharomyces
cerevisiae proteins, are associated with the human exosome. By mammalian
two-hybrid and GST pull-down assays, we show that the hCsl4p protein interacts
directly with two other exosome proteins, hRrp42p and hRrp46p. Mutants of hCsl4p
that fail to interact with either hRrp42p or hRrp46p are also not able to
associate with exosome complexes in vivo. These results indicate that the
association of hCsl4p with the exosome is mediated by protein-protein
interactions with hRrp42p and hRrp46p. Copyright 2002 Academic Press.
PMID: 11812149 [PubMed - indexed for MEDLINE]
13: Nature 2002 Jan 10;415(6868):180-3
Comment in:
Nature. 2002 Jan 10;415(6868):123-4.
Systematic identification of protein complexes in Saccharomyces cerevisiae by
mass spectrometry.
Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L, Adams SL, Millar A, Taylor P,
Bennett K, Boutilier K, Yang L, Wolting C, Donaldson I, Schandorff S, Shewnarane
J, Vo M, Taggart J, Goudreault M, Muskat B, Alfarano C, Dewar D, Lin Z,
Michalickova K, Willems AR, Sassi H, Nielsen PA, Rasmussen KJ, Andersen JR,
Johansen LE, Hansen LH, Jespersen H, Podtelejnikov A, Nielsen E, Crawford J,
Poulsen V, Sorensen BD, Matthiesen J, Hendrickson RC, Gleeson F, Pawson T, Moran
MF, Durocher D, Mann M, Hogue CW, Figeys D, Tyers M.
MDS Proteomics, 251 Attwell Drive, Toronto, Canada M9W 7H4, and
Staermosegaardsvej 6, DK-5230 Odense M, Denmark.
The recent abundance of genome sequence data has brought an urgent need for
systematic proteomics to decipher the encoded protein networks that dictate
cellular function. To date, generation of large-scale protein-protein
interaction maps has relied on the yeast two-hybrid system, which detects binary
interactions through activation of reporter gene expression. With the advent of
ultrasensitive mass spectrometric protein identification methods, it is feasible
to identify directly protein complexes on a proteome-wide scale. Here we report,
using the budding yeast Saccharomyces cerevisiae as a test case, an example of
this approach, which we term high-throughput mass spectrometric protein complex
identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as
baits, we detected 3,617 associated proteins covering 25% of the yeast proteome.
Numerous protein complexes were identified, including many new interactions in
various signalling pathways and in the DNA damage response. Comparison of the
HMS-PCI data set with interactions reported in the literature revealed an
average threefold higher success rate in detection of known complexes compared
with large-scale two-hybrid studies. Given the high degree of connectivity
observed in this study, even partial HMS-PCI coverage of complex proteomes,
including that of humans, should allow comprehensive identification of cellular
networks.
PMID: 11805837 [PubMed - indexed for MEDLINE]
14: Nature 2002 Jan 10;415(6868):141-7
Comment in:
Nature. 2002 Jan 10;415(6868):123-4.
Functional organization of the yeast proteome by systematic analysis of protein
complexes.
Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, Bauer A, Schultz J, Rick JM,
Michon AM, Cruciat CM, Remor M, Hofert C, Schelder M, Brajenovic M, Ruffner H,
Merino A, Klein K, Hudak M, Dickson D, Rudi T, Gnau V, Bauch A, Bastuck S, Huhse
B, Leutwein C, Heurtier MA, Copley RR, Edelmann A, Querfurth E, Rybin V, Drewes
G, Raida M, Bouwmeester T, Bork P, Seraphin B, Kuster B, Neubauer G,
Superti-Furga G.
Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
anne-claude.gavin@cellzome.com
Most cellular processes are carried out by multiprotein complexes. The
identification and analysis of their components provides insight into how the
ensemble of expressed proteins (proteome) is organized into functional units. We
used tandem-affinity purification (TAP) and mass spectrometry in a large-scale
approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We
processed 1,739 genes, including 1,143 human orthologues of relevance to human
biology, and purified 589 protein assemblies. Bioinformatic analysis of these
assemblies defined 232 distinct multiprotein complexes and proposed new cellular
roles for 344 proteins, including 231 proteins with no previous functional
annotation. Comparison of yeast and human complexes showed that conservation
across species extends from single proteins to their molecular environment. Our
analysis provides an outline of the eukaryotic proteome as a network of protein
complexes at a level of organization beyond binary interactions. This
higher-order map contains fundamental biological information and offers the
context for a more reasoned and informed approach to drug discovery.
PMID: 11805826 [PubMed - indexed for MEDLINE]
15: Mol Cell 2002 Jan;9(1):31-44
Comment in:
Mol Cell. 2002 Jan;9(1):8-9.
Composition and functional characterization of the yeast spliceosomal
penta-snRNP.
Stevens SW, Ryan DE, Ge HY, Moore RE, Young MK, Lee TD, Abelson J.
California Institute of Technology, Division of Biology 147-75, Pasadena, CA
91125, USA.
Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For
each round of splicing, the spliceosome assembles de novo in a series of
ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein
interactions. As currently understood, spliceosome assembly proceeds by addition
of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form
functional splicing complexes. We characterized a 45S yeast penta-snRNP which
contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The
particle is functional in extracts and, when supplied with soluble factors, is
capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate
prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise
addition of discrete snRNPs.
PMID: 11804584 [PubMed - indexed for MEDLINE]
16: J Cell Sci 2002 Jan 1;115(Pt 1):195-206
Essential functions of Sds22p in chromosome stability and nuclear localization
of PP1.
Peggie MW, MacKelvie SH, Bloecher A, Knatko EV, Tatchell K, Stark MJ.
Division of Gene Regulation and Expression, School of Life Sciences, MSI/WTB
Complex, University of Dundee, Dundee, DD1 5EH, UK.
Sds22p is a conserved, leucine-rich repeat protein that interacts with the
catalytic subunit of protein phosphatase 1 (PP1(C)) and which has been proposed
to regulate one or more functions of PP1(C) during mitosis. Here we show that
Saccharomyces cerevisiae Sds22p is a largely nuclear protein, most of which is
present as a sTable 1:1 complex with yeast PP1(C) (Glc7p). Temperature-sensitive
(Ts(-)) S. cerevisiae sds22 mutants show profound chromosome instability at
elevated growth temperatures but do not confer a cell cycle stage-specific
arrest. In the sds22-6 Ts(-) mutant, nuclear Glc7p is both reduced in level and
aberrantly localized at 37 degrees C and the interaction between Glc7p and
Sds22p in vitro is reduced at higher temperatures, consistent with the in vivo
Ts(-) growth defect. Like some glc7 mutations, sds22-6 can suppress the Ts(-)
growth defect associated with ipl1-2, a loss of function mutation in a protein
kinase that is known to work in opposition to PP1 on at least two nuclear
substrates. This, together with reciprocal genetic interactions between GLC7 and
SDS22, suggests that Sds22p functions positively with Glc7p to promote
dephosphorylation of nuclear substrates required for faithful transmission of
chromosomes during mitosis, and this role is at least partly mediated by effects
of Sds22p on the nuclear distribution of Glc7p
PMID: 11801737 [PubMed - in process]
17: Genome Inform Ser Workshop Genome Inform 2001;12:123-34
The potential use of SUISEKI as a protein interaction discovery tool.
Blaschke C, Valencia A.
Protein Design Group, CNB/CSIC, Campus Universidad Autonoma, 28049 Madrid,
Spain. blaschke@cnb.uam.es
Relevant information about protein interactions is stored in textual sources.
This sources are commonly used not only as archives of what is already known but
also as information for generating new knowledge, particularly to pose
hypothesis about new possible interactions that can be inferred from the
existing ones. This task is the more creative part of scientific work in
experimental systems. We present a large-scale analysis for the prediction of
new interactions based on the interaction network for the ones already known and
detected automatically in the literature. During the last few years it has
became clear that part of the information about protein interactions could be
extracted with automatic tools, even if these tools are still far from perfect
and key problems such as detection of protein names are not completely solved.
We have developed a integrated automatic approach, called SUISEKI (System for
Information Extraction on Interactions), able to extract protein interactions
from collections of Medline abstracts. Previous experiments with the system have
shown that it is able to extract almost 70% of the interactions present in
relatively large text corpus, with an accuracy of approximately 80% (for the
best defined interactions) that makes the system usable in real scenarios, both
at the level of extraction of protein names and at the level of extracting
interaction between them. With the analysis of the interaction map of
Saccharomyces cerevisiae we show that interactions published in the years
2000/2001 frequently correspond to proteins or genes that were already very
close in the interaction network deduced from the literature published before
these years and that they are often connected to the same proteins. That is,
discoveries are commonly done among highly connected entities. Some biologically
relevant examples illustrate how interactions described in the year 2000 could
have been proposed as reasonable working hypothesis with the information
previously available in the automatically extracted network of interactions.
PMID: 11791231 [PubMed - in process]
18: Biochem Biophys Res Commun 2002 Jan 18;290(2):676-81
Saccharomyces cerevisiae Pra1p/Yip3p interacts with Yip1p and Rab proteins.
Calero M, Collins RN.
Department of Molecular Medicine, Cornell University, Ithaca, New York
14853-6401, USA.
The regulation of membrane traffic involves the Rab family of Ras-related
GTPases, of which there are a total of 11 members in the yeast Saccharomyces
cerevisiae. Previous work has identified PRA1 as a dual prenylated Rab GTPase
and VAMP2 interacting protein [Martinic et al. (1999) J. Biol. Chem. 272,
26991-26998]. In this study we demonstrate that the yeast counterpart of PRA1
interacts with Rab proteins and with Yip1p, a membrane protein of unknown
function that has been reported to interact specifically with the Rab proteins
Ypt1p and Ypt31p. Yeast Pra1p/Yip3p is a factor capable of biochemical
interaction with a panel of different Rab proteins and does not show in vitro
specificity for any particular Rab. The interactions between Pra1p/Yip3p and Rab
proteins are dependent on the presence of the Rab protein C-terminal cysteines
and require C-terminal prenylation.
PMID: 11785952 [PubMed - indexed for MEDLINE]
19: Plant Mol Biol 2001 Dec;47(6):771-83
Identification of a S-ribonuclease-binding protein in Petunia hybrida.
Sims TL, Ordanic M.
Department of Biological Sciences and Plant Molecular Biology Center, Northern
Illinois University, DeKalb, 60115-2861, USA. tsmis@niu.edu
To investigate protein-protein interactions in gametophytic
self-incompatibility, we used a yeast two-hybrid assay to identify proteins that
could interact with the S-ribonuclease protein. These assays identified a
pollen-expressed protein, which we have named PhSBP1, that appears to bind with
a high degree of specificity to the Petunia hybrida S-ribonuclease. Although
PhSBP1 activates reporter gene expression only when expressed in tandem with a
S-RNAse bait protein, binding is not allele-specific. Sequence analysis
demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that
includes a RING-HC domain. Because many RING-finger domain proteins appear to
function as E3 ubiquitin ligases, our results suggest that ubiquitination and
protein degradation may play a role in regulating self-incompatibility
interactions. Together, these results suggest that PhSBPI may be a candidate for
the recently proposed general inhibitor (RI) of self-incompatibility
ribonucleases.
PMID: 11785938 [PubMed - indexed for MEDLINE]
20: Mol Cell Biol 2002 Feb;22(3):927-34
The Sur7p family defines novel cortical domains in Saccharomyces cerevisiae,
affects sphingolipid metabolism, and is involved in sporulation.
Young ME, Karpova TS, Brugger B, Moschenross DM, Wang GK, Schneiter R, Wieland
FT, Cooper JA.
Department of Cell Biology and Physiology, Washington University, St. Louis,
Missouri 63110, USA.
We have discovered a novel cortical patch structure in Saccharomyces cerevisiae
defined by a family of integral plasma membrane proteins, including Sur7p,
Ynl194p, and Ydl222p. Sur7p-family patches localized as cortical patches that
were immobile and stable. These patches were polarized to regions of the cell
with a mature cell wall; they were absent from small buds and the tips of many
medium-sized buds. These patches were distinct from other known cortical
structures. Digestion of the cell wall caused Sur7p patches to disassemble,
indicating that Sur7p requires cell wall-dependent extracellular interactions
for its localization as patches. sur7Delta, ydl222Delta, and ynl194Delta mutants
had reduced sporulation efficiencies. SUR7 was originally described as a
multicopy suppressor of rvs167, whose product is an actin patch component. This
suppression is probably mediated by sphingolipids, since deletion of SUR7,
YDL222, and YNL194 altered the sphingolipid content of the yeast plasma
membrane, and other SUR genes suppress rvs167 via effects on sphingolipid
synthesis. In particular, the sphingoid base length and number of hydroxyl
groups in inositol phosphorylceramides were altered in sur7Delta, ydl222Delta,
and yne194Delta strains.
PMID: 11784867 [PubMed - indexed for MEDLINE]
21: Mol Cell Biol 2002 Feb;22(3):693-703
Histone-dependent association of Tup1-Ssn6 with repressed genes in vivo.
Davie JK, Trumbly RJ, Dent SY.
Department of Biochemistry and Molecular Biology, University of Texas M. D.
Anderson Cancer Center, Houston, Texas 77030, USA.
The Tup1-Ssn6 complex regulates diverse classes of genes in Saccharomyces
cerevisiae and serves as a model for corepressor functions in many organisms.
Tup1-Ssn6 does not directly bind DNA but is brought to target genes through
interactions with sequence-specific DNA binding factors. Full repression by
Tup1-Ssn6 appears to require interactions with both the histone tails and
components of the general transcription machinery, although the relative
contribution of these two pathways is not clear. Here, we map Tup1 locations on
two classes of Tup1-Ssn6-regulated genes in vivo via chromatin
immunoprecipitations. Distinct profiles of Tup1 are observed on a cell-specific
genes and DNA damage-inducible genes, suggesting that alternate repressive
architectures may be created on different classes of repressed genes. In both
cases, decreases in acetylation of histone H3 colocalize with Tup1. Strikingly,
although loss of the Srb10 mediator protein had no effect on Tup1 localization,
both histone tail mutations and histone deacetylase mutations crippled the
association of Tup1 with target loci. Together with previous findings that
Tup1-Ssn6 physically associates with histone deacetylase activities, these
results indicate that the repressor complex alters histone modification states
to facilitate interactions with histones and that these interactions are
required to maintain a stable repressive state.
PMID: 11784848 [PubMed - indexed for MEDLINE]
22: RNA 2001 Dec;7(12):1693-701
A genome-wide survey of RS domain proteins.
Boucher L, Ouzounis CA, Enright AJ, Blencowe BJ.
Banting and Best Department of Medical Research, C.H. Best Institute, University
of Toronto, Ontario, Canada.
Domains rich in alternating arginine and serine residues (RS domains) are
frequently found in metazoan proteins involved in pre-mRNA splicing. The RS
domains of splicing factors associate with each other and are important for the
formation of protein-protein interactions required for both constitutive and
regulated splicing. The prevalence of the RS domain in splicing factors suggests
that it might serve as a useful signature for the identification of new proteins
that function in pre-mRNA processing, although it remains to be determined
whether RS domains also participate in other cellular functions. Using database
search and sequence clustering methods, we have identified and categorized RS
domain proteins encoded within the entire genomes of Homo sapiens, Drosophila
melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae. This
genome-wide survey revealed a surprising complexity of RS domain proteins in
metazoans with functions associated with chromatin structure, transcription by
RNA polymerase II, cell cycle, and cell structure, as well as pre-mRNA
processing. Also identified were RS domain proteins in S. cerevisiae with
functions associated with cell structure, osmotic regulation, and cell cycle
progression. The results thus demonstrate an effective strategy for the genomic
mining of RS domain proteins. The identification of many new proteins using this
strategy has provided a database of factors that are candidates for forming RS
domain-mediated interactions associated with different steps in pre-mRNA
processing, in addition to other cellular functions.
PMID: 11780626 [PubMed - indexed for MEDLINE]
23: Genetics 2001 Dec;159(4):1539-45
(CA/TG) microsatellite sequences escape the inhibition of recombination by
mismatch repair in Saccharomyces cerevisiae.
Gendrel CG, Dutreix M.
UMR-CNRS 2027, Institut Curie-section de Recherche, Universite Paris-Sud,
F-91405 Orsay, France.
Sequence divergence reduces the frequency of recombination, a process that is
dependent on the activity of the mismatch repair system. In the yeast
Saccharomyces cerevisiae, repair of mismatches results in gene conversion or
restoration, whereas failure to repair mismatches results in postmeiotic
segregation (PMS). By examining the conversion and PMS in yeast strains
deficient in various MMR genes and heterozygous for large inserts (107 bp) with
either a mixed sequence or a 39 (CA/TG) repetitive microsatellite sequence, we
demonstrate that: (1) the inhibition of conversion by large inserts depends upon
a complex containing both Msh2 and Pms1 proteins; (2) conversion is not
inhibited if the single-stranded DNA loop in the heteroduplex is the
microsatellite sequence; and (3) large heteroduplex loops with random sequence
or repetitive sequence might be repaired by two complexes, containing either
Msh2 or Pms1. Our results suggest that inhibition of recombination by
heterologous inserts and large loop repair are not processed by the same MMR
complexes. We propose that the inhibition of conversion by large inserts is due
to recognition by the Msh2/Pms1 complex of mismatches created by intrastrand
interactions in the heteroduplex loop.
PMID: 11779795 [PubMed - indexed for MEDLINE]
24: Genetics 2001 Dec;159(4):1435-48
Overactivation of the protein kinase C-signaling pathway suppresses the defects
of cells lacking the Rho3/Rho4-GAP Rgd1p in Saccharomyces cerevisiae.
de Bettignies G, Thoraval D, Morel C, Peypouquet MF, Crouzet M.
Laboratoire de Biologie Moleculaire et de Sequencage, UMR CNRS 5095, Bordeaux
Cedex, France.
The nonessential RGD1 gene encodes a Rho-GTPase activating protein for the Rho3
and Rho4 proteins in Saccharomyces cerevisiae. Previous studies have revealed
genetic interactions between RGD1 and the SLG1 and MID2 genes, encoding two
putative sensors for cell integrity signaling, and VRP1 encoding an actin and
myosin interacting protein involved in polarized growth. To better understand
the role of Rgd1p, we isolated multicopy suppressor genes of the cell lethality
of the double mutant rgd1Delta mid2Delta. RHO1 and RHO2 encoding two small
GTPases, MKK1 encoding one of the MAP-kinase kinases in the protein kinase C
(PKC) pathway, and MTL1, a MID2-homolog, were shown to suppress the rgd1Delta
defects strengthening the functional links between RGD1 and the cell integrity
pathway. Study of the transcriptional activity of Rlm1p, which is under the
control of Mpk1p, the last kinase of the PKC pathway, and follow-up of the PST1
transcription, which is positively regulated by Rlm1p, indicate that the lack of
RGD1 function diminishes the PKC pathway activity. We hypothesize that the
rgd1Delta inactivation, at least through the hyperactivation of the small
GTPases Rho3p and Rho4p, alters the secretory pathway and/or the actin
cytoskeleton and decreases activity of the PKC pathway.
PMID: 11779787 [PubMed - indexed for MEDLINE]
25: Invest Ophthalmol Vis Sci 2002 Jan;43(1):176-82
Protein interactions with myocilin.
Wentz-Hunter K, Ueda J, Yue BY.
Department of Ophthalmology and Visual Sciences, University of Illinois at
Chicago College of Medicine, Chicago, Illinois 60612, USA.
PURPOSE: To identify factors that interact in vivo with myocilin, a glaucoma
gene product. METHODS: The yeast two-hybrid system with myocilin as the bait and
a human skeletal muscle cDNA library as the prey was used to identify potential
factors that interact with myocilin. Interactions were also examined in bovine
trabecular meshwork (TM) cells through a mammalian two-hybrid system.
Biochemical coimmunoprecipitation from both human TM cell lysate and in vitro
translated proteins was also used to confirm results obtained from yeast
analysis. RESULTS: Twenty positive clones isolated through yeast two-hybrid
screening were deemed potential myocilin partners. Sequence analysis determined
that two of them encoded for myocilin from amino acids 64 to 268. Myocilin was
also found to interact with a component of the myosin motor protein, myosin
regulatory light chain (RLC). The myocilin-myocilin and myocilin-RLC
interactions revealed by the yeast system were further confirmed and
demonstrated in cultured TM cells, by means of a mammalian two-hybrid system,
and through biochemical coimmunoprecipitation, subcellular fractionation,
immunofluorescence, and immunogold double labeling. CONCLUSIONS: These results
indicate that myocilin can form homomultimers in vivo, independent of the
olfactomedin-like domain. Further analysis established that the leucine zipper
motif of myocilin may be necessary for the myocilin-RLC interaction. The
interaction of myocilin with RLC, a component of the myosin motor protein
complex, implies a role for myocilin in the actomyosin system, linking in turn
this novel protein to functional status of the TM.
PMID: 11773029 [PubMed - indexed for MEDLINE]
26: Biochem J 2002 Jan 15;361(Pt 2):243-54
Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho
GDP dissociation inhibitor and their extraction from biological membranes.
Forget MA, Desrosiers RR, Gingras D, Beliveau R.
Laboratoire de medecine moleculaire, Hopital Sainte-Justine-Universite du Quebec
a Montreal, P.O. Box 8888, Centre-ville station, Montreal, Quebec, Canada H3C
3P8.
The Rho GDP dissociation inhibitor (RhoGDI) regulates the
activation-inactivation cycle of Rho small GTPases, such as Cdc42 and RhoA, by
extracting them from the membrane. To study the roles of Mg(2+),
phosphatidylinositol 4,5-bisphosphate (PIP(2)), ionic strength and
phosphorylation on the interactions of RhoGDI with Cdc42 and RhoA, we developed
a new, efficient and reliable method to produce prenylated Rho proteins using
the yeast Saccharomyces cerevisiae. It has been previously reported that protein
kinase A (PKA)-treatment of isolated membranes increased RhoA extraction from
membranes by RhoGDI [Lang, Gesbert, Delespine-Carmagnat, Stancou, Pouchelet and
Bertoglio (1996) EMBO J. 16, 510-519]. In the present study, we used an in vitro
affinity chromatography system to show that phosphorylation of RhoA and Cdc42
significantly increased their interaction with RhoGDI under physiological
conditions of ionic strength. This increase was independent of the nucleotide
(GDP or guanosine 5'-[gamma-thio]triphosphate) loaded on to the Rho proteins, as
well as of Mg(2+) and PIP(2). Moreover, dephosphorylation of rat brain membranes
by alkaline phosphatase significantly decreased the extraction of RhoA and Cdc42
by RhoGDI. Subsequent re-phosphorylation by PKA restored the extraction levels,
indicating the reversibility of this process. These results clearly demonstrate
that the phosphorylation states of Cdc42 and RhoA regulate their interactions
with RhoGDI and, consequently, their extraction from rat brain membranes. We
therefore suggest that phosphorylation is a mechanism of regulation of Cdc42 and
RhoA activity that is independent of GDP-GTP cycling.
PMID: 11772396 [PubMed - indexed for MEDLINE]
27: J Biol Chem 2002 Feb 8;277(6):3813-22
Active site mutations in DNA topoisomerase I distinguish the cytotoxic
activities of camptothecin and the indolocarbazole, rebeccamycin.
Woo MH, Vance JR, Marcos AR, Bailly C, Bjornsti MA.
Department of Molecular Pharmacology, St. Jude Children's Research Hospital,
Memphis, Tennessee 38105, USA.
DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the
cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that
target Top1p, such as indolocarbazoles, are under clinical development. However,
whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning
remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin
R-3 activity was examined in vitro and in yeast. Using a series of Top1p
mutants, where substitution of residues around the active site tyrosine has
well-defined effects on enzyme catalysis, we show that catalytically active,
CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not
inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA
complexes. Similar results were obtained with human Top1p, when Ser or His were
substituted for Asn-722. The mutations altered enzyme function and sensitivity
to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1delta,
rad52delta yeast cells expressing human Top1p, but not catalytically inactive
Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular
target of R-3 and indicate that distinct drug-enzyme interactions at the active
site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance
to one poison may potentiate cell sensitivity to structurally distinct compounds
that also target Top1p.
PMID: 11733535 [PubMed - indexed for MEDLINE]
28: Biochemistry 2001 Dec 25;40(51):15562-9
Identification of yeast cofilin residues specific for actin monomer and PIP2
binding.
Ojala PJ, Paavilainen V, Lappalainen P.
Program in Cellular Biotechnology, Institute of Biotechnology, University of
Helsinki, P.O. Box 56, 00014 Helsinki, Finland.
Cofilin/ADF is a ubiquitous actin-binding protein that is important for rapid
actin dynamics in vivo. The long alpha-helix (helix 3 in yeast cofilin) forms
the most highly conserved region in cofilin/ADF proteins, and residues in the
NH2-terminal half of this alpha-helix have been shown to be essential for actin
binding in cofilin/ADF. Recent studies also suggested that the basic residues in
the COOH-terminal half of this alpha-helix would play an important role in
F-actin binding. In contrast to these studies, we show here that the charged
residues in the COOH-terminal half of helix 3 are not important for actin
filament binding in yeast cofilin. Mutations in these residues, however, result
in a small defect in actin monomer interactions. We also show that yeast cofilin
can differentiate between various phosphatidylinositides, and mapped the
PI(4,5)P2 binding site by using a collection of cofilin mutants. The PI(4,5)P2
binding site of yeast cofilin is a large positively charged surface that
consists of residues in helix 3 as well as residues in other parts of the
cofilin molecule. This suggests that cofilin/ADF proteins probably interact
simultaneously with more than one PI(4,5)P2 molecule. The PI(4,5)P2-binding site
overlaps with areas that are important for F-actin binding, explaining why the
actin-related activities of cofilin/ADF are inhibited by PI(4,5)P2. The
biological roles of actin and PI(4,5)P2 interactions of cofilin are discussed in
light of phenotypes of specific yeast strains carrying mutations in residues
that are important for actin and PI(4,5)P2 binding.
PMID: 11747431 [PubMed - indexed for MEDLINE]
29: Biochem J 2002 Jan 1;361(Pt 1):27-34
Direct interactions between molecular chaperones heat-shock protein (Hsp) 70 and
Hsp40: yeast Hsp70 Ssa1 binds the extreme C-terminal region of yeast Hsp40 Sis1.
Qian X, Hou W, Zhengang L, Sha B.
School of Life Sciences, University of Science and Technology of China, Hefei,
People's Republic of China 230026.
Heat-shock protein 40 (Hsp40) enables Hsp70 to play critical roles in a number
of cellular processes, such as protein folding, assembly, degradation and
translocation in vivo. Hsp40 recognizes and binds non-native polypeptides and
delivers them to Hsp70. Then Hsp40 stimulates the ATPase activity of Hsp70 to
fold the polypeptides. By using yeast Hsp40 Sis1 and yeast Hsp70 Ssa1 as our
model proteins, we found that the Sis1 peptide-binding fragment interacts
directly with the full-length Ssa1 in vitro. Further studies showed that the
C-terminal lid domain of Ssa1 could interact with Sis1 peptide-binding domain
physically in vitro. The Sis1 peptide-binding fragment forms a stable complex
with the Ssa1 C-terminal lid domain in solution. The interactions between these
two proteins appear to be charge-charge interactions because high-ionic-strength
buffer can dissociate the complex. Further mapping studies showed that the Sis1
peptide-binding fragment binds the extreme C-terminal 15 amino acid residues of
Ssa1. A flexible glycine-rich region is followed by these 15 residues in the
Ssa1 primary sequence. Atomic force microscopy of the Sis1-Ssa1 complex showed
that only one end of the Ssa1 lid domain binds the Sis1 peptide-binding-fragment
dimer at the upper level of the huge groove within the Sis1 dimer. Based on the
data, we propose an "anchoring and docking" model to illustrate the mechanisms
by which Hsp40 interacts with Hsp70 and delivers the non-native polypeptide to
Hsp70.
PMID: 11743879 [PubMed - indexed for MEDLINE]
30: Chem Res Toxicol 2001 Dec;14(12):1584-9
Mechanisms of nitrogen oxide-mediated disruption of metalloprotein function: an
examination of the copper-responsive yeast transcription factor Ace1.
Shinyashiki M, Pan CJ, Switzer CH, Fukuto JM.
Department of Pharmacology, UCLA School of Medicine, Center for the Health
Sciences, Los Angeles, California 90095-1735, USA.
Nitric oxide (NO) has been found to inhibit the copper-responsive yeast
transcription factor Ace1 in an oxygen-dependent manner. However, the mechanism
responsible for NO-dependent inhibition of Ace1 remains unestablished. In the
present study, the chemical interaction of nitrogen oxide species with Ace1 was
examined using a yeast reporter system. Exposure of yeast to various nitrogen
oxides, under a variety of conditions, revealed that the oxygen-dependent
inhibition of Ace1 is due to the reaction of NO with O(2). The nitrosating
nitrogen oxide species N(2)O(3) is likely to be the disrupter of Ace1 activity.
Considering the similarity of metal-thiolate ligation in Ace1 with other
mammalian metalloproteins such as metallothionein, metal chaperones, and
zinc-finger proteins, these results help to understand the biochemical
interactions of NO with those mammalian metalloproteins.
PMID: 11743740 [PubMed - indexed for MEDLINE]
31: J Mol Biol 2001 Dec 14;314(5):1053-66
Beyond synexpression relationships: local clustering of time-shifted and
inverted gene expression profiles identifies new, biologically relevant
interactions.
Qian J, Dolled-Filhart M, Lin J, Yu H, Gerstein M.
Department of Molecular Biophysics and Biochemistry, Yale University, 266
Whitney Avenue, PO Box 208114, New Haven, CT 06520-8114, USA.
The complexity of biological systems provides for a great diversity of
relationships between genes. The current analysis of whole-genome expression
data focuses on relationships based on global correlation over a whole
time-course, identifying clusters of genes whose expression levels
simultaneously rise and fall. There are, of course, other potential
relationships between genes, which are missed by such global clustering. These
include activation, where one expects a time-delay between related expression
profiles, and inhibition, where one expects an inverted relationship. Here, we
propose a new method, which we call local clustering, for identifying these
time-delayed and inverted relationships. It is related to conventional
gene-expression clustering in a fashion analogous to the way local sequence
alignment (the Smith-Waterman algorithm) is derived from global alignment
(Needleman-Wunsch). An integral part of our method is the use of random score
distributions to assess the statistical significance of each cluster. We applied
our method to the yeast cell-cycle expression dataset and were able to detect a
considerable number of additional biological relationships between genes, beyond
those resulting from conventional correlation. We related these new
relationships between genes to their similarity in function (as determined from
the MIPS scheme) or their having known protein-protein interactions (as
determined from the large-scale two-hybrid experiment); we found that genes
strongly related by local clustering were considerably more likely than random
to have a known interaction or a similar cellular role. This suggests that local
clustering may be useful in functional annotation of uncharacterized genes. We
examined many of the new relationships in detail. Some of them were already
well-documented examples of inhibition or activation, which provide
corroboration for our results. For instance, we found an inverted expression
profile relationship between genes YME1 and YNT20, where the latter has been
experimentally documented as a bypass suppressor of the former. We also found
new relationships involving uncharacterized yeast genes and were able to suggest
functions for many of them. In particular, we found a time-delayed expression
relationship between J0544 (which has not yet been functionally characterized)
and four genes associated with the mitochondria. This suggests that J0544 may be
involved in the control or activation of mitochondrial genes. We have also
looked at other, less extensive datasets than the yeast cell-cycle and found
further interesting relationships. Our clustering program and a detailed website
of clustering results is available at
http://www.bioinfo.mbb.yale.edu/expression/cluster (or
http://www.genecensus.org/expression/cluster). Copyright 2001 Academic Press.
PMID: 11743722 [PubMed - indexed for MEDLINE]
32: Science 2001 Dec 14;294(5550):2364-8
Systematic genetic analysis with ordered arrays of yeast deletion mutants.
Tong AH, Evangelista M, Parsons AB, Xu H, Bader GD, Page N, Robinson M,
Raghibizadeh S, Hogue CW, Bussey H, Andrews B, Tyers M, Boone C.
Banting and Best Department of Medical Research, University of Toronto, Toronto
ON, Canada M5G 1L6.
In Saccharomyces cerevisiae, more than 80% of the approximately 6200 predicted
genes are nonessential, implying that the genome is buffered from the phenotypic
consequences of genetic perturbation. To evaluate function, we developed a
method for systematic construction of double mutants, termed synthetic genetic
array (SGA) analysis, in which a query mutation is crossed to an array of
approximately 4700 deletion mutants. Inviable double-mutant meiotic progeny
identify functional relationships between genes. SGA analysis of genes with
roles in cytoskeletal organization (BNI1, ARP2, ARC40, BIM1), DNA synthesis and
repair (SGS1, RAD27), or uncharacterized functions (BBC1, NBP2) generated a
network of 291 interactions among 204 genes. Systematic application of this
approach should produce a global map of gene function.
PMID: 11743205 [PubMed - indexed for MEDLINE]
33: Science 2002 Jan 11;295(5553):321-4
Comment in:
Science. 2002 Jan 11;295(5553):284-7.
A combined experimental and computational strategy to define protein interaction
networks for peptide recognition modules.
Tong AH, Drees B, Nardelli G, Bader GD, Brannetti B, Castagnoli L, Evangelista
M, Ferracuti S, Nelson B, Paoluzi S, Quondam M, Zucconi A, Hogue CW, Fields S,
Boone C, Cesareni G.
Banting and Best Department of Medical Research and Department of Molecular and
Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5G 1L6.
Peptide recognition modules mediate many protein-protein interactions critical
for the assembly of macromolecular complexes. Complete genome sequences have
revealed thousands of these domains, requiring improved methods for identifying
their physiologically relevant binding partners. We have developed a strategy
combining computational prediction of interactions from phage-display ligand
consensus sequences with large-scale two-hybrid physical interaction tests.
Application to yeast SH3 domains generated a phage-display network containing
394 interactions among 206 proteins and a two-hybrid network containing 233
interactions among 145 proteins. Graph theoretic analysis identified 59 highly
likely interactions common to both networks. Las17 (Bee1), a member of the
Wiskott-Aldrich Syndrome protein (WASP) family of actin-assembly proteins,
showed multiple SH3 interactions, many of which were confirmed in vivo by
coimmunoprecipitation.
PMID: 11743162 [PubMed - indexed for MEDLINE]
34: EMBO J 2001 Dec 17;20(24):7096-107
Subunit interaction maps for the regulatory particle of the 26S proteasome and
the COP9 signalosome.
Fu H, Reis N, Lee Y, Glickman MH, Vierstra RD.
Institute of Botany, Academia Sinica, 128, Sec 2, Academy Road, Taipei, Taiwan
115, Republic of China. hongyong@gate.sinica.edu.tw
The 26S proteasome plays a major role in eukaryotic protein breakdown,
especially for ubiquitin-tagged proteins. Substrate specificity is conferred by
the regulatory particle (RP), which can dissociate into stable lid and base
subcomplexes. To help define the molecular organization of the RP, we tested all
possible paired interactions among subunits from Saccharomyces cerevisiae by
yeast two-hybrid analysis. Within the base, a Rpt4/5/3/6 interaction cluster was
evident. Within the lid, a structural cluster formed around Rpn5/11/9/8.
Interactions were detected among synonymous subunits (Csn4/5/7/6) from the
evolutionarily related COP9 signalosome (CSN) from Arabidopsis, implying a
similar quaternary arrangement. No paired interactions were detected between
lid, base or core particle subcomplexes, suggesting that stable contacts between
them require prior assembly. Mutational analysis defined the ATPase,
coiled-coil, PCI and MPN domains as important for RP assembly. A single residue
in the vWA domain of Rpn10 is essential for amino acid analog resistance, for
degrading a ubiquitin fusion degradation substrate and for stabilizing lid-base
association. Comprehensive subunit interaction maps for the 26S proteasome and
CSN support the ancestral relationship of these two complexes.
PMID: 11742986 [PubMed - indexed for MEDLINE]
35: Mol Cell 2001 Nov;8(5):1075-83
Targeting an mRNA for decapping: displacement of translation factors and
association of the Lsm1p-7p complex on deadenylated yeast mRNAs.
Tharun S, Parker R.
Department of Molecular and Cellular Biology, Howard Hughes Medical Institute,
University of Arizona, 1007 E. Lowell, Tucson, AZ 85721, USA.
tharun@u.arizona.edu
The major pathway of eukaryotic mRNA decay involves deadenylation-dependent
decapping followed by 5' to 3' exonucleolytic degradation. By examining
interactions among mRNA decay factors, the mRNA, and key translation factors, we
have identified a critical transition in mRNP organization that leads to
decapping and degradation of yeast mRNAs. This transition occurs after
deadenylation and includes loss of Pab1p, eIF4E, and eIF4G from the mRNA and
association of the decapping activator complex, Lsm1p-7p, which enhances the
coimmunoprecipitation of a decapping enzyme complex (Dcp1p and Dcp2p) with the
mRNA. These results define an important rearrangement in mRNP organization and
suggest that deadenylation promotes mRNA decapping by both the loss of Pab1p and
the recruitment of the Lsm1p-7p complex.
PMID: 11741542 [PubMed - indexed for MEDLINE]
36: Genome Res 2001 Dec;11(12):1971-3
Is there a bias in proteome research?
Mrowka R, Patzak A, Herzel H.
Johannes-Muller-Institut fur Physiologie, Humboldt-Universitat zu Berlin,
Berlin, Germany. mrowka@rz.hu-berlin.de
Advances in technology have enabled us to take a fresh look at data acquired by
traditional single experiments and to compare them with genomewide data. The
differences can be tremendous, as we show here, in the field of proteomics. We
have compared data sets of protein-protein interactions in Saccharomyces
cerevisiae that were detected by an identical underlying technical method, the
yeast two-hybrid system. We found that the individually identified
protein-protein interactions are considerably different from those identified by
two genomewide scans. Interacting proteins in the pooled database from single
publications are much more closely related to each other with respect to
transcription profiles when compared to genomewide data. This difference may
have been introduced by two factors: by a selection process in individual
publications and by false positives in the whole-genome scans. If we assume that
the differences are a result of false positives in the whole-genome data, the
scans would contain 47%, 44%, and 91% of false positives for the UETZ, ITO-core,
and ITO-full data, respectively. If, however, the true fraction of false
positives is considerably lower than estimated here, the data from
hypothesis-driven experiments must have been subjected to a serious selection
process.
PMID: 11731485 [PubMed - indexed for MEDLINE]
37: Microbiol Mol Biol Rev 2001 Dec;65(4):570-94, table of contents
Transport into and out of the nucleus.
Macara IG.
Center for Cell Signaling, University of Virginia, Charlottesville, Virginia
22908-0577, USA. imacara@virginia.edu
A defining characteristic of eukaryotic cells is the possession of a nuclear
envelope. Transport of macromolecules between the nuclear and cytoplasmic
compartments occurs through nuclear pore complexes that span the double membrane
of this envelope. The molecular basis for transport has been revealed only
within the last few years. The transport mechanism lacks motors and pumps and
instead operates by a process of facilitated diffusion of soluble carrier
proteins, in which vectoriality is provided by compartment-specific assembly and
disassembly of cargo-carrier complexes. The carriers recognize localization
signals on the cargo and can bind to pore proteins. They also bind a small
GTPase, Ran, whose GTP-bound form is predominantly nuclear. Ran-GTP dissociates
import carriers from their cargo and promotes the assembly of export carriers
with cargo. The ongoing discovery of numerous carriers, Ran-independent
transport mechanisms, and cofactors highlights the complexity of the nuclear
transport process. Multiple regulatory mechanisms are also being identified that
control cargo-carrier interactions. Circadian rhythms, cell cycle,
transcription, RNA processing, and signal transduction are all regulated at the
level of nucleocytoplasmic transport. This review focuses on recent discoveries
in the field, with an emphasis on the carriers and cofactors involved in
transport and on possible mechanisms for movement through the nuclear pores.
Publication Types:
Review
Review, Academic
PMID: 11729264 [PubMed - indexed for MEDLINE]
38: Genetics 2001 Nov;159(3):1291-8
Probabilistic prediction of unknown metabolic and signal-transduction networks.
Gomez SM, Lo SH, Rzhetsky A.
Columbia Genome Center, Columbia University, New York, New York 10032, USA.
Regulatory networks provide control over complex cell behavior in all kingdoms
of life. Here we describe a statistical model, based on representing proteins as
collections of domains or motifs, which predicts unknown molecular interactions
within these biological networks. Using known protein-protein interactions of
Saccharomyces cerevisiae as training data, we were able to predict the links
within this network with only 7% false-negative and 10% false-positive error
rates. We also use Markov chain Monte Carlo simulation for the prediction of
networks with maximum probability under our model. This model can be applied
across species, where interaction data from one (or several) species can be used
to infer interactions in another. In addition, the model is extensible and can
be analogously applied to other molecular data (e.g., DNA sequences).
PMID: 11729170 [PubMed - indexed for MEDLINE]
39: EMBO J 2001 Dec 3;20(23):6660-71
Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion
with repair.
Hartsuiker E, Vaessen E, Carr AM, Kohli J.
Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1
9RR, UK.
To study the role of Rad50 in the DNA damage response, we cloned and deleted the
Schizosaccharomyces pombe RAD50 homologue. The deletion is sensitive to a range
of DNA-damaging agents and shows dynamic epistatic interactions with other
recombination-repair genes. We show that Rad50 is necessary for recombinational
repair of the DNA lesion at the mating-type locus and that rad50Delta shows slow
DNA replication. We also find that Rad50 is not required for slowing down S
phase in response to hydroxy urea or methyl methanesulfonate (MMS) treatment.
Interestingly, in rad50Delta cells, the recombination frequency between two
homologous chromosomes is increased at the expense of sister chromatid
recombination. We propose that Rad50, an SMC-like protein, promotes the use of
the sister chromatid as the template for homologous recombinational repair. In
support of this, we found that Rad50 functions in the same pathway for the
repair of MMS-induced damage as Rad21, the homologue of the Saccharomyces
cerevisiae Scc1 cohesin protein. We speculate that Rad50 interacts with the
cohesin complex during S phase to assist repair and possibly re-initiation of
replication after replication fork collapse.
PMID: 11726502 [PubMed - indexed for MEDLINE]
40: EMBO J 2001 Dec 3;20(23):6591-600
Specific roles of protein-phospholipid interactions in the yeast cytochrome bc1
complex structure.
Lange C, Nett JH, Trumpower BL, Hunte C.
Max-Planck-Institut fur Biophysik, Heinrich-Hoffmann-Strasse 7, D-60528
Frankfurt, Germany.
Biochemical data have shown that specific, tightly bound phospholipids are
essential for activity of the cytochrome bc1 complex (QCR), an integral membrane
protein of the respiratory chain. However, the structure and function of such
phospholipids are not yet known. Here we describe five phospholipid molecules
and one detergent molecule in the X-ray structure of yeast QCR at 2.3 A
resolution. Their individual binding sites suggest specific roles in
facilitating structural and functional integrity of the enzyme. Interestingly, a
phosphatidylinositol molecule is bound in an unusual interhelical position near
the flexible linker region of the Rieske iron-sulfur protein. Two possible
proton uptake pathways at the ubiquinone reduction site have been identified:
the E/R and the CL/K pathway. Remarkably, cardiolipin is positioned at the
entrance to the latter. We propose that cardiolipin ensures structural integrity
of the proton-conducting protein environment and takes part directly in proton
uptake. Site-directed mutagenesis of ligating residues confirmed the importance
of the phosphatidylinositol- and cardiolipin-binding sites.
PMID: 11726495 [PubMed - indexed for MEDLINE]
41: J Cell Biol 2001 Nov 26;155(5):763-74
Functional cooperation of Dam1, Ipl1, and the inner centromere protein
(INCENP)-related protein Sli15 during chromosome segregation.
Kang J, Cheeseman IM, Kallstrom G, Velmurugan S, Barnes G, Chan CS.
Section of Molecular Genetics and Microbiology, Institute for Cellular and
Molecular Biology, The University of Texas, Austin, TX 78712, USA.
We have shown previously that Ipl1 and Sli15 are required for chromosome
segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1
protein kinase and these two proteins colocalize to the mitotic spindle. We show
here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of
Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle.
The Ipl1-binding and -stimulating activities of Sli15 both reside within a
region containing homology to the metazoan inner centromere protein (INCENP).
Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for
mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most
likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins
efficiently in vitro, and the in vivo phosphorylation of both proteins is
reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1
and sli15 mutants, thus providing evidence that Dam1 interactions with
Ipl1-Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15
each bind to microtubules directly in vitro, and they are associated with yeast
centromeric DNA in vivo. Given their dual association with microtubules and
kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating
chromosome-spindle interactions or in the movement of kinetochores along
microtubules.
PMID: 11724818 [PubMed - indexed for MEDLINE]
42: Curr Biol 2001 Nov 13;11(22):1794-8
Promoter-specific activation defects by a novel yeast TBP mutant compromised for
TFIIB interaction.
Virbasius CM, Holstege FC, Young RA, Green MR.
Howard Hughes Medical Institute, Programs in Gene Function and Expression,
University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA
01605, USA.
TFIIB is an RNA polymerase II general transcription factor (GTF) that has also
been implicated in the mechanism of action of certain promoter-specific
activators (see, for examples, [1-11]). TFIIB enters the preinitiation complex
(PIC) primarily through contact with the TATA box binding protein (TBP), an
interaction mediated by three TBP residues [12-14]. To study the role of TFIIB
in transcription activation in vivo, we randomly mutagenized these three
residues in yeast TBP and screened for promoter-specific activation mutants. One
mutant bearing a single conservative substitution, TBP-E186D, is the focus of
this study. As expected, TBP-E186D binds normally to the TATA box but fails to
support the entry of TFIIB into the PIC. Cells expressing TBP-E186D are viable
but have a severe slow-growth phenotype. Whole-genome expression analysis
indicates that transcription of 17% of yeast genes are compromised by this
mutation. Chimeric promoter analysis indicates that the region of the gene that
confers sensitivity to the TBP-E186D mutation is the UAS (upstream activating
sequence), which contains the activator binding sites. Most interestingly, other
TBP mutants that interfere with different interactions (TFIIB, TFIIA, or the
TATA box) and a TFIIB mutant defective for interaction with TBP all manifest
distinct and selective promoter-specific activation defects. Our results
implicate the entry of TFIIB into the PIC as a critical step in the activation
of certain promoters and reveal diverse mechanisms of transcription activation.
PMID: 11719223 [PubMed - indexed for MEDLINE]
43: J Biol Chem 2002 Feb 1;277(5):3673-9
Differential ATP binding and intrinsic ATP hydrolysis by amino-terminal domains
of the yeast Mlh1 and Pms1 proteins.
Hall MC, Shcherbakova PV, Kunkel TA.
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research
Triangle Park, North Carolina 27709, USA.
MutL homologs belong to a family of proteins that share a conserved ATP binding
site. We demonstrate that amino-terminal domains of the yeast MutL homologs Mlh1
and Pms1 required for DNA mismatch repair both possess independent, intrinsic
ATPase activities. Amino acid substitutions in the conserved ATP binding sites
concomitantly reduce ATP binding, ATP hydrolysis, and DNA mismatch repair in
vivo. The ATPase activities are weak, consistent with the hypothesis that ATP
binding is primarily responsible for modulating interactions with other MMR
components. Three approaches, ATP hydrolysis assays, limited proteolysis
protection, and equilibrium dialysis, provide evidence that the amino-terminal
domain of Mlh1 binds ATP with >10-fold higher affinity than does the
amino-terminal domain of Pms1. This is consistent with a model wherein ATP may
first bind to Mlh1, resulting in events that permit ATP binding to Pms1 and
later steps in DNA mismatch repair.
PMID: 11717305 [PubMed - indexed for MEDLINE]
44: J Cell Biol 2001 Nov 12;155(4):581-92
Yeast Cdc42 functions at a late step in exocytosis, specifically during
polarized growth of the emerging bud.
Adamo JE, Moskow JJ, Gladfelter AS, Viterbo D, Lew DJ, Brennwald PJ.
Department of Cell and Developmental Biology, University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599, USA.
The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal
organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of
cell growth includes polarization of the actin cytoskeleton, which delivers
secretory vesicles to growth sites at the plasma membrane. We now describe a
novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in
docking and fusion of secretory vesicles that is independent of its role in
actin polarization. cdc42-6 mutants can polarize actin and deliver secretory
vesicles to the bud, but fail to fuse those vesicles with the plasma membrane.
This defect is manifested only during the early stages of bud formation when
growth is most highly polarized, and appears to reflect a requirement for Cdc42
to maintain maximally active exocytic machinery at sites of high vesicle
throughput. Extensive genetic interactions between cdc42-6 and mutations in
exocytic components support this hypothesis, and indicate a functional overlap
with Rho3, which also regulates both actin organization and exocytosis.
Localization data suggest that the defect in cdc42-6 cells is not at the level
of the localization of the exocytic apparatus. Rather, we suggest that Cdc42
acts as an allosteric regulator of the vesicle docking and fusion apparatus to
provide maximal function at sites of polarized growth.
PMID: 11706050 [PubMed - indexed for MEDLINE]
45: Biochemistry 2001 Nov 20;40(46):13933-40
Site-specific mutations in the myosin binding sites of actin affect structural
transitions that control myosin binding.
Prochniewicz E, Thomas DD.
Department of Biochemistry, Molecular Biology, and Biophysics, University of
Minnesota, Minneapolis 55455, USA. ewa@ddt.biochem.umn.edu
We have examined the effects of actin mutations on myosin binding, detected by
cosedimentation, and actin structural dynamics, detected by spectroscopic
probes. Specific mutations were chosen that have been shown to affect the
functional interactions of actin and myosin, two mutations (4Ac and E99A/E100A)
in the proposed region of weak binding to myosin and one mutation (I341A) in the
proposed region of strong binding. In the absence of nucleotide and salt, S1
bound to both wild-type and mutant actins with high affinity (K(d) < microM),
but either ADP or increased ionic strength decreased this affinity. This
decrease was more pronounced for actins with mutations that inhibit functional
interaction with myosin (E99A/E100A and I341A) than for a mutation that enhances
the interaction (4Ac). The mutations E99A/E100A and I341A affected the
microsecond time scale dynamics of actin in the absence of myosin, but the 4Ac
mutation did not have any effect. The binding of myosin eliminated these effects
of mutations on structural dynamics; i.e., the spectroscopic signals from mutant
actins bound to S1 were the same as those from wild-type actin. These results
indicate that mutations in the myosin binding sites affect structural
transitions within actin that control strong myosin binding, without affecting
the structural dynamics of the strongly bound actomyosin complex.
PMID: 11705383 [PubMed - indexed for MEDLINE]
46: Plant Cell 2001 Nov;13(11):2455-70
The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a
domain conserved between plants and animals.
Bellaoui M, Pidkowich MS, Samach A, Kushalappa K, Kohalmi SE, Modrusan Z, Crosby
WL, Haughn GW.
Botany Department and Biotechnology Laboratory, 6270 University Boulevard,
University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
Interactions between TALE (three-amino acid loop extension) homeodomain proteins
play important roles in the development of both fungi and animals. Although in
plants, two different subclasses of TALE proteins include important
developmental regulators, the existence of interactions between plant TALE
proteins has remained unexplored. We have used the yeast two-hybrid system to
demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can
selectively heterodimerize with specific KNAT homeodomain proteins. Interaction
is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences
including the MEINOX domain. These findings validate the hypothesis that the
MEINOX domain has been conserved between plants and animals as an interaction
domain for developmental regulators. In yeast, BEL1 and KNAT proteins can
activate transcription only as a heterodimeric complex, suggesting a role for
such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT
MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role
for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence
meristem.
PMID: 11701881 [PubMed - indexed for MEDLINE]
47: Cell 2001 Nov 2;107(3):373-86
Structure of the 80S ribosome from Saccharomyces cerevisiae--tRNA-ribosome and
subunit-subunit interactions.
Spahn CM, Beckmann R, Eswar N, Penczek PA, Sali A, Blobel G, Frank J.
Howard Hughes Medical Institute, Health Research Inc., Albany, NY 12201, USA.
A cryo-EM reconstruction of the translating yeast 80S ribosome was analyzed.
Computationally separated rRNA and protein densities were used for docking of
appropriately modified rRNA models and homology models of yeast ribosomal
proteins. The core of the ribosome shows a remarkable degree of conservation.
However, some significant differences in functionally important regions and
dramatic changes in the periphery due to expansion segments and additional
ribosomal proteins are evident. As in the bacterial ribosome, bridges between
the subunits are mainly formed by RNA contacts. Four new bridges are present at
the periphery. The position of the P site tRNA coincides precisely with its
prokaryotic counterpart, with mainly rRNA contributing to its molecular
environment. This analysis presents an exhaustive inventory of an eukaryotic
ribosome at the molecular level.
PMID: 11701127 [PubMed - indexed for MEDLINE]
48: Annu Rev Genet 2001;35:193-208
Chromatin insulators and boundaries: effects on transcription and nuclear
organization.
Gerasimova TI, Corces VG.
Department of Biology, The Johns Hopkins University, 3400 North Charles Street,
Baltimore, Maryland 21218, UDA. tgerasimova@jhu.edu
Chromatin boundaries and insulators are transcriptional regulatory elements that
modulate interactions between enhancers and promoters and protect genes from
silencing effects by the adjacent chromatin. Originally discovered in
Drosophila, insulators have now been found in a variety of organisms, ranging
from yeast to humans. They have been found interspersed with regulatory
sequences in complex genes and at the boundaries between active and inactive
chromatin. Insulators might modulate transcription by organizing the chromatin
fiber within the nucleus through the establishment of higher-order domains of
chromatin structure.
Publication Types:
Review
Review, Tutorial
PMID: 11700282 [PubMed - indexed for MEDLINE]
49: J Mol Biol 2001 Nov 9;313(5):955-63
UBA domains mediate protein-protein interactions between two DNA
damage-inducible proteins.
Bertolaet BL, Clarke DJ, Wolff M, Watson MH, Henze M, Divita G, Reed SI.
Department of Molecular Biology, The Scripps Research Institute, 10550 North
Torrey Pines Road, La Jolla, CA 92037, USA.
The Saccharomyces cerevisiae genes RAD23 and DDI1 were identified in a screen
for multicopy suppressors of the temperature-sensitivity of a mutant allele of
S. cerevisiae PDS1. Pds1 is a regulator of anaphase that needs to accumulate and
then be degraded by the ubiquitin-proteasome pathway at the metaphase-anaphase
transition for cells to progress normally through mitosis. Both the Rad23 and
Ddi1 pds1 suppression phenotypes depend on a shared motif known as a UBA domain
found in a variety of proteins associated with ubiquitin metabolism. UBA domains
were found to be essential for homodimerization of Rad23 and heterodimerization
between Rad23 and Ddi1, but not for homodimerization of Ddi1. This observation,
coupled with the findings that Rad23 and Ddi1 UBA domains bind ubiquitin and
that dimerization of Rad23 blocks ubiquitin binding, suggests a possible
mechanism for regulating Rad23 and Ddi1 function. Copyright 2001 Academic Press.
PMID: 11700052 [PubMed - indexed for MEDLINE]
50: Proc Natl Acad Sci U S A 2001 Nov 20;98(24):13675-80
Yeast Dam1p has a role at the kinetochore in assembly of the mitotic spindle.
Jones MH, He X, Giddings TH, Winey M.
Department of Molecular, Cellular, and Developmental Biology, Campus Box 347,
University of Colorado, Boulder, CO 80309-0347, USA.
During mitosis, replicated chromosomes are separated to daughter cells by the
microtubule-based mitotic spindle. Chromosomes attach to the mitotic spindle
through specialized DNA/protein structures called kinetochores, but the
mechanism of attachment is not well understood. We show here that the yeast
microtubule-binding protein, Dam1p, associates physically and functionally with
kinetochores, suggesting a role in kinetochore attachment to the spindle. An
epitope-tagged version of Dam1p colocalizes with the integral kinetochore
component Ndc10p/Cbf2p in immunofluorescence analysis of chromosome spreads. In
addition, Dam1p is associated preferentially with centromeric DNA as shown by
chromatin immunoprecipitation experiments, and this association depends on
Ndc10p/Cbf2p. We also demonstrate genetic interactions between DAM1 and CTF19 or
SLK19 genes encoding kinetochore proteins. Although the defect caused by the
dam1-1 mutation leads to activation of the spindle checkpoint surveillance
system and consequent persistence of sister chromatid cohesion, the metaphase
arrest spindle abnormally elongates, resulting in virtually complete chromosome
missegregation. Execution point experiments indicate that Dam1p has a role in
formation of a metaphase spindle and in anaphase spindle elongation. Finally, we
have observed that the protein encoded by the dam1-1 allele becomes delocalized
at the nonpermissive temperature, correlating with the subsequent onset of the
mutant phenotype. Our studies are consistent with a role for Dam1p in attachment
of sister chromatids through the kinetochore to the mitotic spindle before
chromosome segregation.
PMID: 11698664 [PubMed - indexed for MEDLINE]
51: Exp Cell Res 2001 Nov 15;271(1):142-51
Functional conservation of 14-3-3 isoforms in inhibiting bad-induced apoptosis.
Subramanian RR, Masters SC, Zhang H, Fu H.
Department of Pharmacology, Emory University School of Medicine, Atlanta,
Georgia 30322, USA.
14-3-3 proteins are a family of homologous eukaryotic molecules with seven
distinct isoforms in mammalian cells. Isoforms of 14-3-3 proteins interact with
diverse ligands and are involved in the regulation of mitogenesis, cell cycle
progression, and apoptosis. However, whether different 14-3-3 isoforms are
responsible for distinct functions remains elusive. Here we report that multiple
isoforms of 14-3-3 proteins were capable of binding to several ligands, Bad,
Raf-1, and Cbl. In a functional assay of 14-3-3 isoforms, all mammalian 14-3-3
isoforms could inhibit Bad-induced apoptosis. Thus, 14-3-3 function in
regulating one of its ligands, Bad, is conserved among mammalian isoforms. We
addressed whether 14-3-3 isoforms are differentially expressed in tissues, which
may in part determine isoform-specific interactions. In situ hybridization
revealed that 14-3-3zeta was present in most tissues tested, but sigma was
preferentially expressed in epithelial cells. Thus, isoforms of 14-3-3 can
interact and control the function of selected protein ligands, and differential
tissue distribution of 14-3-3 isoforms may contribute to their specific
interactions and subsequent downstream signaling events. Copyright 2001 Academic
Press.
PMID: 11697890 [PubMed - indexed for MEDLINE]
52: Curr Biol 2001 Oct 30;11(21):1711-5
The DECD box putative ATPase Sub2p is an early mRNA export factor.
Jensen TH, Boulay J, Rosbash M, Libri D.
Howard Hughes Medical Institute, Department of Biology, Brandeis University,
Waltham, MA 02454, USA. thj@mbio.aau.dk
Nuclear mRNA metabolism relies on the interplay between transcription,
processing, and nuclear export. RNA polymerase II transcripts experience major
rearrangements within the nucleus, which include alterations in the structure of
the mRNA precursors as well as the addition and perhaps even removal of proteins
prior to transport across the nuclear membrane. Such mRNP-remodeling steps are
thought to require the activity of RNA helicases/ATPases. One such protein, the
DECD box RNA-dependent ATPase Sub2p/UAP56, is involved in both early and late
steps of spliceosome assembly. Here, we report a more general function of
Saccharomyces cerevisiae Sub2p in mRNA nuclear export. We observe a rapid and
dramatic nuclear accumulation of poly(A)(+) RNA in strains carrying mutant
alleles of sub2. Strikingly, an intronless transcript, HSP104, also accumulates
in nuclei, suggesting that Sub2p function is not restricted to splicing events.
The HSP104 transcripts are localized in a single nuclear focus that is suggested
to be at or near their site of transcription. Intriguingly, Sub2p shows strong
genetic and functional interactions with the RNA polymerase II-associated
DNA/DNA:RNA helicase Rad3p as well as the nuclear RNA exosome component Rrp6p,
which was independently implicated in the retention of mRNAs at transcription
sites. Taken together, our data suggest that Sub2p functions at an early step in
the mRNA export process.
PMID: 11696331 [PubMed - indexed for MEDLINE]
53: Nat Genet 2001 Dec;29(4):482-6
Correlation between transcriptome and interactome mapping data from
Saccharomyces cerevisiae.
Ge H, Liu Z, Church GM, Vidal M.
Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School,
Boston, Massachusetts 02115, USA.
Genomic and proteomic approaches can provide hypotheses concerning function for
the large number of genes predicted from genome sequences. Because of the
artificial nature of the assays, however, the information from these
high-throughput approaches should be considered with caution. Although it is
possible that more meaningful hypotheses could be formulated by integrating the
data from various functional genomic and proteomic projects, it has yet to be
seen to what extent the data can be correlated and how such integration can be
achieved. We developed a 'transcriptome-interactome correlation mapping'
strategy to compare the interactions between proteins encoded by genes that
belong to common expression-profiling clusters with those between proteins
encoded by genes that belong to different clusters. Using this strategy with
currently available data sets for Saccharomyces cerevisiae, we provide the first
global evidence that genes with similar expression profiles are more likely to
encode interacting proteins. We show how this correlation between transcriptome
and interactome data can be used to improve the quality of hypotheses based on
the information from both approaches. The strategy described here may help to
integrate other functional genomic and proteomic data, both in yeast and in
higher organisms.
PMID: 11694880 [PubMed - indexed for MEDLINE]
54: Mol Biol Cell 2001 Nov;12(11):3668-79
In vivo role for actin-regulating kinases in endocytosis and yeast epsin
phosphorylation.
Watson HA, Cope MJ, Groen AC, Drubin DG, Wendland B.
Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218,
USA.
The yeast actin-regulating kinases Ark1p and Prk1p are signaling proteins
localized to cortical actin patches, which may be sites of endocytosis.
Interactions between the endocytic proteins Pan1p and End3p may be regulated by
Prk1p-dependent threonine phosphorylation of Pan1p within the consensus sequence
[L/I]xxQxTG. We identified two Prk1p phosphorylation sites within the
Pan1p-binding protein Ent1p, a yeast epsin homologue, and demonstrate
Prk1p-dependent phosphorylation of both threonines. Converting both threonines
to either glutamate or alanine mimics constitutively phosphorylated or
dephosphorylated Ent1p, respectively. Synthetic growth defects were observed in
a pan1-20 ENT1(EE) double mutant, suggesting that Ent1p phosphorylation
negatively regulates the formation/activity of a Pan1p-Ent1p complex.
Interestingly, pan1-20 ent2 Delta but not pan1-20 ent1 Delta double mutants had
improved growth and endocytosis over the pan1-20 mutant. We found that
actin-regulating Ser/Thr kinase (ARK) mutants exhibit endocytic defects and that
overexpressing either wild-type or alanine-substituted Ent1p partially
suppressed phenotypes associated with loss of ARK kinases, including growth,
endocytosis, and actin localization defects. Consistent with synthetic growth
defects of pan1-20 ENT1(EE) cells, overexpressing glutamate-substituted Ent1p
was deleterious to ARK mutants. Surprisingly, overexpressing the related Ent2p
protein could not suppress ARK kinase mutant phenotypes. These results suggest
that Ent1p and Ent2p are not completely redundant and may perform opposing
functions in endocytosis. These data support the model that, as for
clathrin-dependent recycling of synaptic vesicles, yeast endocytic protein
phosphorylation inhibits endocytic functions.
PMID: 11694597 [PubMed - indexed for MEDLINE]
55: Mol Cell Biol 2001 Dec;21(23):8082-94
Multiple interactions in Sir protein recruitment by Rap1p at silencers and
telomeres in yeast.
Moretti P, Shore D.
Department of Microbiology, College of Physicians & Surgeons of Columbia
University, New York, New York 10032, USA.
Initiation of transcriptional silencing at mating type loci and telomeres in
Saccharomyces cerevisiae requires the recruitment of a Sir2/3/4 (silent
information regulator) protein complex to the chromosome, which occurs at least
in part through its association with the silencer- and telomere-binding protein
Rap1p. Sir3p and Sir4p are structural components of silent chromatin that can
self-associate, interact with each other, and bind to the amino-terminal tails
of histones H3 and H4. We have identified a small region of Sir3p between amino
acids 455 and 481 that is necessary and sufficient for association with the
carboxyl terminus of Rap1p but not required for Sir complex formation or histone
binding. SIR3 mutations that delete this region cause a silencing defect at HMR
and telomeres. However, this impairment of repression is considerably less than
that displayed by Rap1p carboxy-terminal truncations that are defective in Sir3p
binding. This difference may be explained by the ability of the Rap1p carboxyl
terminus to interact independently with Sir4p, which we demonstrate by in vitro
binding and two-hybrid assays. Significantly, the Rap1p-Sir4p two-hybrid
interaction does not require Sir3p and is abolished by mutation of the carboxyl
terminus of Rap1p. We propose that both Sir3p and Sir4p can directly and
independently bind to Rap1p at mating type silencers and telomeres and suggest
that Rap1p-mediated recruitment of Sir proteins operates through multiple
cooperative interactions, at least some of which are redundant. The physical
separation of the Rap1p interaction region of Sir3p from parts of the protein
required for Sir complex formation and histone binding raises the possibility
that Rap1p can participate directly in the maintenance of silent chromatin
through the stabilization of Sir complex-nucleosome interactions.
PMID: 11689698 [PubMed - indexed for MEDLINE]
56: Mol Cell Biol 2001 Dec;21(23):7981-94
Structural requirements for function of yeast GGAs in vacuolar protein sorting,
alpha-factor maturation, and interactions with clathrin.
Mullins C, Bonifacino JS.
Cell Biology and Metabolism Branch, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland 20892-5430, USA.
The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding proteins) are a
family of multidomain adaptor proteins involved in protein sorting at the
trans-Golgi network of eukaryotic cells. Here we present results from a
functional characterization of the two Saccharomyces cerevisiae GGAs, Gga1p and
Gga2p. We show that deletion of both GGA genes causes defects in sorting of
carboxypeptidase Y (CPY) and proteinase A to the vacuole, vacuolar morphology,
and maturation of alpha-factor. A structure-function analysis reveals a
requirement of the VHS, GAT, and hinge for function, while the GAE domain is
less important. We identify putative clathrin-binding motifs in the hinge domain
of both yeast GGAs. These motifs are shown to mediate clathrin binding in vitro.
While mutation of these motifs alone does not block function of the GGAs in
vivo, combining these mutations with truncations of the hinge and GAE domains
diminishes function, suggesting functional cooperation between different
clathrin-binding elements. Thus, these observations demonstrate that the yeast
GGAs play important roles in the CPY pathway, vacuole biogenesis, and
alpha-factor maturation and identify structural determinants that are critical
for these functions.
PMID: 11689690 [PubMed - indexed for MEDLINE]
57: J Virol 2001 Dec;75(23):11344-53
Functional interactions of human immunodeficiency virus type 1 integrase with
human and yeast HSP60.
Parissi V, Calmels C, De Soultrait VR, Caumont A, Fournier M, Chaignepain S,
Litvak S.
REGER, UMR-5097 Centre National de la Recherche Scientifique (CNRS)-Universite
Victor Segalen Bordeaux 2, Bordeaux, France.
vincent.parissi@reger.u-bordeaux2.fr
Integration of human immunodeficiency virus type 1 (HIV-1) proviral DNA in the
nuclear genome is catalyzed by the retroviral integrase (IN). In addition to IN,
viral and cellular proteins associated in the high-molecular-weight
preintegration complex have been suggested to be involved in this process. In an
attempt to define host factors interacting with IN, we used an in vitro system
to identify cellular proteins in interaction with HIV-1 IN. The yeast
Saccharomyces cerevisiae was chosen since (i) its complete sequence has been
established and the primary structure of all the putative proteins from this
eucaryote has been deduced, (ii) there is a significant degree of homology
between human and yeast proteins, and (iii) we have previously shown that the
expression of HIV-1 IN in yeast induces a lethal phenotype. Strong evidences
suggest that this lethality is linked to IN activity in infected human cells
where integration requires the cleavage of genomic DNA. Using IN-affinity
chromatography we identified four yeast proteins interacting with HIV-1 IN,
including the yeast chaperonin yHSP60, which is the counterpart of human hHSP60.
Yeast lethality induced by HIV-1 IN was abolished when a mutated HSP60 was
coexpressed, therefore suggesting that both proteins interact in vivo. Besides
interacting with HIV-1 IN, the hHSP60 was able to stimulate the in vitro
processing and joining activities of IN and protected this enzyme from thermal
denaturation. In addition, the functional human HSP60-HSP10 complex in the
presence of ATP was able to recognize the HIV-1 IN as a substrate.
PMID: 11689615 [PubMed - indexed for MEDLINE]
58: Hum Mol Genet 2001 Oct 1;10(21):2463-8
The phylogenetic distribution of frataxin indicates a role in iron-sulfur
cluster protein assembly.
Huynen MA, Snel B, Bork P, Gibson TJ.
Biocomputing, EMBL/Max-Delbrueck-Center fur molecular medicin, Berlin-Buch,
Germany. huynen@cmbi.kun.nl
Much has been learned about the cellular pathology of Friedreich's ataxia, a
recessive neurodegenerative disease resulting from insufficient expression of
the mitochondrial protein frataxin. However, the biochemical function of
frataxin has remained obscure, hampering attempts at therapeutic intervention.
To predict functional interactions of frataxin with other proteins we
investigated whether its gene specifically co-occurs with any other genes in
sequenced genomes. In 56 available genomes we identified two genes with
identical phylogenetic distributions to the frataxin/cyaY gene: hscA and
hscB/JAC1. These genes have not only emerged in the same evolutionary lineage as
the frataxin gene, they have also been lost at least twice with it, and they
have been horizontally transferred with it in the evolution of the mitochondria.
The proteins encoded by hscA and hscB, the chaperone HSP66 and the co-chaperone
HSP20, have been shown to be required for the synthesis of 2Fe-2S clusters on
ferredoxin in proteobacteria. JAC1, an ortholog of hscB, and SSQ1, a paralog of
hscA, have been shown to be required for iron-sulfur cluster assembly in
mitochondria of Saccharomyces cerevisiae. Combining data on the co-occurrence of
genes in genomes with experimental and predicted cellular localization data of
their proteins supports the hypothesis that frataxin is directly involved in
iron-sulfur cluster protein assembly. They indicate that frataxin is
specifically involved in the same sub-process as HSP20/Jac1p.
PMID: 11689493 [PubMed - indexed for MEDLINE]
59: Gene 2001 Aug 22;274(1-2):169-77
Erratum in:
Gene 2001 Oct 31;278(1-2):265
Identification of interaction partners for two closely-related members of the
ETS protein family, FLI and ERG.
Deramaudt TB, Remy P, Stiegler P.
FRE 2168 du CNRS, Mecanismes Moleculaires de la Division Cellulaire et du
Developpement, Institut de Physiologie et de Chimie Biologique, 21 rue Rene
Descartes, 67084 Strasbourg Cedex, France.
Fli and erg are two members of the ETS gene family that encodes transcription
factors related to the c-ets-1 proto-oncogene. The products of the ETS genes act
as transcriptional effectors in cell proliferation, differentiation, and
oncogenic transformation. FLI and ERG, two closely-related proteins, bind, as do
all the ETS proteins characterized so far, to DNA sequences with an invariable
central GGA core flanked by preferred nucleotides. Nevertheless,
promoter-specific responses to FLI or ERG may be driven by mechanisms involving
multicomponent complexes. Using a yeast two-hybrid screen, we have identified
several proteins that physically interact with either FLI or ERG proteins used
as bait. The Xenopus developmentally implicated Xvent-2 and Xvent-2B proteins,
and the Xenopus splicing factor RNP-C/U1C physically interact with Xl-FLI and
Xl-ERG, both in the yeast two-hybrid system and in vitro. We also report the
potential interaction of FLI and ERG with Sox-D, a stabilizing protein that may
modulate their transcriptional activity. Furthermore, the possible involvement
of the transcriptional effectors FLI and ERG in mRNA processing, hematopoiesis
or in the control of angiogenesis is suggested through possible interactions
with, respectively, RNA binding proteins and hnRNPs, a repressor of the
hematopoietic pathway (SAP18), and the HAF protein.
PMID: 11675009 [PubMed - indexed for MEDLINE]
60: Biochemistry 2001 Oct 30;40(43):13088-96
Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate
kinase.
Bollenbach TJ, Nowak T.
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame,
Indiana 46556 USA.
Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic
pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and
citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic
data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric
communication between the substrate, PEP, and the allosteric effector, FBP
[Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results
indicate that divalent cations modulate multiligand interactions, and hence
cooperativity with YPK. The nature of multiligand interactions on YPK was
investigated in the presence of the physiological divalent activator Mg(2+). The
binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence
spectroscopy. The binding data were subject to thermodynamic linked-function
analysis to determine the magnitudes of the multiligand interactions governing
the allosteric activation of YPK. The two ligand coupling free energies between
PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75
kcal/mol, respectively. The two-ligand coupling free energies between PEP and
Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the
cation. This indicates that the interactions between the divalent cation and PEP
with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not
simply electrostatic in nature, as originally hypothesized. The magnitude of the
heterotropic interaction between the metal and FBP is similar with Mg(2+) and
Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by
3.21 kcal/mol compared to independent binding. This complex is destabilized by
3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex.
Interpretation of K(d) values when cooperative binding occurs must be done with
care as these are not simple thermodynamic constants. These data demonstrate
that the divalent metal, which activates phosphoryl transfer in YPK, plays a key
role in modulating the various multiligand interactions that define the overall
allosteric properties of the enzyme.
PMID: 11669647 [PubMed - indexed for MEDLINE]
61: J Biol Chem 2002 Feb 15;277(7):5290-8
Unusual binding properties of the SH3 domain of the yeast actin-binding protein
Abp1: structural and functional analysis.
Fazi B, Cope MJ, Douangamath A, Ferracuti S, Schirwitz K, Zucconi A, Drubin DG,
Wilmanns M, Cesareni G, Castagnoli L.
Department of Biology, University of Rome Tor Vergata, Via della Ricerca
Scientifica, 00133 Roma, Italy.
Abp1p is an actin-binding protein that plays a central role in the organization
of Saccharomyces cerevisiae actin cytoskeleton. By a combination of two-hybrid
and phage-display approaches, we have identified six new ligands of the Abp1-SH3
domain. None of these SH3-mediated novel interactions was detected in recent all
genome high throughput protein interaction projects. Here we show that the
SH3-mediated association of Abp1p with the Ser/Thr kinases Prk1p and Ark1p is
essential for their localization to actin cortical patches. The Abp1-SH3 domain
has a rather unusual binding specificity, because its target peptides contain
the tetrapentapeptide +XXXPXXPX+PXXL with positive charges flanking the
polyproline core on both sides. Here we present the structure of the Abp1-SH3
domain solved at 1.3-A resolution. The peptide-binding pockets in the SH3 domain
are flanked by two acidic residues that are uncommon at those positions in the
SH3 domain family. We have shown by site-directed mutagenesis that one of these
negatively charged side chains may be the key determinant for the preference for
non-classical ligands.
PMID: 11668184 [PubMed - indexed for MEDLINE]
62: Inorg Chem 1996 Mar 13;35(6):1692-1700
New Type 2 Copper-Cysteinate Proteins. Copper Site Histidine-to-Cysteine Mutants
of Yeast Copper-Zinc Superoxide Dismutase.
Lu Y, Roe JA, Bender CJ, Peisach J, Banci L, Bertini I, Gralla EB, Valentine JS.
Department of Chemistry and Biochemistry, University of California, Los Angeles,
Los Angeles, California 90095, Department of Molecular Pharmacology, Albert
Einstein College of Medicine of Yeshiva University, Bronx, New York 10461,
Department of Chemistry and Biochemistry, Loyola Marymount University, Los
Angeles, California 90045, and Department of Chemistry, University of Florence,
Florence, Italy.
Preparation and characterization of two new site-directed mutant copper-zinc
superoxide dismutase proteins from Saccharomyces cerevisiae, i.e., His46Cys
(H46C) and His120Cys (H120C), in which individual histidyl ligands in the
copper-binding site were replaced by cysteine, are reported here. These two
mutant CuZnSOD proteins may be described as type 2 (or normal) rather than type
1 (or blue) copper-cysteinate proteins and are characterized by their yellow
rather than blue color, resulting from intense copper-to-sulfur charge transfer
bands around 400 nm, their type 2 EPR spectra, with large rather than small
nuclear hyperfine interactions, and their characteristic type 2 d-d electronic
absorption spectra. An interesting difference between these two copper site
His-to-Cys mutations is that the imidazolate bridge between the two metal sites
that is characteristic of the wild-type protein remains intact in the case of
the H46C mutant but is not present in the case of the H120C mutant.
PMID: 11666393 [PubMed - as supplied by publisher]
63: J Biol Chem 2001 Dec 7;276(49):46225-9
Asymmetric recognition of DNA local distortion. Structure-based functional
studies of eukaryotic Msh2-Msh6.
Drotschmann K, Yang W, Brownewell FE, Kool ET, Kunkel TA.
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research
Triangle Park, North Carolina 27709, USA.
Crystal structures of bacterial MutS homodimers bound to mismatched DNA reveal
asymmetric interactions of the two subunits with DNA. A phenylalanine and
glutamate of one subunit make mismatched base-specific interactions, and
residues of both subunits contact the DNA backbone surrounding the mismatched
base, but asymmetrically. A number of amino acids in MutS that contact the DNA
are conserved in the eukaryotic Msh2-Msh6 heterodimer. We report here that yeast
strains with amino acids substituted for residues inferred to interact with the
DNA backbone or mismatched base have elevated spontaneous mutation rates
consistent with defective mismatch repair. Purified Msh2-Msh6 with substitutions
in the conserved Phe(337) and Glu(339) in Msh6 thought to stack or hydrogen
bond, respectively, with the mismatched base do have reduced DNA binding
affinity but normal ATPase activity. Moreover, wild-type Msh2-Msh6 binds with
lower affinity to mismatches with thymine replaced by difluorotoluene, which
lacks the ability to hydrogen bond. The results suggest that yeast Msh2-Msh6
interacts asymmetrically with the DNA through base-specific stacking and
hydrogen bonding interactions and backbone contacts. The importance of these
contacts decreases with increasing distance from the mismatch, implying that
interactions at and near the mismatch are important for binding in a kinked DNA
conformation.
PMID: 11641390 [PubMed - indexed for MEDLINE]
64: Genetics 2001 Oct;159(2):487-97
Genetic interactions of Spt4-Spt5 and TFIIS with the RNA polymerase II CTD and
CTD modifying enzymes in Saccharomyces cerevisiae.
Lindstrom DL, Hartzog GA.
Department of Molecular, Cell and Developmental Biology, University of
California, Santa Cruz, 95064, USA.
Genetic and biochemical studies have identified many factors thought to be
important for transcription elongation. We investigated relationships between
three classes of these factors: (1) transcription elongation factors Spt4-Spt5,
TFIIS, and Spt16; (2) the C-terminal heptapeptide repeat domain (CTD) of RNA
polymerase II; and (3) protein kinases that phosphorylate the CTD and a
phosphatase that dephosphorylates it. We observe that spt4 and spt5 mutations
cause strong synthetic phenotypes in combination with mutations that shorten or
alter the composition of the CTD; affect the Kin28, Bur1, or Ctk1 CTD kinases;
and affect the CTD phosphatase Fcp1. We show that Spt5 co-immunoprecipitates
with RNA polymerase II that has either a hyper- or a hypophosphorylated CTD.
Furthermore, mutation of the CTD or of CTD modifying enzymes does not affect the
ability of Spt5 to bind RNA polymerase II. We find a similar set of genetic
interactions between the CTD, CTD modifying enzymes, and TFIIS. In contrast, an
spt16 mutation did not show these interactions. These results suggest that the
CTD plays a key role in modulating elongation in vivo and that at least a subset
of elongation factors are dependent upon the CTD for their normal function.
PMID: 11606527 [PubMed - indexed for MEDLINE]
65: Biochemistry 2001 Oct 23;40(42):12704-11
MDP-1 is a new and distinct member of the haloacid dehalogenase family of
aspartate-dependent phosphohydrolases.
Selengut JD.
Laboratory of Biochemistry, National Heart, Lung and Blood Institute, Building
50-2347, National Institutes of Health, 50 South Drive, Bethesda, Maryland
20892-8012, USA. selengut@nih.gov
MDP-1 is a eukaryotic magnesium-dependent acid phosphatase with little sequence
homology to previously characterized phosphatases. The presence of a conserved
motif (Asp-X-Asp-X-Thr) in the N terminus of MDP-1 suggested a relationship to
the haloacid dehalogenase (HAD) superfamily, which contains a number of
magnesium-dependent acid phosphatases. These phosphatases utilize an aspartate
nucleophile and contain a number of conserved active-site residues and
hydrophobic patches, which can be plausibly aligned with conserved residues in
MDP-1. Seven site-specific point mutants of MDP-1 were produced by modifying the
catalytic aspartate, serine, and lysine residues to asparagine or glutamate,
alanine, and arginine, respectively. The activity of these mutants confirms the
assignment of MDP-1 as a member of the HAD superfamily. Detailed comparison of
the sequence of the 15 MDP-1 sequences from various organisms with other HAD
superfamily sequences suggests that MDP-1 is not closely related to any
particular member of the superfamily. The crystal structures of several HAD
family enzymes identify a domain proximal to the active site responsible for
important interactions with low molecular weight substrates. The absence of this
domain or any other that might perform the same function in MDP-1 suggests an
"open" active site capable of interactions with large substrates such as
proteins. This suggestion was experimentally confirmed by demonstration that
MDP-1 is competent to catalyze the dephosphorylation of tyrosine-phosphorylated
proteins.
PMID: 11601995 [PubMed - indexed for MEDLINE]
66: EMBO J 2001 Oct 15;20(20):5626-35
Apocytochrome c requires the TOM complex for translocation across the
mitochondrial outer membrane.
Diekert K, de Kroon AI, Ahting U, Niggemeyer B, Neupert W, de Kruijff B, Lill R.
Institut fur Zytobiologie und Zytopathologie der Philipps-Universitat Marburg,
Robert-Koch-Strasse 5, 35033 Marburg, Germany.
The import of proteins into the mitochondrial intermembrane space differs in
various aspects from the classical import pathway into the matrix. Apocytochrome
c defines one of several pathways known to reach the intermembrane space, yet
the components and pathways involved in outer membrane translocation are poorly
defined. Here, we report the reconstitution of the apocytochrome c import
reaction using proteoliposomes harbouring purified components. Import
specifically requires the protease-resistant part of the TOM complex and is
driven by interactions of the apoprotein with internal parts of the complex
(involving Tom40) and the 'trans-side receptor' cytochrome c haem lyase. Despite
the necessity of TOM complex function, the translocation pathway of
apocytochrome c does not overlap with that of presequence-containing
preproteins. We conclude that the TOM complex is a universal preprotein
translocase that mediates membrane passage of apocytochrome c and other
preproteins along distinct pathways. Apocytochrome c may provide a paradigm for
the import of other small proteins into the intermembrane space such as factors
used in apoptosis and protection from stress.
PMID: 11598006 [PubMed - indexed for MEDLINE]
67: Physiol Genomics 2001 Oct 10;7(1):27-34
Two-hybrid analysis of the Saccharomyces cerevisiae 26S proteasome.
Cagney G, Uetz P, Fields S.
Departments of Genetics and Medicine, Howard Hughes Medical Institute,
University of Washington, Seattle, Washington 98195-7360, USA.
A two-hybrid screen against an activation domain array of Saccharomyces
cerevisiae proteins was carried out for 31 yeast proteasome proteins. Fifty-five
putative interactions were identified: 21 between components of the proteasome
complex and 34 between proteasome proteins and other proteins. Many of these
latter interactions involved either proteins of the ubiquitin pathway, cell
cycle proteins, protein kinases or a translation initiation factor subunit. The
role of eleven proteins associated with proteasome function by these screens was
analyzed by examining the corresponding deletion strains for temperature
sensitivity and canavanine sensitivity and for the stability of a
ubiquitin-beta-galactosidase fusion protein. These assays additionally
implicated three proteins, Bim1, Ump1, and YKL171W, in proteasome function. This
study demonstrates the utility of genome-wide two-hybrid assays as an entry
point for the further analysis of a large protein complex.
PMID: 11595789 [PubMed - indexed for MEDLINE]
68: J Biol Chem 2001 Dec 14;276(50):46745-50
Cdc42 interacts with the exocyst and regulates polarized secretion.
Zhang X, Bi E, Novick P, Du L, Kozminski KG, Lipschutz JH, Guo W.
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Polarized delivery and incorporation of proteins and lipids to specific domains
of the plasma membrane is fundamental to a wide range of biological processes
such as neuronal synaptogenesis and epithelial cell polarization. The exocyst
complex is specifically localized to sites of active exocytosis and plays
essential roles in secretory vesicle targeting and docking at the plasma
membrane. Sec3p, a component of the exocyst, is thought to be a spatial landmark
for polarized exocytosis. In a search for proteins that regulate the
localization of the exocyst in the budding yeast Saccharomyces cerevisiae, we
found that certain cdc42 mutants affect the polarized localization of the
exocyst proteins. In addition, we found that these mutant cells have a
randomized protein secretion pattern on the cell surface. Biochemical
experiments indicated that Sec3p directly interacts with Cdc42 in its GTP-bound
form. Genetic studies demonstrated synthetically lethal interactions between
cdc42 and several exocyst mutants. These results have revealed a role for Cdc42
in exocytosis. We propose that Cdc42 coordinates the vesicle docking machinery
and the actin cytoskeleton for polarized secretion.
PMID: 11595741 [PubMed - indexed for MEDLINE]
69: Gene 2001 Aug 8;273(2):207-14
Molecular cloning and characterization of a steroid receptor-binding regulator
of G-protein signaling protein cDNA.
Ikeda M, Hirokawa M, Satani N, Kinoshita T, Watanabe Y, Inoue H, Tone S,
Ishikawa T, Minatogawa Y.
Department of Biochemistry, Kawasaki Medical School, 577 Matsushima Kurashiki,
701-0192, Okayama, Japan. ikeda@bcc.kawasaki-m.ac.jp
Steroid hormone receptors are composed of six major functional domains, i.e. the
A/B domains as the activation function 1 domain (AF-1), domain C as the
DNA-binding domain, domain D as a hinge domain and domain E/F as the
ligand-dependent transcriptional domain (AF-2). They regulate gene transcription
through interactions with various nuclear factors of their domains, such as AF-1
and AF-2. We have insufficient knowledge of the function of the DNA-binding
domain (domain C) except for its DNA-binding function or the hinge domain
(domain D). Therefore, we attempted to identify factors interacting with the
domains by using a yeast two-hybrid system. Domains C and D of estrogen receptor
alpha were used as a bait to isolate cDNA clones from a rat ovary cDNA library.
We isolated the cDNA clone of a novel steroid receptor-binding protein bearing
the regulator of G-protein signaling (RGS) designated as SRB-RGS. The protein
repressed the transcriptional activity of estrogen receptor alpha, suggesting
cross-talk of steroid hormones and peptide hormones (or growth factors) for
signal transductions mediated by SRB-RGS.
PMID: 11595167 [PubMed - indexed for MEDLINE]
70: Structure (Camb) 2001 Oct;9(10):897-904
Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a
novel role for the ubiquitin tail.
Hamilton KS, Ellison MJ, Barber KR, Williams RS, Huzil JT, McKenna S, Ptak C,
Glover M, Shaw GS.
Department of Biochemistry, The University of Alberta, Edmonton, Alberta T6G
2H7, Canada.
BACKGROUND: Ubiquitin-conjugating enzymes (E2s) are central enzymes involved in
ubiquitin-mediated protein degradation. During this process, ubiquitin (Ub) and
the E2 protein form an unstable E2-Ub thiolester intermediate prior to the
transfer of ubiquitin to an E3-ligase protein and the labeling of a substrate
for degradation. A series of complex interactions occur among the target
substrate, ubiquitin, E2, and E3 in order to efficiently facilitate the transfer
of the ubiquitin molecule. However, due to the inherent instability of the E2-Ub
thiolester, the structural details of this complex intermediate are not known.
RESULTS: A three-dimensional model of the E2-Ub thiolester intermediate has been
determined for the catalytic domain of the E2 protein Ubc1 (Ubc1(Delta450)) and
ubiquitin from S. cerevisiae. The interface of the E2-Ub intermediate was
determined by kinetically monitoring thiolester formation by 1H-(15)N HSQC
spectra by using combinations of 15N-labeled and unlabeled Ubc1(Delta450) and Ub
proteins. By using the surface interface as a guide and the X-ray structures of
Ub and the 1.9 A structure of Ubc1(Delta450) determined here, docking
simulations followed by energy minimization were used to produce the first model
of a E2-Ub thiolester intermediate. CONCLUSIONS: Complementary surfaces were
found on the E2 and Ub proteins whereby the C terminus of Ub wraps around the E2
protein terminating in the thiolester between C88 (Ubc1(Delta450)) and G76 (Ub).
The model supports in vivo and in vitro experiments of E2 derivatives carrying
surface residue substitutions. Furthermore, the model provides insights into the
arrangement of Ub, E2, and E3 within a ternary targeting complex.
PMID: 11591345 [PubMed - indexed for MEDLINE]
71: Mol Cell Neurosci 2001 Sep;18(3):307-19
Doublecortin interacts with mu subunits of clathrin adaptor complexes in the
developing nervous system.
Friocourt G, Chafey P, Billuart P, Koulakoff A, Vinet MC, Schaar BT, McConnell
SK, Francis F, Chelly J.
Laboratoire de Genetique et Physiopathologie des retards mentaux, ICGM, INSERM,
CHU, Cochin, 24, rue du Faubourg Saint Jacques, Paris, 75014, France.
Doublecortin is a microtubule-associated protein required for normal
corticogenesis in the developing brain. We carried out a yeast two-hybrid screen
to identify interacting proteins. One of the isolated clones encodes the mu1
subunit of the adaptor complex AP-1 involved in clathrin-dependent protein
sorting. We found that Doublecortin also interacts in yeast with mu2 from the
AP-2 complex. Mutagenesis and pull-down experiments showed that these
interactions were mediated through a tyrosine-based sorting signal (YLPL) in the
C-terminal part of Doublecortin. The functional relevance of these interactions
was suggested by the coimmunoprecipitation of Doublecortin with AP-1 and AP-2
from mouse brain extracts. This interaction was further supported by RNA in situ
hybridization and immunofluorescence studies. Taken together these data indicate
that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in
vivo and are consistent with a potential involvement of Doublecortin in protein
sorting or vesicular trafficking. Copyright 2001 Academic Press.
PMID: 11591131 [PubMed - indexed for MEDLINE]
72: Int J Food Microbiol 2001 Sep 19;69(1-2):101-11
Saccharomyces cerevisiae as a starter culture in Mycella.
Hansen TK, Tempel TV, Cantor MD, Jakobsen M.
Department of Dairy and Food Science, Food Microbiology, The Royal Veterinary
and Agricultural University, Frederiksberg, Denmark. tkh@kvl.dk
The potential use of Saccharomyces cerevisiae FB7 as an additional starter
culture for the production of Mycella, a Danish Gorgonzola type cheese, was
investigated. Two dairy productions of Mycella, each containing batches of
experimental cheeses with S. cerevisiae added and reference cheeses without
yeast added were carried out. For both experimental and reference cheeses,
chemical analysis (pH, a(w), NaCl, water and fat content) were carried out
during the ripening period, but no significant differences were found. The
evolution of lactic acid bacteria was almost identical in both the experimental
and reference cheeses and similar results were found for the number of yeast. S.
cerevisiae FB7 was found to be predominant in the core of the experimental
cheeses throughout the ripening period, while Debaryomyces hansenii dominated in
the reference cheese and on the surface of the experimental cheeses. In the
cheeses with S. cerevisiae FB7, an earlier sporulation and an improved growth of
Penicillium roqueforti was observed compared to the reference cheeses.
Furthermore, in the experimental cheese, synergistic interactions were also
found in the aroma analysis, the degradation of casein and by the sensory
analysis. The observed differences indicate a positive contribution to the
overall quality of Mycella by S. cerevisiae FB7.
PMID: 11589548 [PubMed - indexed for MEDLINE]
73: Europ J Paediatr Neurol 2001;5 Suppl A:89-93
Analysis of CLN3-protein interactions using the yeast two-hybrid system.
Leung KY, Greene ND, Munroe PB, Mole SE.
Heart Science Centre, Harefield Hospital, Middlesex, UK.
kit-yi.leung@harefield.nthames.nhs.uk
Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a childhood
neurodegenerative disease that is caused by mutations in the CLN3 gene. The
protein encoded by CLN3 has no homology with any proteins of known function and
its cellular role remains elusive. In order to investigate the role played by
the CLN3 protein we aimed to identify interacting proteins. Here, we describe
the yeast two-hybrid system as the approach taken to investigate such
protein-protein interactions. CLN3 was expressed as a fusion protein with a
DNA-binding domain and used to screen a library of human fetal brain cDNAs fused
to a transcriptional activation domain. Owing to low level expression of the
full length CLN3 fusion protein, truncated regions corresponding to the
predicted hydrophilic regions were also tested. No proteins that interact with
CLN3 were detected, nor was there any evidence for CLN3-CLN3 interactions.
Potential interaction of CLN3 with subunit c of mitochondrial ATP synthase, the
major component of the storage material that accumulates in Batten disease
patients, was also tested. No interaction was detected suggesting that the
accumulation of subunit c does not result from loss of a process that requires a
direct interaction with CLN3. We conclude that either CLN3 does not interact
with other proteins or such interactions cannot be detected using the two-hybrid
system.
PMID: 11589015 [PubMed - indexed for MEDLINE]
74: Biochem Biophys Res Commun 2001 Oct 12;287(5):1083-7
The PUB domain: a putative protein-protein interaction domain implicated in the
ubiquitin-proteasome pathway.
Suzuki T, Park H, Till EA, Lennarz WJ.
Department of Biochemistry and Cell Biology, State University of New York at
Stony Brook, Stony Brook, New York, 11794-5215, USA.
Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which
may be involved in the proteasome-dependent pathway for degradation of misfolded
glycoproteins formed in the endoplasmic reticulum (ER) that are exported into
the cytoplasm. A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is
widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al.
J. Cell Biol. 149, 1039-1051, 2000). The recently uncovered complete genome
sequence of Arabidopsis thaliana prompted us to search for the protein homologue
of Png1p in this organism. Interestingly, when the mouse Png1p homologue
sequence was used as a query, not only a Png1p homologue containing a
transglutaminase-like domain that is believed to contain a catalytic triad for
PNGase activity, but also four proteins which had a domain of 46 amino acids in
length that exhibited significant similarity to the N-terminus of mouse Png1p
were identified. Moreover, three of these homologous proteins were also found to
possess a UBA or UBX domain, which are found in various proteins involved in the
ubiquitin-related pathway. We name this newly found homologous region the PUB
(Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that
this domain may mediate protein-protein interactions. Copyright 2001 Academic
Press.
PMID: 11587532 [PubMed - indexed for MEDLINE]
75: Genome Biol 2001;2(9):RESEARCH0039
Abundant protein domains occur in proportion to proteome size.
Malek JA.
Agencourt Bioscience Corporation, 100 Cummings Center, Suite 107J, Beverly, MA
01915, USA. jamalek@agencourt.com
BACKGROUND: Conserved domains in proteins have crucial roles in protein
interactions, DNA binding, enzyme activity and other important cellular
processes. It will be of interest to determine the proportions of genes
containing such domains in the proteomes of different eukaryotes. RESULTS: The
average proportion of conserved domains in each of five eukaryote genomes was
calculated. In pairwise genome comparisons, the ratio of genes containing a
given conserved domain in the two genomes on average reflected the ratio of the
predicted total gene numbers of the two genomes. These ratios have been verified
using a repository of databases and one of its subdivisions of conserved
domains. CONCLUSIONS: Many conserved domains occur as a constant proportion of
proteome size across the five sequenced eukaryotic genomes. This raises the
possibility that this proportion is maintained because of functional constraints
on interacting domains. The universality of the ratio in the five eukaryotic
genomes attests to its potential importance.
PMID: 11574058 [PubMed - indexed for MEDLINE]
76: Cell 2001 Sep 21;106(6):723-33
A plant viral "reinitiation" factor interacts with the host translational
machinery.
Park HS, Himmelbach A, Browning KS, Hohn T, Ryabova LA.
Friedrich Miescher-Institute, P.O. Box 2543, CH-4002, Basel, Switzerland.
The cauliflower mosaic virus transactivator, TAV, controls translation
reinitiation of major open reading frames on polycistronic RNA. We show here
that TAV function depends on its association with polysomes and eukaryotic
initiation factor eIF3 in vitro and in vivo. TAV physically interacts with eIF3
and the 60S ribosomal subunit. Two proteins mediating these interactions were
identified: eIF3g and 60S ribosomal protein L24. Transient expression of eIF3g
and L24 in plant protoplasts strongly affects TAV-mediated reinitiation
activity. We demonstrate that TAV/eIF3/40S and eIF3/TAV/60S ternary complexes
form in vitro, and propose that TAV mediates efficient recruitment of eIF3 to
polysomes, allowing translation of polycistronic mRNAs by reinitiation,
overcoming the normal cell barriers to this process.
PMID: 11572778 [PubMed - indexed for MEDLINE]
77: EMBO J 2001 Sep 17;20(18):5290-301
The structure of an AspRS-tRNA(Asp) complex reveals a tRNA-dependent control
mechanism.
Moulinier L, Eiler S, Eriani G, Gangloff J, Thierry JC, Gabriel K, McClain WH,
Moras D.
UPR 9004, Laboratoire de Biologie et Genomique Structurales, Institut de
Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, 1 rue
Laurent Fries, BP 163, 67404 Illkirch Cedex, France.
The 2.6 A resolution crystal structure of an inactive complex between yeast
tRNA(Asp) and Escherichia coli aspartyl-tRNA synthetase reveals the molecular
details of a tRNA-induced mechanism that controls the specificity of the
reaction. The dimer is asymmetric, with only one of the two bound tRNAs entering
the active site cleft of its subunit. However, the flipping loop, which controls
the proper positioning of the amino acid substrate, acts as a lid and prevents
the correct positioning of the terminal adenosine. The structure suggests that
the acceptor stem regulates the loop movement through sugar phosphate backbone-
protein interactions. Solution and cellular studies on mutant tRNAs confirm the
crucial role of the tRNA three-dimensional structure versus a specific
recognition of bases in the control mechanism.
PMID: 11566892 [PubMed - indexed for MEDLINE]
78: EMBO J 2001 Sep 17;20(18):5207-18
Structure of the yeast nucleosome core particle reveals fundamental changes in
internucleosome interactions.
White CL, Suto RK, Luger K.
Department of Biochemistry and Molecular Biology, Colorado State University,
Fort Collins, CO 80523-1870, USA.
Chromatin is composed of nucleosomes, the universally repeating protein-DNA
complex in eukaryotic cells. The crystal structure of the nucleosome core
particle from Saccharomyces cerevisiae reveals that the structure and function
of this fundamental complex is conserved between single-cell organisms and
metazoans. Our results show that yeast nucleosomes are likely to be subtly
destabilized as compared with nucleosomes from higher eukaryotes, consistent
with the idea that much of the yeast genome remains constitutively open during
much of its life cycle. Importantly, minor sequence variations lead to dramatic
changes in the way in which nucleosomes pack against each other within the
crystal lattice. This has important implications for our understanding of the
formation of higher order chromatin structure and its modulation by
post-translational modifications. Finally, the yeast nucleosome core particle
provides a structural context by which to interpret genetic data obtained from
yeast. Coordinates have been deposited with the Protein Data Bank under
accession number 1ID3.
PMID: 11566884 [PubMed - indexed for MEDLINE]
79: Mol Cell Biol 2001 Oct;21(20):6782-95
Human STAGA complex is a chromatin-acetylating transcription coactivator that
interacts with pre-mRNA splicing and DNA damage-binding factors in vivo.
Martinez E, Palhan VB, Tjernberg A, Lymar ES, Gamper AM, Kundu TK, Chait BT,
Roeder RG.
Laboratories of Biochemistry and Molecular Biology, The Rockefeller University,
New York, New York 10021, USA.
GCN5 is a histone acetyltransferase (HAT) originally identified in Saccharomyces
cerevisiae and required for transcription of specific genes within chromatin as
part of the SAGA (SPT-ADA-GCN5 acetylase) coactivator complex. Mammalian cells
have two distinct GCN5 homologs (PCAF and GCN5L) that have been found in three
different SAGA-like complexes (PCAF complex, TFTC [TATA-binding-protein-free
TAF(II)-containing complex], and STAGA [SPT3-TAF(II)31-GCN5L acetylase]). The
composition and roles of these mammalian HAT complexes are still poorly
characterized. Here, we present the purification and characterization of the
human STAGA complex. We show that STAGA contains homologs of most yeast SAGA
components, including two novel human proteins with histone-like folds and
sequence relationships to yeast SPT7 and ADA1. Furthermore, we demonstrate that
STAGA has acetyl coenzyme A-dependent transcriptional coactivator functions from
a chromatin-assembled template in vitro and associates in HeLa cells with
spliceosome-associated protein 130 (SAP130) and DDB1, two structurally related
proteins. SAP130 is a component of the splicing factor SF3b that associates with
U2 snRNP and is recruited to prespliceosomal complexes. DDB1 (p127) is a
UV-damaged-DNA-binding protein that is involved, as part of a complex with DDB2
(p48), in nucleotide excision repair and the hereditary disease xeroderma
pigmentosum. Our results thus suggest cellular roles of STAGA in chromatin
modification, transcription, and transcription-coupled processes through direct
physical interactions with sequence-specific transcription activators and with
components of the splicing and DNA repair machineries.
PMID: 11564863 [PubMed - indexed for MEDLINE]
80: Genes Dev 2001 Sep 15;15(18):2445-56
Mechanisms controlling differential promoter-occupancy by the yeast forkhead
proteins Fkh1p and Fkh2p: implications for regulating the cell cycle and
differentiation.
Hollenhorst PC, Pietz G, Fox CA.
Department of Biomolecular Chemistry, University of Wisconsin, Madison,
Wisconsin 53706, USA.
The roles of DNA and Mcm1p interactions in determining the overlapping and
distinct functions of the yeast cell cycle regulatory transcription factors
Fkh1p and Fkh2p were examined. Full-length recombinant Fkh1p and Fkh2p were
purified and their binding to bona fide promoters examined in vitro. Each
protein bound a variety of target promoters with similar specificity in vitro,
consistent with the observation that these proteins bind common promoters in
vivo. However, in vivo, the Fkh1p and Fkh2p occupied different target promoters
to different extents, suggesting that each was primarily responsible for
controlling a different set of genes. Additional in vitro studies provided a
mechanistic explanation for this differential promoter-occupancy. Specifically,
the Fkh2p, but not the Fkh1p, was capable of binding cooperatively with Mcm1p.
The Mcm1p-Fkh2p cooperative binding was enhanced by, but did not require, the
presence of a Mcm1p-binding site within a target promoter. Consistent with these
data, Mcm1p was present at Fkh-controlled promoters in vivo regardless of
whether they contained Mcm1p-binding sites, suggesting a role for Mcm1p at
promoters not thought previously to be under Mcm1p control. Analysis of Fkh1p
and Fkh2p binding to promoter targets in vivo by use of mutant strains indicated
that the two proteins compete for promoter-occupancy at a number of target
promoters. We postulate that Fkh1p and a stable Fkh2p/Mcm1p complex compete for
binding to target promoters and that the levels and/or binding activity of
Fkh1p, but not Fkh2p, are most limiting for promoter-occupancy in vivo.
Interestingly, the in vitro DNA-binding assays, using a variety of promoter
targets, revealed that bona fide Fkh target promoters contained two or more
Fkh-binding sites that allowed the Fkh1p and Fkh2p proteins to form multiple
protein-DNA complexes in vitro. Multiple Fkh-binding sites may be a
distinguishing feature of bona fide Fkh promoters in yeast and other organisms.
PMID: 11562353 [PubMed - indexed for MEDLINE]
81: Genetics 2001 Sep;159(1):91-105
Multiple interactions among the components of the recombinational DNA repair
system in Schizosaccharomyces pombe.
Tsutsui Y, Khasanov FK, Shinagawa H, Iwasaki H, Bashkirov VI.
Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871,
Japan.
Schizosaccharomyces pombe Rhp55 and Rhp57 are RecA-like proteins involved in
double-strand break (DSB) repair. Here we demonstrate that Rhp55 and Rhp57
proteins strongly interact in vivo, similar to Saccharomyces cerevisiae Rad55p
and Rad57p. Mutations in the conserved ATP-binding/hydrolysis folds of both the
Rhp55 and Rhp57 proteins impaired their function in DNA repair but not in cell
proliferation. However, when combined, ATPase fold mutations in Rhp55p and
Rhp57p resulted in severe defects of both functions, characteristic of the
deletion mutants. Yeast two-hybrid analysis also revealed other multiple in vivo
interactions among S. pombe proteins involved in recombinational DNA repair.
Similar to S. cerevisiae Rad51p-Rad54p, S. pombe Rhp51p and Rhp54p were found to
interact. Both putative Rad52 homologs in S. pombe, Rad22p and Rti1p, were found
to interact with the C-terminal region of Rhp51 protein. Moreover, Rad22p and
Rti1p exhibited mutual, as well as self-, interactions. In contrast to the S.
cerevisiae interacting pair Rad51p-Rad55p, S. pombe Rhp51 protein strongly
interacted with Rhp57 but not with Rhp55 protein. In addition, the Rti1 and
Rad22 proteins were found to form a complex with the large subunit of S. pombe
RPA. Our data provide compelling evidence that most, but not all, of the
protein-protein interactions found in S. cerevisiae DSB repair are
evolutionarily conserved.
PMID: 11560889 [PubMed - indexed for MEDLINE]
82: Genetics 2001 Sep;159(1):77-89
The Ras/PKA signaling pathway of Saccharomyces cerevisiae exhibits a functional
interaction with the Sin4p complex of the RNA polymerase II holoenzyme.
Howard SC, Chang YW, Budovskaya YV, Herman PK.
Department of Molecular Genetics, The Ohio State University, Columbus, Ohio
43210, USA.
Saccharomyces cerevisiae cells enter into the G(0)-like resting state,
stationary phase, in response to specific types of nutrient limitation. We have
initiated a genetic analysis of this resting state and have identified a
collection of rye mutants that exhibit a defective transcriptional response to
nutrient deprivation. These transcriptional defects appear to disrupt the
control of normal growth because the rye mutants are unable to enter into a
normal stationary phase upon nutrient deprivation. In this study, we examined
the mutants in the rye1 complementation group and found that rye1 mutants were
also defective for stationary phase entry. Interestingly, the RYE1 gene was
found to be identical to SIN4, a gene that encodes a component of the yeast
Mediator complex within the RNA polymerase II holoenzyme. Moreover, mutations
that affected proteins within the Sin4p module of the Mediator exhibited
specific genetic interactions with the Ras protein signaling pathway. For
example, mutations that elevated the levels of Ras signaling, like RAS2(val19),
were synthetic lethal with sin4. In all, our data suggest that specific proteins
within the RNA polymerase II holoenzyme might be targets of signal transduction
pathways that are responsible for coordinating gene expression with cell growth.
PMID: 11560888 [PubMed - indexed for MEDLINE]
83: J Virol 2001 Oct;75(20):9613-22
Functional interaction map of lyssavirus phosphoprotein: identification of the
minimal transcription domains.
Jacob Y, Real E, Tordo N.
Laboratoire des Lyssavirus, Institut Pasteur, 75724 Paris Cedex 15, France.
yjacob@pasteur.fr
Lyssaviruses, the causative agents of rabies encephalitis, are distributed in
seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype
1) and Mokola virus (genotype 3) were used to develop a strategy to identify
functional homologous interactive domains from two proteins (P and N) which
participate in the viral ribonucleoprotein (RNP) transcription-replication
complex. This strategy combined two-hybrid and green fluorescent protein-reverse
two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein
interactions and a reverse genetic assay in mammalian cells to study the
transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins
contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa)
176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal
portion of P (aa 52 to 189) also contains a homomultimerization site. Here we
demonstrate that N-P interactions, although weaker, are maintained between
proteins of the different genotypes. A minimal transcriptional module of the P
protein was obtained by fusing the first 60 N-terminal aa containing the L
protein binding site to the C-terminal strong N-BD. Random mutation of the
strong N-BD on P protein identified three highly conserved K residues crucial
for N-P interaction. Their mutagenesis in full-length P induced a
transcriptionally defective RNP. The analysis of homologous interactive domains
presented here and previously reported dissections of the P protein allowed us
to propose a model of the functional interaction network of the lyssavirus P
protein. This model underscores the central role of P at the interface between L
protein and N-RNA template.
PMID: 11559793 [PubMed - indexed for MEDLINE]
84: J Biol Chem 2001 Nov 9;276(45):42003-10
The structural and functional organization of the yeast mediator complex.
Kang JS, Kim SH, Hwang MS, Han SJ, Lee YC, Kim YJ.
National Creative Research Center for Genome Regulation, Department of
Biochemistry, Yonsei University, Seoul 120-749, Korea.
The Mediator complex of Saccharomyces cerevisiae is required for diverse aspects
of transcription by RNA polymerase II (pol II). Mediator is composed of two
functionally distinct subcomplexes, Rgr1 and Srb4. To identify the structures
and functions of each subcomplex, we expressed recombinant proteins for each
subunit and assayed their interactions with each other and with basal
transcription proteins. The Rgr1 subcomplex is composed of the Gal11 module,
which binds activators, and the Med9/10 module. The Med9/10 module is required
for both transcriptional activation and repression, and these activities appear
to be carried out by two submodules. Proteins in the Med9 submodule interact
physically and genetically with Srb10/11, suggesting that the Med9 submodule
mediates the repression of pol II. Purified recombinant Srb4 subcomplex
stimulated basal transcription of pol II but had little effect on activated
transcription and phosphorylation of the C-terminal domain of the Rpb1 subunit
of pol II. Both subcomplexes of Mediator interacted with a distinct set of basal
transcription factors and pol II. The modular organization of Mediator and the
associated functions suggest that the Mediator complex may recruit and/or
stabilize the preinitiation complex through several points of contact with
transcriptional regulators and basal transcription factors.
PMID: 11555651 [PubMed - indexed for MEDLINE]
85: Mol Biol Cell 2001 Sep;12(9):2870-80
Control of microtubule dynamics by Stu2p is essential for spindle orientation
and metaphase chromosome alignment in yeast.
Kosco KA, Pearson CG, Maddox PS, Wang PJ, Adams IR, Salmon ED, Bloom K, Huffaker
TC.
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY
14853-2703, USA.
Stu2p is a member of a conserved family of microtubule-binding proteins and an
essential protein in yeast. Here, we report the first in vivo analysis of
microtubule dynamics in cells lacking a member of this protein family. For these
studies, we have used a conditional Stu2p depletion strain expressing
alpha-tubulin fused to green fluorescent protein. Depletion of Stu2p leads to
fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase
cells. The reduction in cytoplasmic microtubule dynamics is due primarily to
decreases in both the catastrophe and rescue frequencies and an increase in the
fraction of time microtubules spend pausing. These changes have significant
consequences for the cell because they impede the ability of cytoplasmic
microtubules to orient the spindle. In addition, recovery of fluorescence after
photobleaching indicates that kinetochore microtubules are no longer dynamic in
the absence of Stu2p. This deficiency is correlated with a failure to properly
align chromosomes at metaphase. Overall, we provide evidence that Stu2p promotes
the dynamics of microtubule plus-ends in vivo and that these dynamics are
critical for microtubule interactions with kinetochores and cortical sites in
the cytoplasm.
PMID: 11553724 [PubMed - indexed for MEDLINE]
86: Mol Biol Cell 2001 Sep;12(9):2756-66
The GTPase effector domain sequence of the Dnm1p GTPase regulates self-assembly
and controls a rate-limiting step in mitochondrial fission.
Fukushima NH, Brisch E, Keegan BR, Bleazard W, Shaw JM.
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.
Dnm1p belongs to a family of dynamin-related GTPases required to remodel
different cellular membranes. In budding yeast, Dnm1p-containing complexes
assemble on the cytoplasmic surface of the outer mitochondrial membrane at sites
where mitochondrial tubules divide. Our previous genetic studies suggested that
Dnm1p's GTPase activity was required for mitochondrial fission and that Dnm1p
interacted with itself. In this study, we show that bacterially expressed Dnm1p
can bind and hydrolyze GTP in vitro. Coimmunoprecipitation studies and yeast
two-hybrid analysis suggest that Dnm1p oligomerizes in vivo. With the use of the
yeast two-hybrid system, we show that this Dnm1p oligomerization is mediated, in
part, by a C-terminal sequence related to the GTPase effector domain (GED) in
dynamin. The Dnm1p interactions characterized here are similar to those reported
for dynamin and dynamin-related proteins that form higher order structures in
vivo, suggesting that Dnm1p assembles to form rings or collars that surround
mitochondrial tubules. Based on previous findings, a K705A mutation in the Dnm1p
GED is predicted to interfere with GTP hydrolysis, stabilize active Dnm1p-GTP,
and stimulate a rate-limiting step in fission. Here we show that expression of
the Dnm1 K705A protein in yeast enhances mitochondrial fission. Our results
provide evidence that the GED region of a dynamin-related protein modulates a
rate-limiting step in membrane fission.
PMID: 11553714 [PubMed - indexed for MEDLINE]
87: Mol Biol Cell 2001 Sep;12(9):2601-13
Dad1p, third component of the Duo1p/Dam1p complex involved in kinetochore
function and mitotic spindle integrity.
Enquist-Newman M, Cheeseman IM, Van Goor D, Drubin DG, Meluh PB, Barnes G.
Department of Molecular and Cell Biology, University of California, Berkeley, CA
94720-3202, USA.
We showed recently that a complex between Duo1p and Dam1p is required for both
spindle integrity and kinetochore function in the budding yeast Saccharomyces
cerevisiae. To extend our understanding of the functions and interactions of the
Duo1p/Dam1p complex, we analyzed the novel gene product Dad1p (for Duo1 and Dam1
interacting). Dad1p physically associates with Duo1p by two-hybrid analysis,
coimmunoprecipitates with Duo1p and Dam1p out of yeast protein extracts, and
shows interdependent localization with Duo1p and Dam1p to the mitotic spindle.
These results indicate that Dad1p functions as a component of the Duo1p/Dam1p
complex. Like Duo1p and Dam1p, Dad1p also localizes to kinetochore regions in
chromosomes spreads. Here, we also demonstrate by chromatin immunoprecipitation
that Duo1p, Dam1p, and Dad1p associate specifically with centromeric DNA in a
manner that is dependent upon Ndc10 and partially dependent upon the presence of
microtubules. To explore the functions of Dad1p in vivo, we generated a
temperature-sensitive allele, dad1-1. This allele shows spindle defects and a
mitotic arrest phenotype that is dependent upon the spindle assembly checkpoint.
In addition, dad1-1 mutants undergo chromosome mis-segregation at the
restrictive temperature, resulting in a dramatic decrease in viability.
PMID: 11553702 [PubMed - indexed for MEDLINE]
88: J Clin Endocrinol Metab 2001 Sep;86(9):4416-23
Pitfalls in characterizing P450c17 mutations associated with isolated
17,20-lyase deficiency.
Gupta MK, Geller DH, Auchus RJ.
Division of Endocrinology and Metabolism, Department of Internal Medicine,
University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857, USA.
The cytochrome P450c17 enzyme system performs both the 17alpha-hydroxylase and
17,20-lyase reactions in the human adrenal glands and gonads. This 17,20-lyase
activity is required for the biosynthesis of dehydroepiandrosterone, the C(19)
precursor of sex steroids. Considerable evidence supports the idea that the
17,20-lyase activity of this system is particularly sensitive to alterations in
the interactions between P450c17 and its cofactor proteins P450-oxidoreductase
and cytochrome b(5). We have described two patients with the clinical phenotype
of isolated 17,20-lyase deficiency in whom single amino acid replacement
mutations in the redox partner binding site of P450c17 (R347H and R358Q)
selectively ablate 17,20-lyase activity while preserving most
17alpha-hydroxylase activity. We have shown by computer modeling and detailed
biochemical studies that mutations R347H and R358Q impair the interactions of
P450c17 with P450-oxidoreductase and cytochrome b(5) (redox partners). Another
mutation reported to cause isolated 17,20-lyase deficiency (F417C) does not map
within the redox partner binding site, but might nonetheless alter the
interaction of the mutant protein with redox partners. To study the interaction
of the F417C mutation with P450 oxidoreductase and cytochrome b(5), we expressed
the cDNA for this protein in yeast microsomes, a heterologous expression system
in which the composition of redox partner proteins can be varied systematically.
Although the full-length protein was expressed in quantities comparable to those
of wild-type P450c17 in this system, the F417C mutation did not form a classical
P450 difference spectrum and was devoid of both 17alpha-hydroxylase and
17,20-lyase activities. To ensure that this result was not unique to the yeast
expression system, we also expressed wild-type P450c17 and the F417C mutation in
COS-7 cells, and we again found that the F417C mutation was expressed, but was
not active. To conclusively demonstrate that a particular mutation in P450c17
causes isolated 17,20-lyase deficiency, accurate enzymatic studies of the mutant
protein must reproducibly show activities consistent with the diagnosis.
Mutations R347H and R358Q are the only two such mutations found in humans proven
to cause isolated 17,20-lyase deficiency.
PMID: 11549685 [PubMed - indexed for MEDLINE]
89: Eur Biophys J 2001 Aug;30(4):273-83
Fluoroalcohol-induced structural changes of proteins: some aspects of
cosolvent-protein interactions.
Gast K, Siemer A, Zirwer D, Damaschun G.
Max-Delbruck-Centrum fur Molekulare Medizin Berlin-Buch, Germany.
gast@mdc-berlin.de
The conformational transitions of bovine beta-lactoglobulin A and
phosphoglycerate kinase from yeast induced by hexafluoroisopropanol (HFIP) and
trifluoroethanol (TFE) have been studied by dynamic light scattering and
circular dichroism spectroscopy in order to elucidate the potential of
fluoroalcohols to bring about structural changes of proteins. Moreover, pure
fluoroalcohol-water mixed solvents were investigated to prove the relation
between cluster formation and the effects on proteins. The results demonstrate
that cluster formation is mostly an accompanying phenomenon because important
structural changes of the proteins occur well below the critical concentration
of fluoroalcohol at which the formation of clusters sets in. According to our
light scattering experiments, the remarkable potential of HFIP is a consequence
of extensive preferential binding. Surprisingly, preferential binding seems to
play a vanishing role in the case of TFE. However, the comparable Stokes radii
of both proteins in the highly helical state induced by either HFIP or TFE point
to a similar degree of solvation in both mixed solvents. This shows that direct
binding or an indirect mechanism must be equally taken into consideration to
explain the effects of alcohols on proteins. The existence of a compact helical
intermediate with non-native secondary structure on the transition of
beta-lactoglobulin A from the native to the highly helical state is clearly
demonstrated.
PMID: 11548130 [PubMed - indexed for MEDLINE]
90: Oncogene 2001 Aug 30;20(38):5279-90
Toxicity of human adenovirus E4orf4 protein in Saccharomyces cerevisiae results
from interactions with the Cdc55 regulatory B subunit of PP2A.
Roopchand DE, Lee JM, Shahinian S, Paquette D, Bussey H, Branton PE.
Department of Biochemistry, McGill University, McIntyre Medical Building,
Montreal, Quebec, Canada, H3G 1Y6.
The E4orf4 protein of human adenovirus induces p53-independent apoptosis, a
process that may promote cell death and viral spread. When expressed alone,
E4orf4 kills transformed cells but not normal human cells. The only clear target
of E4orf4 in mammalian cells is the Balpha (B55) subunit of protein phosphatase
2A (PP2A), a member of one of three classes of regulatory B subunits. Here we
report the effects of E4orf4 in Saccharomyces cerevisiae, which encodes two PP2A
regulatory B subunits, CDC55 and RTS1, that share homology with mammalian B and
B' subunits, respectively. E4orf4 expression was found to be toxic in yeast,
resulting in the accumulation of cells in G2/M phase that failed to grow upon
removal of E4orf4. E4orf4-expressing yeast also displayed an elongated cell
morphology similar to cdc55 deletion strains. E4orf4 required CDC55 to elicit
its effect, whereas RTS1 was dispensable. The recruitment of the PP2A holoenzyme
by E4orf4 was entirely dependent on Cdc55. These studies indicate that
E4orf4-induced apoptosis in mammalian cells and cell death in yeast require
functional interactions with B-type subunits of PP2A. However, some inhibition
of growth by E4orf4 was observed in the cdc55 strain and with an E4orf4 mutant
that fails to interact with Cdc55, indicating that E4orf4 may possess a second
Cdc55-independent function affecting cell growth.
PMID: 11536041 [PubMed - indexed for MEDLINE]
91: Biochem J 2001 Sep 15;358(Pt 3):727-35
Binding of the merlin-I product of the neurofibromatosis type 2 tumour
suppressor gene to a novel site in beta-fodrin is regulated by association
between merlin domains.
Neill GW, Crompton MR.
Centre for Cutaneous Research, St Bartholomew's and the Royal London, Queen Mary
and Westfield College, 2 Newark Street, London E1 2AT, UK.
The mechanism underlying the tumour-suppressor activity of the neurofibromatosis
type 2 (NF2) gene product, merlin, is largely undefined but there is evidence
that the biological function of the protein might be mediated partly through
interactions with the cytoskeleton. Merlin is expressed predominantly as two
isoforms that differ at their C-termini owing to alternative splicing of exon
16. By expressing merlin isoform I as bait in a yeast two-hybrid screen, we
isolated a clone encoding a region of the cytoskeletal protein beta-fodrin.
Confirmation of the merlin-fodrin interaction was provided by using the
mammalian two-hybrid system and binding assays in vitro. In addition, these
assays and co-immunoprecipitation from mammalian cells revealed that the binding
site for fodrin is located in the C-terminal half of merlin at a site that is
masked in the native protein. Co-expression of the N-terminus of merlin
decreased the interaction of its C-terminus with fodrin, implicating homophilic
interactions of merlin isoform I in masking the fodrin-binding site. The effect
of three disease-associated mutations on the merlin-fodrin interaction and
merlin dimerization was also investigated. The mutation L535P, but not L360P or
K413E, significantly decreased the merlin-fodrin interaction but not
dimerization, indicating that the tumour suppressor ability of merlin might
reside partly in its ability to interact with the cytoskeleton via fodrin.
PMID: 11535133 [PubMed - indexed for MEDLINE]
92: Mol Cell Biol 2001 Oct;21(19):6606-14
Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by
interactions between integrase and Sir4p.
Xie W, Gai X, Zhu Y, Zappulla DC, Sternglanz R, Voytas DF.
Department of Zoology and Genetics, Iowa State University, Ames, Iowa
50011-3260, USA.
The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially
into regions of silent chromatin at the telomeres and silent mating loci (HMR
and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid
residues near the C terminus of integrase (LXSSXP). The targeting domain
establishes silent chromatin when it is tethered to a weakened HMR-E silencer,
and it disrupts telomeric silencing when it is overexpressed. As determined by
both yeast two-hybrid and in vitro binding assays, the targeting domain
interacts with the C terminus of Sir4p, a structural component of silent
chromatin. This interaction is abrogated by mutations in the targeting domain
that disrupt integration into silent chromatin, suggesting that recognition of
Sir4p by the targeting domain is the primary determinant in Ty5 target
specificity.
PMID: 11533248 [PubMed - indexed for MEDLINE]
93: Mol Cell Biol 2001 Oct;21(19):6429-39
A novel upstream RNA polymerase III promoter element becomes essential when the
chromatin structure of the yeast U6 RNA gene is altered.
Martin MP, Gerlach VL, Brow DA.
Department of Biomolecular Chemistry, University of Wisconsin Medical School,
Madison, Wisconsin 53706-1532, USA.
The Saccharomyces cerevisiae U6 RNA gene, SNR6, possesses upstream sequences
that allow productive binding in vitro of the RNA polymerase III (Pol III)
transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other
assembly factors. TFIIIC-independent transcription of SNR6 in vitro is highly
sensitive to point mutations in a consensus TATA box at position -30. In
contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently
because TFIIIC bound to the intragenic A block and downstream B block can
recruit TFIIIB via protein-protein interactions. A mutant allele of SNR6 with
decreased spacing between the A and B blocks, snr6-Delta42, exhibits increased
dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo
expression of snr6-Delta42 is much more sensitive to mutations in a (dT-dA)(7)
tract between the TATA box and transcription start site than to mutations in the
TATA box itself. Inversion of single base pairs in the center of the dT-dA tract
nearly abolishes transcription of snr6-Delta42, yet inversion of all 7 base
pairs has little effect on expression, indicating that the dA-dT tract is
relatively orientation independent. Although it is within the TFIIIB footprint,
point mutations in the dT-dA tract do not inhibit TFIIIB binding or
TFIIIC-independent transcription of SNR6 in vitro. In the absence of the
chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when
A-to-B block spacing is wild type. We conclude that the (dT-dA)(7) tract and
Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in
vivo.
PMID: 11533232 [PubMed - indexed for MEDLINE]
94: EMBO J 2001 Sep 3;20(17):4935-43
The chromatin remodelling factor Brg-1 interacts with beta-catenin to promote
target gene activation.
Barker N, Hurlstone A, Musisi H, Miles A, Bienz M, Clevers H.
Department of Immunology, University Medical Center Utrecht, Heidelberglaan 100,
3584 CX Utrecht, The Netherlands.
Wnt-induced formation of nuclear Tcf-beta-catenin complexes promotes
transcriptional activation of target genes involved in cell fate decisions.
Inappropriate expression of Tcf target genes resulting from mutational
activation of this pathway is also implicated in tumorigenesis. The C-terminus
of beta-catenin is indispensable for the transactivation function, which
probably reflects the presence of binding sites for essential transcriptional
coactivators such as p300/CBP. However, the precise mechanism of transactivation
remains unclear. Here we demonstrate an interaction between beta-catenin and
Brg-1, a component of mammalian SWI/SNF and Rsc chromatin-remodelling complexes.
A functional consequence of reintroduction of Brg-1 into Brg-1-deficient cells
is enhanced activity of a Tcf-responsive reporter gene. Consistent with this,
stable expression of inactive forms of Brg-1 in colon carcinoma cell lines
specifically inhibits expression of endogenous Tcf target genes. In addition, we
observe genetic interactions between the Brg-1 and beta-catenin homologues in
flies. We conclude that beta-catenin recruits Brg-1 to Tcf target gene
promoters, facilitating chromatin remodelling as a prerequisite for
transcriptional activation.
PMID: 11532957 [PubMed - indexed for MEDLINE]
95: EMBO J 2001 Sep 3;20(17):4684-93
Ski7p G protein interacts with the exosome and the Ski complex for 3'-to-5' mRNA
decay in yeast.
Araki Y, Takahashi S, Kobayashi T, Kajiho H, Hoshino S, Katada T.
Department of Physiological Chemistry, Graduate School of Pharmaceutical
Sciences, University of Tokyo, Tokyo 113-0033, Japan.
Two cytoplasmic mRNA-decay pathways have been characterized in yeast, and both
are initiated by shortening of the 3'-poly(A) tail. In the major 5'-to-3' decay
pathway, the deadenylation triggers removal of the 5'-cap, exposing the
transcript body for 5'-to-3' degradation. An alternative 3'-to-5' decay pathway
also follows the deadenylation and requires two multi-complexes: the exosome
containing various 3'-exonucleases and the Ski complex consisting of the RNA
helicase Ski2p, Ski3p and Ski8p. In addition, Ski7p, which has an N-terminal
domain and a C-terminal elongation factor 1alpha-like GTP-binding domain, is
involved in the 3'-to-5' decay. However, physical interaction between the
exosome and the Ski complex, together with the function of Ski7p, has remained
unknown. Here we report that the N domain of Ski7p is required and sufficient
for the 3'-to-5' decay. Furthermore, the exosome and the Ski complex interact
with the different regions of Ski7p N domain, and both interactions are required
for the 3'-to-5' decay. Thus, Ski7p G protein appears to function as a
signal-coupling factor between the two multi-complexes operating in the 3'-to-5'
mRNA-decay pathway.
PMID: 11532933 [PubMed - indexed for MEDLINE]
96: Virology 2001 Sep 1;287(2):266-74
Hepatitis B virus X protein interferes with cell viability through interaction
with the p127-kDa UV-damaged DNA-binding protein.
Lin-Marq N, Bontron S, Leupin O, Strubin M.
Department of Genetics and Microbiology, University Medical Centre, Rue
Michel-Servet 1, Geneva 4, 1211, Switzerland.
The hepatitis B virus X protein (HBx) is essential for establishing natural
viral infection and has been implicated in the development of liver cancer
associated with chronic infection. The basis for HBx function in either process
is not understood. In cell culture, HBx exhibits pleiotropic activities
affecting transcription, DNA repair, cell growth, and apoptotic cell death.
Numerous cellular proteins including the p127-kDa subunit of UV-damaged
DNA-binding activity have been reported to interact with HBx but the functional
significance of these interactions remains unclear. Here we show that the
binding of HBx to p127 interferes with cell viability. Mutational analysis
reveals that HBx contacts p127 via a region to which no function has been
assigned previously. An HBx variant bearing a single-charge reversal
substitution within this region loses p127 binding and concomitant cytotoxicity.
This mutant regains activity when directly fused to p127. These studies confirm
that p127 is an important cellular target of HBx, and they indicate that HBx
does not exert its effect by sequestering p127, and thereby preventing its
normal function, but instead by conferring to p127 a deleterious activity.
Copyright 2001 Academic Press.
PMID: 11531405 [PubMed - indexed for MEDLINE]
97: Methods Mol Biol 2001;177:319-28
Membrane recruitment systems for analysis of protein-protein interactions.
Aronheim A.
B. Rappaport Faculty of Medicine, Israel Institute of Technology, Haifa, Israel.
PMID: 11530615 [PubMed - indexed for MEDLINE]
98: Methods Mol Biol 2001;177:261-70
The split-hybrid system. Uncoding multiprotein networks and defining mutations
that affect protein interactions.
Goldman PS, DeMaggio AJ, Goodman RH, Hoekstra MF.
Icos Corporation, Bothell, WA, USA.
PMID: 11530611 [PubMed - indexed for MEDLINE]
99: Methods Mol Biol 2001;177:241-59
One-hybrid systems for detecting protein-DNA interactions.
Alexander MK, Bourns BD, Zakian VA.
Lewis Thomas Lab, Princeton University, Princeton, NJ, USA.
PMID: 11530610 [PubMed - indexed for MEDLINE]
100: Methods Mol Biol 2001;177:179-98
Protein interactions important in eukaryotic translation initiation.
Asano K, Hinnebusch AG.
Laboratory of Gene Regulation and Development, National Institute of Child
Health and Development, Bethesda, MD, USA.
PMID: 11530606 [PubMed - indexed for MEDLINE]
101: Methods Mol Biol 2001;177:151-9
Two-hybrid interactions confirmed by coimmunoprecipitation of epitope-tagged
clones.
Naumovski L.
Department of Pediatrics, Stanford University School of Medicine, Stanford, CA,
USA.
PMID: 11530604 [PubMed - indexed for MEDLINE]
102: Methods Mol Biol 2001;177:135-50
Confirming yeast two-hybrid protein interactions using in vitro
glutathione-S-transferase pulldowns.
Kraichely DM, MacDonald PN.
Proctor and Gamble, Cincinnati, OH, USA.
PMID: 11530602 [PubMed - indexed for MEDLINE]
103: Nucleic Acids Res 2001 Sep 1;29(17):3566-75
DNA-binding activity and subunit interaction of the mariner transposase.
Zhang L, Dawson A, Finnegan DJ.
Institute of Cell and Molecular Biology, University of Edinburgh, King's
Buildings, Edinburgh EH9 3JR, UK.
Mos1 is a member of the mariner/Tc1 family of transposable elements originally
identified in Drosophila mauritiana. It has 28 bp terminal inverted repeats and
like other elements of this type it transposes by a cut and paste mechanism,
inserts at TA dinucleotides and codes for a transposase. This is the only
protein required for transposition in vitro. We have investigated the DNA
binding properties of Mos1 transposase and the role of transposase-transposase
interactions in transposition. Purified transposase recognises the terminal
inverted repeats of Mos1 due to a DNA-binding domain in the N-terminal 120 amino
acids. This requires a putative helix-turn-helix motif between residues 88 and
108. Binding is preferentially to the right hand end, which differs at four
positions from the repeat at the left end. Cleavage of Mos1 by transposase is
also preferentially at the right hand end. Wild-type transposase monomers
interact with each other in a yeast two-hybrid assay and we have used this to
isolate mutations resulting in reduced interaction. These mutations lie along
the length of the protein, indicating that transposase-transposase interactions
are not due to a single interaction domain. One such mutation which retains both
DNA-binding and catalytic activity has greatly reduced ability to excise Mos1
from plasmid DNA through coordinate cleavage of the two ends and transposition
in vitro is lowered to a level 20-fold below that of the wild-type. This
suggests that transposase-transposase interaction is required to form a synaptic
complex necessary for coordinate cleavage at the ends of Mos1 during
transposition. This mutant enzyme allows insertion at dinucleotides other than
TA, including sequences with GC base pairs. This is the first example of a
mariner/Tc1 transposase with altered target specificity.
PMID: 11522826 [PubMed - indexed for MEDLINE]
104: Nucleic Acids Res 2001 Sep 1;29(17):3513-9
A relationship between gene expression and protein interactions on the proteome
scale: analysis of the bacteriophage T7 and the yeast Saccharomyces cerevisiae.
Grigoriev A.
GPC Biotech, Fraunhoferstrasse 20, Martinsried 82152, Germany.
andrei.grigoriev@gpc-biotech.com
The relationship between the similarity of expression patterns for a pair of
genes and interaction of the proteins they encode is demonstrated both for the
simple genome of the bacteriophage T7 and the considerably more complex genome
of the yeast Saccharomyces cerevisiae. Statistical analysis of large-scale gene
expression and protein interaction data shows that protein pairs encoded by
co-expressed genes interact with each other more frequently than with random
proteins. Furthermore, the mean similarity of expression profiles is
significantly higher for respective interacting protein pairs than for random
ones. Such coupled analysis of gene expression and protein interaction data may
allow evaluation of the results of large-scale gene expression and protein
interaction screens as demonstrated for several publicly available datasets. The
role of this link between expression and interaction in the evolution from
monomeric to oligomeric protein structures is also discussed.
PMID: 11522820 [PubMed - indexed for MEDLINE]
105: J Biol Chem 2001 Oct 26;276(43):39945-9
An accessible hydrophobic surface is a key element of the molecular chaperone
action of Atp11p.
Sheluho D, Ackerman SH.
Department of Surgery, Wayne State University School of Medicine, 1225 Ellman
Bldg., 421 E. Canfield Ave., Detroit, MI 48201, USA.
Atp11p is a soluble protein of mitochondria that binds unassembled beta subunits
of the F(1)-ATPase and prevents them from aggregating in the matrix. In this
report, we show that Atp11p protects the insulin B chain from aggregating in
vitro and therefore acts as a molecular chaperone. The chaperone action of
Atp11p is mediated by hydrophobic interactions. An accessible hydrophobic
surface in Atp11p was identified with the environment-sensitive fluorescent
probe 1,1'-bis(4-anilino-5-napththalenesulfonic acid (bis-ANS). The spectral
changes of bis-ANS in the presence of Atp11p indicate that the probe binds to a
nonpolar region of the protein. Furthermore, the dye quenches the fluorescence
of Atp11p tryptophan residues in a concentration-dependent manner. Although up
to three molecules of bis-ANS can bind cooperatively to Atp11p, the binding of
only one dye molecule is sufficient to virtually eliminate the chaperone
activity of the protein.
PMID: 11522798 [PubMed - indexed for MEDLINE]
106: Biochim Biophys Acta 2001 Aug 17;1506(2):89-102
Analysis of suppressor mutation reveals long distance interactions in the bc(1)
complex of Saccharomyces cerevisiae.
Brasseur G, Di Rago JP, Slonimski PP, Lemesle-Meunier D.
Laboratoire de Bioenergetique et Ingenierie des Proteines, CNRS, Marseilles,
France. brasseur@ibsm.cnrs-mrs.fr
Four totally conserved glycines are involved in the packing of the two
cytochrome b hemes, b(L) and b(H), of the bc(1) complex. The conserved glycine
131 is involved in the packing of heme b(L) and is separated by only 3 A from
this heme in the bc(1) complex structure. The cytochrome b respiratory deficient
mutant G131S is affected in the assembly of the bc(1) complex. An intragenic
suppressor mutation was obtained at position 260, in the ef loop, where a
glycine was replaced by an alanine. This respiratory competent revertant
exhibited a low bc(1) complex activity and was affected in the electron transfer
at the Q(P) site. The k(min) for the substrate DBH(2) was diminished by an order
of magnitude and EPR spectra showed a partially empty Q(P) site. However, the
binding of the Q(P) site inhibitors stigmatellin and myxothiazol remained
unchanged in the suppressor strain. Optical spectroscopy revealed that heme b(L)
is red shifted by 0.8 nm and that the E(m) of heme b(L) was slightly increased
(+20 mV) in the revertant strain as compared to wild type strain values.
Addition of a methyl group at position 260 is thus sufficient to allow the
assembly of the bc(1) complex and the insertion of heme b(L) despite the
presence of the serine at position 131. Surprisingly, reversion at position 260
was located 13 A away from the original mutation and revealed a long distance
interaction in the yeast bc(1) complex.
PMID: 11522251 [PubMed - indexed for MEDLINE]
107: J Biol Chem 2001 Oct 19;276(42):38394-9
Differential utilization of enzyme-substrate interactions for acylation but not
deacylation during the catalytic cycle of Kex2 protease.
Rockwell NC, Fuller RS.
Department of Biological Chemistry, University of Michigan Medical Center, Ann
Arbor, Michigan 48109, USA.
Kex2 protease from Saccharomyces cerevisiae is the prototype for a family of
eukaryotic proprotein processing proteases belonging to the subtilase
superfamily of serine proteases. Kex2 can be distinguished from degradative
subtilisins on the basis of stringent substrate specificity and distinct
pre-steady-state behavior. To better understand these mechanistic differences,
we have examined the effects of substrate residues at P(1) and P(4) on
individual steps in the Kex2 catalytic cycle with a systematic series of
isosteric peptidyl amide and ester substrates. The results demonstrate that
substrates based on known, physiological cleavage sites exhibit high acylation
rates (> or =550 s(-1)) with Kex2. Substitution of Lys for the physiologically
correct Arg at P(1) resulted in a > or =200-fold drop in acylation rate with
almost no apparent effect on binding or deacylation. In contrast, substitution
of the physiologically incorrect Ala for Nle at P(4) resulted in a much smaller
defect in acylation and a modest but significant effect on binding with Lys at
P(1). This substitution also had no effect on deacylation. These results
demonstrate that Kex2 utilizes enzyme-substrate interactions in different ways
at different steps in the catalytic cycle, with the S(1)-P(1) contact providing
a key specificity determinant at the acylation step.
PMID: 11514565 [PubMed - indexed for MEDLINE]
108: FEBS Lett 2001 Aug 17;503(2-3):196-200
The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with
two diacid inhibitors.
Erskine PT, Coates L, Newbold R, Brindley AA, Stauffer F, Wood SP, Warren MJ,
Cooper JB, Shoolingin-Jordan PM, Neier R.
Division of Biochemistry and Molecular Biology, School of Biological Sciences,
University of Southampton, UK.
The structures of 5-aminolaevulinic acid dehydratase complexed with two
irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been
solved at high resolution. Both inhibitors bind by forming a Schiff base link
with Lys 263 at the active site. Previous inhibitor binding studies have defined
the interactions made by only one of the two substrate moieties (P-side
substrate) which bind to the enzyme during catalysis. The structures reported
here provide an improved definition of the interactions made by both of the
substrate molecules (A- and P-side substrates). The most intriguing result is
the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the
enzyme involving Lys 210. It has been known for many years that P-side substrate
forms a Schiff base (with Lys 263) but until now there has been no evidence that
binding of A-side substrate involves formation of a Schiff base with the enzyme.
A catalytic mechanism involving substrate linked to the enzyme through Schiff
bases at both the A- and P-sites is proposed.
PMID: 11513881 [PubMed - indexed for MEDLINE]
109: Mol Cell Biol 2001 Sep;21(18):6270-9
H2A.Z is required for global chromatin integrity and for recruitment of RNA
polymerase II under specific conditions.
Adam M, Robert F, Larochelle M, Gaudreau L.
Departement de Biologie, Universite de Sherbrooke, Sherbrooke, Quebec J1K 2R1,
Canada.
Evolutionarily conserved variant histone H2A.Z has been recently shown to
regulate gene transcription in Saccharomyces cerevisiae. Here we show that loss
of H2A.Z in this organism negatively affects the induction of GAL genes.
Importantly, fusion of the H2A.Z C-terminal region to S phase H2A without its
corresponding C-terminal region can mediate the variant histone's specialized
function in GAL1-10 gene induction, and it restores the slow-growth phenotype of
cells with a deletion of HTZ1. Furthermore, we show that the C-terminal region
of H2A.Z can interact with some components of the transcriptional apparatus. In
cells lacking H2A.Z, recruitment of RNA polymerase II and TATA-binding protein
to the GAL1-10 promoters is significantly diminished under inducing conditions.
Unexpectedly, we also find that H2A.Z is required to globally maintain chromatin
integrity under GAL gene-inducing conditions. We hypothesize that H2A.Z can
positively regulate gene transcription, at least in part, by modulating
interactions with RNA polymerase II-associated factors at certain genes under
specific cell growth conditions.
PMID: 11509669 [PubMed - indexed for MEDLINE]
110: J Biol Chem 2001 Oct 26;276(43):40254-62
The DNA-binding domain of yeast heat shock transcription factor independently
regulates both the N- and C-terminal activation domains.
Bulman AL, Hubl ST, Nelson HC.
Johnson Research Foundation, Department of Biochemistry and Biophysics,
University of Pennsylvania School of Medicine, 422 Curie Blvd., Philadelphia, PA
19104, USA.
The expression of heat shock proteins in response to cellular stresses is
dependent on the activity of the heat shock transcription factor (HSF). In
yeast, HSF is constitutively bound to DNA; however, the mitigation of negative
regulation in response to stress dramatically increases transcriptional
activity. Through alanine-scanning mutagenesis of the surface residues of the
DNA-binding domain, we have identified a large number of mutants with increased
transcriptional activity. Six of the strongest mutations were selected for
detailed study. Our studies suggest that the DNA-binding domain is involved in
the negative regulation of both the N-terminal and C-terminal activation domains
of HSF. These mutations do not significantly affect DNA binding. Circular
dichroism analysis suggests that a subset of the mutants may have altered
secondary structure, whereas a different subset has decreased thermal stability.
Our findings suggest that the regulation of HSF transcriptional activity (under
both constitutive and stressed conditions) may be partially dependent on the
local topology of the DNA-binding domain. In addition, the DNA-binding domain
may mediate key interactions with ancillary factors and/or other intramolecular
regulatory regions in order to modulate the complex regulation of HSF's
transcriptional activity.
PMID: 11509572 [PubMed - indexed for MEDLINE]
111: J Biol Chem 2001 Nov 2;276(44):41205-12
Molecular interactions of the Gbeta binding domain of the Ste20p/PAK family of
protein kinases. An isolated but fully functional Gbeta binding domain from
Ste20p is only partially folded as shown by heteronuclear NMR spectroscopy.
Song J, Chen Z, Xu P, Gingras R, Ng A, Leberer E, Thomas DY, Ni F.
Biomolecular NMR Laboratory and the Montreal Joint Centre for Structural
Biology, Biotechnology Research Institute, National Research Council of Canada,
Montreal, Quebec H4P 2R2, Canada.
The transmission of the mating signal of the budding yeast Saccharomyces
cerevisiae requires Ste20p, a member of the serine/threonine protein kinases of
the Ste20p/PAK family, to link the Gbeta subunit of the heterotrimeric G protein
to the mitogen-activated protein kinase cascades. The binding site of Ste20p to
the Gbeta subunit was mapped to a consensus sequence of SSLphiPLI/VXphiphibeta
(X for any residue; phi for A, I, L, S or T; beta for basic residues), which was
shown to be a novel Gbeta binding (GBB) motif present only in the noncatalytic
C-terminal domains of the Ste20p/PAK family of protein kinases (Leeuw, T., Wu,
C., Schrag, J. D., Whiteway, M., Thomas, D. Y., and Leberer, E. (1998) Nature
391, 191-195; Leberer, E., Dignard, D., Thomas, D. Y., and Leeuw, T. (2000)
Biol. Chem. 381, 427-431). Here, we report the results of an NMR study on two
GBB motif peptides and the entire C-terminal domain derived from Ste20p. The NMR
data show that the two peptide fragments are not uniquely structured in aqueous
solution, but in the presence of 40% trifluoroethanol, the longer 37-residue
peptide exhibited two well defined, but flexibly linked helical structure
elements. Heteronuclear NMR data indicate that the fully functional 86-residue
C-terminal domain of Ste20p is again unfolded in aqueous solution but has
helical secondary structure preferences similar to those of the two peptide
fragments. The NMR results on the two GBB peptides and the entire GBB domain all
indicate that the two important binding residues, Ser(879) and Ser(880), are
located at the junction between two helical segments. These experimental
observations with the prototype GBB domain of a novel family of Gbeta-controlled
effectors may have important implications in understanding the molecular
mechanisms of the signal transduction from the heterotrimeric G protein to the
mitogen-activated protein kinase cascade.
PMID: 11509560 [PubMed - indexed for MEDLINE]
112: Biometals 2001 Jun;14(2):99-112
Old iron, young copper: from Mars to Venus.
Crichton RR, Pierre JL.
Unite de Biochimie, Universite Catholique de Louvain, Belgium.
Crichton@bioc.ucl.ac.be
Iron and copper are metals which play an important role in the living world.
From a brief consideration of their chemistry and biochemistry we conclude that
the early chemistry of life used water soluble ferrous iron while copper was in
the water-insoluble Cu(I) state as highly insoluble sulphides. The advent of
oxygen was a catastrophic event for most living organisms, and can be considered
to be the first general irreversible pollution of the earth. In contrast to the
oxidation of iron and its loss of bioavailability as insoluble Fe(III), the
oxidation of insoluble Cu(I) led to soluble Cu(II). A new iron biochemistry
became possible after the advent of oxygen, with the development of chelators of
Fe(III), which rendered iron once again accessible, and with the control of the
potential toxicity of iron by its storage in a water soluble, non-toxic,
bio-available storage protein (ferritin). Biology also discovered that whereas
enzymes involved in anaerobic metabolism were designed to operate in the lower
portion of the redox spectrum, the arrival of dioxygen created the need for a
new redox active metal which could attain higher redox potentials. Copper, now
bioavailable, was ideally suited to exploit the oxidizing power of dioxygen. The
arrival of copper also coincided with the development of multicellular organisms
which had extracellular cross-linked matrices capable of resisting attack by
oxygen free radicals. After the initial 'iron age' subsequent evolution moved,
not towards a 'copper age', but rather to an 'iron-copper' age. In the second
part of the review, this symbiosis of iron and copper is examined in yeast. We
then briefly consider iron and copper metabolism in mammals, before looking at
iron-copper interactions in mammals, particularly man, and conclude with the
reflection that, as in Greek and Roman mythology, a better understanding of the
potentially positive interactions between Mars (iron) and Venus (copper) can
only be to the advantage of our species.
Publication Types:
Review
Review, Tutorial
PMID: 11508852 [PubMed - indexed for MEDLINE]
113: EMBO J 2001 Aug 15;20(16):4577-87
Mechanistic aspects of DnaA-RepA interaction as revealed by yeast forward and
reverse two-hybrid analysis.
Sharma R, Kachroo A, Bastia D.
Department of Microbiology, Duke University Medical Center, Durham, NC 27710,
USA.
Using yeast forward and reverse two-hybrid analysis and biochemical techniques,
we present novel and definitive in vivo and in vitro evidence that both the
N-terminal domain I and C-terminal domain IV of the host-encoded DnaA initiator
protein of Escherichia coli interact physically with plasmid-encoded RepA
initiator of pSC101. The N-terminal, but not the C-terminal, region of RepA
interacted with DnaA in vitro. These protein-protein interactions are critical
for two very early steps of replication initiation, namely origin unwinding and
helicase loading. Neither domain I nor IV of DnaA could individually collaborate
with RepA to promote pSC101 replication. However, when the two domains are
co-expressed within a common cell milieu and allowed to associate non-covalently
with each other via a pair of leucine zippers, replication of the plasmid was
supported in vivo. Thus, the result shows that physical tethering, either
non-covalent or covalent, of domain I and IV of DnaA and interaction of both
domains with RepA, are critical for replication initiation. The results also
provide the molecular basis for a novel, potential, replication-based bacterial
two-hybrid system.
PMID: 11500384 [PubMed - indexed for MEDLINE]
114: RNA 2001 Aug;7(8):1084-96
Ribosomal protein L5 helps anchor peptidyl-tRNA to the P-site in Saccharomyces
cerevisiae.
Meskauskas A, Dinman JD.
Department of Molecular Genetics and Microbiology, University of Medicine and
Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854,
USA.
Our previous demonstration that mutants of 5S rRNA called mof9 can specifically
alter efficiencies of programmed ribosomal frameshifting (PRF) suggested a role
for this ubiquitous molecule in the maintenance of translational reading frame,
though the repetitive nature of the 5S rDNA gene (>100 copies/cell) inhibited
more detailed analyses. However, given the known interactions between 5S rRNA
and ribosomal protein L5 (previously called L1 or YL3) encoded by an essential,
single-copy gene, we monitored the effects of a series of well-defined rpl5
mutants on PRF and virus propagation. Consistent with the mof9 results, we find
that the rpl5 mutants promoted increased frameshifting efficiencies in both the
-1 and +1 directions, and conferred defects in the ability of cells to propagate
two endogenous viruses. Biochemical analyses demonstrated that mutant ribosomes
had decreased affinities for peptidyl-tRNA. Pharmacological studies showed that
sparsomycin, a peptidyltransferase inhibitor that specifically increases the
binding of peptidyl-tRNA with ribosomes, was antagonistic to the frameshifting
defects of the most severe mutant, and the extent of sparsomycin resistance
correlated with the severity of the frameshifting defects in all of the mutants.
These results provide biochemical and physiological evidence that one function
of L5 is to anchor peptidyl-tRNA to the P-site. A model is presented describing
how decreased affinity of ribosomes for peptidyl-tRNA can affect both -1 and +1
frameshifting, and for the effects of sparsomycin.
PMID: 11497428 [PubMed - indexed for MEDLINE]
115: Microbiology 2001 Aug;147(Pt 8):2007-19
A GAS-like gene family in the pathogenic fungus Candida glabrata.
Weig M, Haynes K, Rogers TR, Kurzai O, Frosch M, Muhlschlegel FA.
Institut fur Hygiene und Mikrobiologie, Universitat Wurzburg,
Josef-Schneider-Str. 2, 97080 Wurzburg, Germany.
In fungi, the cell wall plays a major role in host-pathogen interactions.
Despite this, little is known about the molecular basis of cell wall assembly in
Candida glabrata, which has emerged as the second most common cause of systemic
candidosis. A C. glabrata gene family, CgGAS1-3, that shares significant
homologies with both the GAS1 gene of Saccharomyces cerevisiae, which is
necessary for cell wall assembly, and the pH-regulated genes PHR1 and PHR2 of
Candida albicans, which are involved in cell wall assembly and required for
virulence, has been cloned. Among the members of this family, CgGAS1-3 display a
unique expression pattern. Both CgGAS1 and CgGAS2 are constitutively expressed.
In contrast, CgGAS3 transcript was not detectable under any of the assayed
conditions. The C. glabrata actin gene, CgACT1, has also been cloned to be used
as a meaningful loading control in Northern blots. CgGAS1 and CgGAS2 were
deleted by two different methodological approaches. A rapid PCR-based strategy
by which gene disruption was achieved with short regions of homology (50 bp) was
applied successfully to C. glabrata. DeltaCggas1 or DeltaCggas2 cells
demonstrated similar aberrant morphologies, displaying an altered bud morphology
and forming floccose aggregates. These phenotypes suggest a role for CgGAS1 and
CgGAS2 in cell wall biosynthesis. Further evidence for this hypothesis was
obtained by successful functional complementation of a gas1 null mutation in S.
cerevisiae with the C. glabrata CgGAS1 or CgGAS2 gene.
PMID: 11495979 [PubMed - indexed for MEDLINE]
116: Oncogene 2001 Jul 5;20(30):3995-4006
Multiple signaling interactions of Abl and Arg kinases with the EphB2 receptor.
Yu HH, Zisch AH, Dodelet VC, Pasquale EB.
The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, California, CA
92037, USA.
The Eph family of receptor tyrosine kinases and the Abl family of non-receptor
tyrosine kinases have both been implicated in tissue morphogenesis. They
regulate the organization of the actin cytoskeleton in the developing nervous
system and participate in signaling pathways involved in axon growth. Both Eph
receptors and Abl are localized in the neuronal growth cone, suggesting that
they play a role in axon pathfinding. Two-hybrid screens identified regions of
Abl and Arg that bind to the EphB2 and EphA4 receptors, suggesting a novel
signaling connection involving the two kinase families. The association of
full-length Abl and Arg with EphB2 was confirmed by co-immunoprecipitation and
found to involve several distinct protein interactions. The SH2 domains of Abl
and Arg bind to tyrosine-phosphorylated motifs in the juxtamembrane region of
EphB2. A second, phosphorylation-independent interaction with EphB2 involves
non-conserved sequences in the C-terminal tails of Abl and Arg. A third
interaction between Abl and EphB2 is probably mediated by an intermediary
protein because it requires tyrosine phosphorylation of EphB2, but not the
binding sites for the Abl SH2 domain. The connection between EphB2 and Abl/Arg
appears to be reciprocal. Activated EphB2 causes tyrosine phosphorylation of Abl
and Arg, and vice versa. Interestingly, treatment of COS cells and B35
neuronal-like cells with ephrin-B1 to activate endogenous EphB2 decreased the
kinase activity of endogenous Abl. These data are consistent with the opposite
effects that Eph receptors and Abl have on neurite ougrowth and suggest that Eph
receptors and Abl family kinases have shared signaling activities.
PMID: 11494128 [PubMed - indexed for MEDLINE]
117: Proc Natl Acad Sci U S A 2001 Aug 14;98(17):9648-53
The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3,
subunit interactions, and expression of subunits in polarized cells.
Matern HT, Yeaman C, Nelson WJ, Scheller RH.
Genentech, Inc., Department of Richard Scheller, South San Francisco, CA
94080-4990, USA.
The yeast exocyst complex (also called Sec6/8 complex in higher eukaryotes) is a
multiprotein complex essential for targeting exocytic vesicles to specific
docking sites on the plasma membrane. It is composed of eight proteins (Sec3,
-5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weights ranging
from 70 to 144 kDa. Mammalian orthologues for seven of these proteins have been
described and here we report the cloning and initial characterization of the
remaining subunit, Sec3. Human Sec3 (hSec3) shares 17% sequence identity with
yeast Sec3p, interacts in the two-hybrid system with other subunits of the
complex (Sec5 and Sec8), and is expressed in almost all tissues tested. In
yeast, Sec3p has been proposed to be a spatial landmark for polarized secretion
(1), and its localization depends on its interaction with Rho1p (2). We
demonstrate here that hSec3 lacks the potential Rho1-binding site and
GFP-fusions of hSec3 are cytosolic. Green fluorescent protein (GFP)-fusions of
nearly every subunit of the mammalian Sec6/8 complex were expressed in
Madin-Darby canine kidney (MDCK) cells, but they failed to assemble into a
complex with endogenous proteins and localized in the cytosol. Of the subunits
tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact
when expressed in MDCK cells. Cells overexpressing GFP-Exo70 fail to form a
tight monolayer, suggesting the Exo70 targeting interaction is critical for
normal development of polarized epithelial cells.
PMID: 11493706 [PubMed - indexed for MEDLINE]
118: J Cell Biol 2001 Aug 6;154(3):549-71
A protein interaction map for cell polarity development.
Drees BL, Sundin B, Brazeau E, Caviston JP, Chen GC, Guo W, Kozminski KG, Lau
MW, Moskow JJ, Tong A, Schenkman LR, McKenzie A 3rd, Brennwald P, Longtine M, Bi
E, Chan C, Novick P, Boone C, Pringle JR, Davis TN, Fields S, Drubin DG.
Department of Molecular and Cell Biology, University of California, Berkeley, CA
94720, USA.
Many genes required for cell polarity development in budding yeast have been
identified and arranged into a functional hierarchy. Core elements of the
hierarchy are widely conserved, underlying cell polarity development in diverse
eukaryotes. To enumerate more fully the protein-protein interactions that
mediate cell polarity development, and to uncover novel mechanisms that
coordinate the numerous events involved, we carried out a large-scale two-hybrid
experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with
the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases
were used to screen an array of yeast transformants that express approximately
90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4
activation domain fusions. 191 protein-protein interactions were detected, of
which 128 had not been described previously. 44 interactions implicated 20
previously uncharacterized proteins in cell polarity development. Further
insights into possible roles of 13 of these proteins were revealed by their
multiple two-hybrid interactions and by subcellular localization. Included in
the interaction network were associations of Cdc42 and Rho1 pathways with
proteins involved in exocytosis, septin organization, actin assembly,
microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other
interactions suggested direct connections between Rho1- and Cdc42-regulated
pathways; the secretory apparatus and regulators of polarity establishment;
actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic
machinery. In total, a network of interactions that provide an integrated
response of signaling proteins, the cytoskeleton, and organelles to the spatial
cues that direct polarity development was revealed.
PMID: 11489916 [PubMed - indexed for MEDLINE]
119: J Biol Chem 2001 Oct 19;276(42):38820-9
Functional analysis of the hydrophobic patch on nuclear transport factor 2
involved in interactions with the nuclear pore in vivo.
Quimby BB, Leung SW, Bayliss R, Harreman MT, Thirumala G, Stewart M, Corbett AH.
Department of Biochemistry, Emory University School of Medicine, Atlanta,
Georgia 30322, USA.
Nuclear transport factor 2 (NTF2) is a small homodimeric protein that interacts
simultaneously with both RanGDP and FxFG nucleoporins. The interaction between
NTF2 and Ran is essential for the import of Ran into the nucleus. Here we use
mutational analysis to dissect the in vivo role of the interaction between NTF2
and nucleoporins. We identify a series of surface residues that form a
hydrophobic patch on NTF2, which when mutated disrupt the NTF2-nucleoporin
interaction. Analysis of these mutants in vivo demonstrates that the strength of
this interaction can be significantly reduced without affecting cell viability.
However, cells cease to be viable if the interaction between NTF2 and
nucleoporins is abolished completely, indicating that this interaction is
essential for the function of NTF2 in vivo. In addition, we have isolated a
dominant negative mutant of NTF2, N77Y, which has increased affinity for
nucleoporins. Overexpression of the N77Y protein blocks nuclear protein import
and concentrates Ran at the nuclear rim. These data support a mechanism in which
NTF2 interacts transiently with FxFG nucleoporins to translocate through the
pore and import RanGDP into the nucleus.
PMID: 11489893 [PubMed - indexed for MEDLINE]
120: Traffic 2001 Aug;2(8):565-76
Clathrin interactions with C-terminal regions of the yeast AP-1 beta and gamma
subunits are important for AP-1 association with clathrin coats.
Yeung BG, Payne GS.
Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA
90095-1737, USA.
Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo
recruitment and clathrin assembly during clathrin-coated vesicle biogenesis. We
have identified, and characterized the physiological significance of
clathrin-binding activities in the two large subunits of the AP-1 complex in
Saccharomyces cerevisiae. Using GST-fusion chromatography, two clathrin-binding
sites were defined in the beta1 subunit that match consensus clathrin-binding
sequences in other mammalian and yeast clathrin-binding proteins. Clathrin
interactions were also identified with the C-terminal region of the gamma
subunit. When introduced into chromosomal genes, point mutations in the beta1
clathrin-binding motifs, or deletion of the gamma C-terminal region, reduced
association of AP-1 with clathrin in coimmunoprecipitation assays. The beta1
mutations or the gamma truncation individually produced minor effects on AP-1
distribution by subcellular fractionation. However, when beta1 and gamma
mutations were combined, severe defects were observed in AP-1 association with
membranes and incorporation into clathrin-coated vesicles. The combination of
subunit mutations accentuated growth and alpha-factor pheromone maturation
defects in chc1-ts cells, though not to the extent caused by complete loss of
AP-1 activity. Our results suggest that both the beta1 and gamma subunits
contribute interactions with clathrin that are important for stable assembly of
AP-1 complexes into clathrin coats in vivo.
PMID: 11489214 [PubMed - indexed for MEDLINE]
121: J Autoimmun 2001 Aug;17(1):51-61
Expression of protein tyrosine phosphatase-like molecule ICA512/IA-2 induces
growth arrest in yeast cells and transfected mammalian cell lines.
Papakonstantinou T, Myers MA, Jois J, Roucou X, Prescott M, Rowley MJ, Mackay
IR.
Department of Biochemistry and Molecular Biology, Monash University, Clayton,
Victoria 3168, Australia.
The ICA512/IA-2 molecule, a protein with similarity to receptor-type protein
tyrosine phosphatases, was discovered during studies to identify autoantigens in
Type 1 diabetes. The biological function of ICA512/IA-2 is unknown. We describe
striking effects of ICA512/IA-2 on viability and growth of both yeast cells and
cultured mammalian cells. In transformed yeast Saccharomyces cerevisiae cells,
expression of ICA512/IA-2 induced growth retardation as judged by measurements
of optical density and counts of colony-forming units. In contrast, expression
of the intracellular domain (amino acids 600-979) of ICA512/IA-2 in yeast or
mammalian cells had no such effects. In investigations on apoptosis, expression
of ICA512/IA-2 in yeast cells caused loss of plasma membrane asymmetry, but not
release of cytochrome c from mitochondria which did occur in a control system
after expression of the pro-apoptotic molecule Bax. Possible interactions
between ICA512/IA-2 and components of the cytoskeleton were not supported by
studies on staining of fixed yeast cells with phalloidin-Texas Red. With
transfected mammalian cell lines COS-7 and NIH3T3, expression of ICA512/IA-2
likewise induced growth arrest, with some of the morphological features of
apoptosis. Thus obligatory expression of ICA512/IA-2 in eukaryotic cells causes
disruption of cellular activities, with growth arrest in yeast and nuclear
pycnosis/fragmentation in mammalian cells. A possible explanation is that growth
inhibition reflects a part of the presently unknown function of ICA512/IA-2.
Copyright 2001 Academic Press.
PMID: 11488637 [PubMed - indexed for MEDLINE]
122: J Biol Chem 2001 Sep 21;276(38):35644-51
Interactions in the error-prone postreplication repair proteins hREV1, hREV3,
and hREV7.
Murakumo Y, Ogura Y, Ishii H, Numata S, Ichihara M, Croce CM, Fishel R,
Takahashi M.
Department of Pathology, Nagoya University Graduate School of Medicine, 65
Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. murakumo@med.nagoya-u.ac.jp
Most mutations after DNA damage in yeast Saccharomyces cerevisiae are induced by
error-prone translesion DNA synthesis employing scRev1 and DNA polymerase zeta
that consists of scRev3 and scRev7 proteins. Recently, the human REV1 (hREV1)
and REV3 (hREV3) genes were identified, and their products were revealed to be
involved in UV-induced mutagenesis, as observed for their yeast counterparts.
Human REV7 (hREV7) was also cloned, and its product was found to interact with
hREV3, but the biological function of hREV7 remained unknown. We report here the
analyses of precise interactions in the human REV proteins. The interaction
between hREV1 and hREV7 was identified by the yeast two-hybrid library screening
using a bait of hREV7, which was confirmed by in vitro and in vivo binding
assays. The homodimerization of hREV7 was also detected in the two-hybrid
analysis. In addition, the precise domains for interaction between hREV7 and
hREV1 or hREV3 and for hREV7 homodimerization were determined. Although hREV7
interacts with both hREV1 and hREV3, a stable complex formation of the three
proteins was undetectable in vitro. These findings suggest the possibility that
hREV7 might play an important role in regulating the enzymatic activities of
hREV1 and hREV3 for mutagenesis in response to DNA damage.
PMID: 11485998 [PubMed - indexed for MEDLINE]
123: Genes Dev 2001 Aug 1;15(15):1957-70
Characterization of U4 and U6 interactions with the 5' splice site using a S.
cerevisiae in vitro trans-splicing system.
Johnson TL, Abelson J.
Division of Biology, California Institute of Technology, Pasadena, California
91125, USA.
Spliceosome assembly has been characterized as the ordered association of the
snRNP particles U1, U2, and U4/U6.U5 onto pre-mRNA. We have used an in vitro
trans-splicing/cross-linking system in Saccharomyces cerevisiae nuclear extracts
to examine the first step of this process, 5' splice site recognition. This
trans-splicing reaction has ATP, Mg(2+), and splice-site sequence requirements
similar to those of cis-splicing reactions. Using this system, we identified and
characterized a novel U4-5' splice site interaction that is ATP-dependent, but
does not require the branch point, the 3' splice site, or the 5' end of the U1
snRNA. Additionally, we identified several ATP-dependent U6 cross-links at the
5' splice site, indicating that different regions of U6 sample it before a U6-5'
splice site interaction is stabilized that persists through the first step of
splicing. This work provides evidence for ATP-dependent U4/U6 association with
the 5' splice site independent of ATP-mediated U2 association with the branch
point. Furthermore, it defines specific nucleotides in U4 and U6 that interact
with the 5' splice site at this early stage, even in the absence of base-pairing
with the U1 snRNA.
PMID: 11485990 [PubMed - indexed for MEDLINE]
124: Biochem J 2001 Aug 15;358(Pt 1):7-16
A large family of endosome-localized proteins related to sorting nexin 1.
Teasdale RD, Loci D, Houghton F, Karlsson L, Gleeson PA.
R.W. Johnson Pharmaceutical Research Institute, 3210 Merryfield Row, San Diego,
CA 92121, USA. r.teasdale@imb.uq.edu.au
Sorting nexin 1 (SNX1), a peripheral membrane protein, has previously been shown
to regulate the cell-surface expression of the human epidermal growth factor
receptor [Kurten, Cadena and Gill (1996) Science 272, 1008-1010]. Searches of
human expressed sequence tag databases with SNX1 revealed eleven related human
cDNA sequences, termed SNX2 to SNX12, eight of them novel. Analysis of
SNX1-related sequences in the Saccharomyces cerevisiae genome clearly shows a
greatly expanded SNX family in humans in comparison with yeast. On the basis of
the predicted protein sequences, all members of this family of hydrophilic
molecules contain a conserved 70-110-residue Phox homology (PX) domain, referred
to as the SNX-PX domain. Within the SNX family, subgroups were identified on the
basis of the sequence similarities of the SNX-PX domain and the overall domain
structure of each protein. The members of one subgroup, which includes human
SNX1, SNX2, SNX4, SNX5 and SNX6 and the yeast Vps5p and YJL036W, all contain
coiled-coil regions within their large C-terminal domains and are found
distributed in both membrane and cytosolic fractions, typical of hydrophilic
peripheral membrane proteins. Localization of the human SNX1 subgroup members in
HeLa cells transfected with the full-length cDNA species revealed a similar
intracellular distribution that in all cases overlapped substantially with the
early endosome marker, early endosome autoantigen 1. The intracellular
localization of deletion mutants and fusions with green fluorescent protein
showed that the C-terminal regions of SNX1 and SNX5 are responsible for their
endosomal localization. On the basis of these results, the functions of these
SNX molecules are likely to be unique to endosomes, mediated in part by
interactions with SNX-specific C-terminal sequences and membrane-associated
determinants.
PMID: 11485546 [PubMed - indexed for MEDLINE]
125: EMBO J 2001 Aug 1;20(15):4041-54
Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway
by interacting with these complexes.
Hoogenraad CC, Akhmanova A, Howell SA, Dortland BR, De Zeeuw CI, Willemsen R,
Visser P, Grosveld F, Galjart N.
MGC Department of Cell Biology, Erasmus University, PO Box 1738, 3000 DR
Rotterdam, The Netherlands.
Genetic analysis in Drosophila suggests that Bicaudal-D functions in an
essential microtubule-based transport pathway, together with cytoplasmic dynein
and dynactin. However, the molecular mechanism underlying interactions of these
proteins has remained elusive. We show here that a mammalian homologue of
Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction
is confirmed by mass spectrometry, immunoprecipitation studies and in vitro
binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex
and has properties of a peripheral coat protein, yet it also co-localizes with
dynactin at microtubule plus ends. Overexpression studies using green
fluorescent protein-tagged forms of BICD2 verify its intracellular distribution
and co-localization with dynactin, and indicate that the C-terminus of BICD2 is
responsible for Golgi targeting. Overexpression of the N-terminal domain of
BICD2 disrupts minus-end-directed organelle distribution and this portion of
BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells
results in an extensive BICD2-dynactin-dynein co-localization. Taken together,
these data suggest that mammalian BICD2 plays a role in the dynein- dynactin
interaction on the surface of membranous organelles, by associating with these
complexes.
PMID: 11483508 [PubMed - indexed for MEDLINE]
126: EMBO J 2001 Aug 1;20(15):4035-40
Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic
vacuole fusion.
Kato M, Wickner W.
Department of Biochemistry, Dartmouth Medical School, Vail Building, Hanover, NH
03755-3844, USA.
In vitro homotypic fusion of yeast vacuoles occurs in three stages: priming, the
Sec18 (NSF)-mediated changes that precede vacuole association; docking, the Ypt7
and SNARE-mediated pairing of vacuoles; and fusion, mediated by
calmodulin/V0/t-SNARE interactions. Defects in catalysts of each stage result in
fragmented (unfused) vacuoles. Strains with deletions in any of ERG genes 3-6,
lacking normal ergosterol biosynthesis, have fragmented vacuoles. The ergosterol
ligands filipin, nystatin and amphotericin B block the in vitro fusion of
vacuoles from wild-type cells. Each of these inhibitors acts at the priming
stage to inhibit Sec17p release from vacuoles. A reversible delay in Sec18p
action prevents vacuoles from acquiring resistance to any of these three drugs,
confirming that their action is on the normal fusion pathway. Ergosterol or
cholesterol delivery to wild-type vacuoles stimulates their in vitro fusion, and
the in vitro fusion of ergDelta vacuoles requires added sterol. The need for
ergosterol for vacuole priming underscores the role of lipids in organizing the
membrane elements of this complex reaction.
PMID: 11483507 [PubMed - indexed for MEDLINE]
127: EMBO J 2001 Aug 1;20(15):3938-46
Novel modular domain PB1 recognizes PC motif to mediate functional
protein-protein interactions.
Ito T, Matsui Y, Ago T, Ota K, Sumimoto H.
Division of Genome Biology, Cancer Research Institute, Kanazawa University, 13-1
Takaramachi, Kanazawa 920-0934, Japan. titolab@kenroku.kanazawa-u.ac.jp
Modular domains mediating specific protein-protein interactions play central
roles in the formation of complex regulatory networks to execute various
cellular activities. Here we identify a novel domain PB1 in the budding yeast
protein Bem1p, which functions in polarity establishment, and mammalian
p67(phox), which activates the microbicidal phagocyte NADPH oxidase. Each of
these specifically recognizes an evolutionarily conserved PC motif to interact
directly with Cdc24p (an essential protein for cell polarization) and p40(phox)
(a component of the signaling complex for the oxidase), respectively. Swapping
the PB1 domain of Bem1p with that of p67(phox), which abolishes its interaction
with Cdc24p, confers on cells temperature- sensitive growth and a bilateral
mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the
PC motif-containing region of p40(phox), which restores the interaction with the
altered Bem1p. This domain-swapping experiment demonstrates that Bem1p function
requires interaction with Cdc24p, in which the PB1 domain and the PC motif
participate as responsible modules.
PMID: 11483497 [PubMed - indexed for MEDLINE]
128: Proc Natl Acad Sci U S A 2001 Aug 14;98(17):9581-6
Kinetic trapping of DNA by transcription factor IIIB.
Cloutier TE, Librizzi MD, Mollah AK, Brenowitz M, Willis IM.
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY
10461, USA.
High levels of RNA polymerase III gene transcription are achieved by facilitated
recycling of the polymerase on transcription factor IIIB (TFIIIB)-DNA complexes
that are stable through multiple rounds of initiation. TFIIIB-DNA complexes in
yeast comprise the TATA-binding protein (TBP), the TFIIB-related factor
TFIIIB70, and TFIIIB90. The high stability of the TFIIIB-DNA complex is
conferred by TFIIIB90 binding to TFIIIB70-TBP-DNA complexes. This stability is
thought to result from compound bends introduced in the DNA by TBP and TFIIIB90
and by protein-protein interactions that obstruct DNA dissociation. Here we
present biochemical evidence that the high stability of TFIIIB-DNA complexes
results from kinetic trapping of the DNA. Thermodynamic analysis shows that the
free energies of formation of TFIIIB70-TBP-DNA (DeltaG degrees = -12.10 +/- 0.12
kcal/mol) and TFIIIB-DNA (DeltaG degrees = -11.90 +/- 0.14 kcal/mol) complexes
are equivalent whereas a kinetic analysis shows that the half-lives of these
complexes (46 +/- 3 min and 95 +/- 6 min, respectively) differ significantly.
The differential stability of these isoenergetic complexes demonstrates that
TFIIIB90 binding energy is used to drive conformational changes and increase the
barrier to complex dissociation.
PMID: 11481428 [PubMed - indexed for MEDLINE]
129: Proc Natl Acad Sci U S A 2001 Aug 14;98(17):9760-5
Interactions of Exo1p with components of MutLalpha in Saccharomyces cerevisiae.
Tran PT, Simon JA, Liskay RM.
Department of Molecular and Medical Genetics, Oregon Health Sciences University,
Portland 97201, USA.
Previously, we reported evidence suggesting that Saccharomyces cerevisiae
MutLalpha, composed of Mlh1p and Pms1p, was a functional member of the gyrase
b/Hsp90/MutL (GHL) dimeric ATPase superfamily characterized by highly conserved
ATPase domains. Similar to other GHL ATPases, these putative ATPase domains of
MutLalpha may be important for the recruitment and/or activation of downstream
effectors. One downstream effector candidate is Exo1p, a 5'-3' double stranded
DNA exonuclease that has previously been implicated in DNA mismatch repair
(MMR). Here we report yeast two-hybrid results suggesting that Exo1p can
interact physically with MutLalpha through the Mlh1p subunit. We also report
epistasis analysis involving MutLalpha ATPase mutations combined with exo1Delta.
One interpretation of our genetic results is that MutLalpha ATPase domains
function to direct Exo1p and other functionally redundant exonucleases during
MMR. Finally, our results show that much of the increase in spontaneous mutation
observed in an exo1Delta strain is REV3-dependent, in turn suggesting that Exo1p
is also involved in one or more MMR-independent mutation avoidance pathways.
PMID: 11481425 [PubMed - indexed for MEDLINE]
130: Nat Genet 2001 Aug;28(4):303-4
Comment on:
Nat Genet. 2001 Aug;28(4):327-34.
To bind or not to bind.
Biggin MD.
Gene expression is regulated by transcription factors binding selectively to
particular portions of the genome. To what extent are these protein-DNA
interactions influenced by the intrinsic sequence-specific recognition
properties at each protein, and to what extent are they affected by other
factors, such as chromatin structure or cooperative interactions with other
proteins. Genome-wide surveys of DNA binding by transcription factors in vivo
are beginning to provide some answers.
Publication Types:
Comment
News
PMID: 11479583 [PubMed - indexed for MEDLINE]
131: J Biol Chem 2001 Sep 28;276(39):36295-302
Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell
nuclear antigen (PCNA) is independent of its in vitro interaction via a
consensus PCNA binding region.
Frank G, Qiu J, Zheng L, Shen B.
Department of Cell and Tumor Biology, City of Hope National Medical Center,
Duarte, California 91010, USA.
Interaction between human flap endonuclease-1 (hFEN-1) and proliferating cell
nuclear antigen (PCNA) represents a good model for interactions between multiple
functional proteins involved in DNA metabolic pathways. A region of 9 conserved
amino acid residues (residues Gln-337 through Lys-345) in the C terminus of
human FEN-1 (hFEN-1) was shown to be responsible for the interaction with PCNA.
Our current study indicates that 4 amino acid residues in hFEN-1 (Leu-340,
Asp-341, Phe-343, and Phe-344) are critical for human PCNA (hPCNA) interaction.
A conserved PCNA interaction motif in various proteins from assorted species has
been defined as Q(1)X(2)X(3)(L/I)(4)X(5)X(6)F(7)(F/Y)(8), although our results
fail to implicate Q(1) (Gln-337 in hFEN-1) as a crucial residue. Surprisingly,
all hFEN-1 mutants, including L340A, D341A, F343A, and F344A, retained
hPCNA-mediated stimulation of both exo- and flap endonuclease activities.
Furthermore, our in vitro assay showed that hPCNA failed to bind to the scRad27
(yeast homolog of FEN-1) nuclease. However, its nuclease activities were
significantly enhanced in the presence of hPCNA. Four additional Saccharomyces
cerevisiae scRad27 mutants, including multiple alanine mutants and a deletion
mutant of the entire PCNA binding region, were constructed to confirm this
result. All of these mutants retained PCNA-driven nuclease activity stimulation.
We therefore conclude that stimulation of eukaryotic hFEN-1 nuclease activities
by PCNA is independent of its in vitro interaction via the PCNA binding region.
PMID: 11477073 [PubMed - indexed for MEDLINE]
132: Science 2001 Sep 14;293(5537):2101-5
Global analysis of protein activities using proteome chips.
Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, Bertone P, Lan N, Jansen R,
Bidlingmaier S, Houfek T, Mitchell T, Miller P, Dean RA, Gerstein M, Snyder M.
Department of Molecular, Cellular, and Developmental Biology, Yale University,
New Haven, CT 06520, USA.
To facilitate studies of the yeast proteome, we cloned 5800 open reading frames
and overexpressed and purified their corresponding proteins. The proteins were
printed onto slides at high spatial density to form a yeast proteome microarray
and screened for their ability to interact with proteins and phospholipids. We
identified many new calmodulin- and phospholipid-interacting proteins; a common
potential binding motif was identified for many of the calmodulin-binding
proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and
screened for diverse biochemical activities. The microarrays can also be used to
screen protein-drug interactions and to detect posttranslational modifications.
PMID: 11474067 [PubMed - indexed for MEDLINE]
133: Nat Struct Biol 2001 Aug;8(8):695-700
Erratum in:
Nat Struct Biol 2002 Mar;9(3):231
A histone fold TAF octamer within the yeast TFIID transcriptional coactivator.
Selleck W, Howley R, Fang Q, Podolny V, Fried MG, Buratowski S, Tan S.
Center for Gene Regulation, Department of Biochemistry & Molecular Biology, The
Pennsylvania State University, University Park, Pennsylvania 16802-1014, USA.
Gene activity in a eukaryotic cell is regulated by accessory factors to RNA
polymerase II, which include the general transcription factor complex TFIID,
composed of TBP and TBP-associated factors (TAFs). Three TAFs that contain
histone fold motifs (yTAF17, yTAF60 and yTAF61) are critical for transcriptional
regulation in the yeast Saccharomyces cerevisiae and are found in both TFIID and
SAGA, a multicomponent histone acetyltransferase transcriptional coactivator.
Although these three TAFs were proposed to assemble into a pseudooctamer
complex, we find instead that yTAF17, yTAF60 and yTAF61 form a specific TAF
octamer complex with a fourth TAF found in TFIID, yTAF48. We have reconstituted
this complex in vitro and established that it is an octamer containing two
copies each of the four components. Point mutations within the histone folds
disrupt the octamer in vitro, and temperature-sensitive mutations in the histone
folds can be specifically suppressed by overexpressing the other TAF octamer
components in vivo. Our results indicate that the TAF octamer is similar both in
stoichiometry and histone fold interactions to the histone octamer component of
chromatin.
PMID: 11473260 [PubMed - indexed for MEDLINE]
134: Biochim Biophys Acta 2001 Jun 29;1532(3):234-47
A genetic screen for ethanolamine auxotrophs in Saccharomyces cerevisiae
identifies a novel mutation in Mcd4p, a protein implicated in
glycosylphosphatidylinositol anchor synthesis.
Storey MK, Wu WI, Voelker DR.
Department of Medicine, Program in Cell Biology, National Jewish Medical and
Research Center, 1400 Jackson Street, Denver, CO 80206, USA.
A genetic screen for ethanolamine auxotrophs has identified a novel mutant
allele of the morphogenesis checkpoint dependent (MCD)-4 gene, designated
mcd4-P301L. In the presence of a null allele for the phosphatidylserine (PtdSer)
decarboxylase 1 gene (psd1 Delta), the mcd4-P301L mutation confers temperature
sensitivity for growth on minimal medium. This growth defect is reversed by
either ethanolamine or choline supplementation. Incubation of mutant cells with
[(3)H]serine followed by analysis of the aminoglycerophospholipids demonstrated
a 60% decrease in phosphatidylethanolamine (PtdEtn) formation compared to
parental cells. Chemical analysis of phospholipid content after culture under
non-permissive conditions also demonstrated a 60% decrease in the PtdEtn pool
compared to the parental strain. Although the morphogenesis checkpoint dependent
(MCD)-4 gene and its homologues have been shown to play a role in
glycosylphosphatidylinositol (GPI) anchor synthesis, the mcd4-P301L strain
displayed normal incorporation of [(3)H]inositol into both proteins and lipids.
Thus, a defect in GPI anchor synthesis does not explain either the ethanolamine
auxotrophy or biochemical phenotype of this mutant. We also examined the growth
characteristics and PtdSer metabolism of a previously described mcd4-174 mutant
strain, with defects in GPI anchor synthesis, protein modification and cell wall
maintenance. The mcd4-174, psd1 Delta strain is a temperature sensitive
ethanolamine auxotroph that requires osmotic support for growth, and displays
normal PtdEtn formation compared to parental cells. These results reveal
important genetic interactions between PSD1 and MCD4 genes, and provide evidence
that Mcd4p can modulate aminoglycerophospholipid metabolism, in a way
independent of its role in GPI anchor synthesis.
PMID: 11470244 [PubMed - indexed for MEDLINE]
135: Biochemistry 2001 Jul 31;40(30):9049-58
Temperature-induced denaturation and renaturation of triosephosphate isomerase
from Saccharomyces cerevisiae: evidence of dimerization coupled to refolding of
the thermally unfolded protein.
Benitez-Cardoza CG, Rojo-Dominguez A, Hernandez-Arana A.
Area de Biofisicoquimica, Departamento de Quimica, Universidad Autonoma
Metropolitana-Iztapalapa, Apartado Postal 55-534, Iztapalapa D.F. 09340, Mexico.
The thermal denaturation of the dimeric enzyme triosephosphate isomerase (TIM)
from Saccharomyces cerevisiae was studied by spectroscopic and calorimetric
methods. At low protein concentration the structural transition proved to be
reversible in thermal scannings conducted at a rate greater than 1.0 degrees C
min(-1). Under these conditions, however, the denaturation-renaturation cycle
exhibited marked hysteresis. The use of lower scanning rates lead to pronounced
irreversibility. Kinetic studies indicated that denaturation of the enzyme
likely consists of an initial first-order reaction that forms thermally unfolded
(U) TIM, followed by irreversibility-inducing reactions which are probably
linked to aggregation of the unfolded protein. As judged from CD measurements, U
possesses residual secondary structure but lacks most of the tertiary
interactions present in native TIM. Furthermore, the large increment in heat
capacity upon denaturation suggests that extensive exposure of surface area
occurs when U is formed. Above 63 degrees C, reactions leading to
irreversibility were much slower than the unfolding process; as a result, U was
sufficiently long-lived as to allow an investigation of its refolding kinetics.
We found that U transforms into nativelike TIM through a second-order reaction
in which association is coupled to the regain of secondary structure. The rate
constants for unfolding and refolding of TIM displayed temperature dependences
resembling those reported for monomeric proteins but with considerably larger
activation enthalpies. Such large temperature dependences seem to be determinant
for the occurrence of kinetically controlled transitions and thus constitute a
simple explanation for the hysteresis observed in thermal scannings.
PMID: 11467968 [PubMed - indexed for MEDLINE]
136: J Bacteriol 2001 Aug;183(16):4761-70
Domain interactions in the yeast ATP binding cassette transporter Ycf1p:
intragenic suppressor analysis of mutations in the nucleotide binding domains.
Falcon-Perez JM, Martinez-Burgos M, Molano J, Mazon MJ, Eraso P.
Instituto de Investigaciones Biomedicas "Alberto Sols," CSIC-UAM, Madrid, Spain.
The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC)
transporter required for heavy metal and drug detoxification. Cluster analysis
shows that Ycf1p is strongly related to the human multidrug-associated protein
(MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may
serve as an excellent model for the study of eukaryotic ABC transporter
structure and function. Identifying intramolecular interactions in these
transporters may help to elucidate energy transfer mechanisms during transport.
To identify regions in Ycf1p that may interact to couple ATPase activity to
substrate binding and/or movement across the membrane, we sought intragenic
suppressors of ycf1 mutations that affect highly conserved residues presumably
involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site
suppressors were identified for the D777N mutation which affects the invariant
Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1).
Two of the suppressor mutations (V543I and F565L) are located in the first
transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R,
G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in
NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity
or functional interactions between NBD1 and the other three domains. The
original D777N mutant protein exhibits a strong defect in the apparent affinity
for ATP and V(max) of transport. The phenotypic characterization of the
suppressor mutants shows that suppression does not result from restoring these
alterations but rather from a change in substrate specificity. We discuss the
possible involvement of Asp777 in coupling ATPase activity to substrate binding
and/or transport across the membrane.
PMID: 11466279 [PubMed - indexed for MEDLINE]
137: Mol Cell Biol 2001 Aug;21(16):5541-53
Yeast RNA polymerase I enhancer is dispensable for transcription of the
chromosomal rRNA gene and cell growth, and its apparent transcription
enhancement from ectopic promoters requires Fob1 protein.
Wai H, Johzuka K, Vu L, Eliason K, Kobayashi T, Horiuchi T, Nomura M.
Department of Biological Chemistry, University of California-Irvine, Irvine,
California 92697-1700, USA.
At the end of the 35S rRNA gene within ribosomal DNA (rDNA) repeats in
Saccharomyces cerevisiae lies an enhancer that has been shown to greatly
stimulate rDNA transcription in ectopic reporter systems. We found, however,
that the enhancer is not necessary for normal levels of rRNA synthesis from
chromosomal rDNA or for cell growth. Yeast strains which have the entire
enhancer from rDNA deleted did not show any defects in growth or rRNA synthesis.
We found that the stimulatory activity of the enhancer for ectopic reporters is
not observed in cells with disrupted nucleolar structures, suggesting that
reporter genes are in general poorly accessible to RNA polymerase I (Pol I)
machinery in the nucleolus and that the enhancer improves accessibility. We also
found that a fob1 mutation abolishes transcription from the enhancer-dependent
rDNA promoter integrated at the HIS4 locus without any effect on transcription
from chromosomal rDNA. FOB1 is required for recombination hot spot (HOT1)
activity, which also requires the enhancer region, and for recombination within
rDNA repeats. We suggest that Fob1 protein stimulates interactions between rDNA
repeats through the enhancer region, thus helping ectopic rDNA promoters to
recruit the Pol I machinery normally present in the nucleolus.
PMID: 11463836 [PubMed - indexed for MEDLINE]
138: Proc Natl Acad Sci U S A 2001 Jul 17;98(15):8447-53
Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA
strand exchange.
Solinger JA, Heyer WD.
Division of Biological Sciences, Section of Microbiology, University of
California, Davis, CA 95616, USA.
Rad54 and Rad51 are important proteins for the repair of double-stranded DNA
breaks by homologous recombination in eukaryotes. As previously shown, Rad51
protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein
directly interacts with such filaments to enhance synapsis, the homologous
pairing with a double-stranded DNA partner. Here we demonstrate that
Saccharomyces cerevisiae Rad54 protein has an additional role in the
postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA
extension of established joint molecules in Rad51/Rpa-mediated DNA strand
exchange. This function depended on the ATPase activity of Rad54 protein and on
specific protein:protein interactions between the yeast Rad54 and Rad51
proteins.
PMID: 11459988 [PubMed - indexed for MEDLINE]
139: Mol Genet Genomics 2001 Jun;265(4):705-10
Genetic interactions within TFIIIC, the promoter-binding factor of yeast RNA
polymerase III.
Rozenfeld S, Thuriaux P.
Service de Biochimie et Genetique Moleculaire, CEA/Saclay, Gif-su-Yvette,
France.
TFIIIC is a heteromultimeric protein, made of six distinct subunits in
Saccharomyces cerevisiae, that binds to RNA polymerase III promoters and
triggers the assembly of the transcription complex. The largest yeast subunit
tau138, encoded by TFC3, binds to the B-box promoter element. This binding is
defective in the temperature-sensitive mutant tfc3-G349E; the mutation
responsible is located in one of two conserved motifs shared with the B-binding
component of human TFIIIC. Rare dominant gain-of-function mutations that restore
growth at high temperature were obtained following ultraviolet mutagenesis of
tfc3-G349E. All of them resulted from single amino acid substitutions that alter
the structure of TFIIIC. Three were due to reversion or intragenic suppression
(TFC3-K754E and TFC3-L804H) events. Three were identical isolates of TFC6-E330K,
a previously described mutation of the tau91 subunit. The remaining suppressors
mapped in TFC4, and resulted in amino acid replacements in the second largest
subunit of TFIIIC (tau131). With the exception TFC4-E711K, these affect
positions that are invariant between the S. cerevisiae and Homo sapiens
proteins, and are localised in conserved tetratricopeptide motifs. These
findings demonstrate a close functional interaction between the two largest
subunits of TFIIIC and underscore the importance of the tetratricopeptide motif
of tau131.
PMID: 11459191 [PubMed - indexed for MEDLINE]
140: J Biol Chem 2001 Sep 14;276(37):34832-9
Modulation of myosin function by isoform-specific properties of Saccharomyces
cerevisiae and muscle tropomyosins.
Strand J, Nili M, Homsher E, Tobacman LS.
Departments of Internal Medicine and Biochemistry, the University of Iowa, Iowa
City, Iowa 52242, USA.
Tropomyosin is an extended coiled-coil protein that influences actin function by
binding longitudinally along thin filaments. The present work compares cardiac
tropomyosin and the two tropomyosins from Saccharomyces cerevisiae, TPM1 and
TPM2, that are much shorter than vertebrate tropomyosins. Unlike cardiac
tropomyosin, the phase of the coiled-coil-forming heptad repeat of TPM2 is
discontinuous; it is interrupted by a 4-residue deletion. TPM1 has two such
deletions, which flank the 38-residue partial gene duplication that causes TPM1
to span five actins instead of the four of TPM2. Each of the three tropomyosin
isoforms modulates actin-myosin interactions, with isoform-specific effects on
cooperativity and strength of myosin binding. These different properties can be
explained by a model that combines opposite effects, steric hindrance between
myosin and tropomyosin when the latter is bound to a subset of its sites on
actin, and also indirect, favorable interactions between tropomyosin and myosin,
mediated by mutually promoted changes in actin. Both of these effects are
influenced by which tropomyosin isoform is present. Finally, the tropomyosins
have isoform-specific effects on in vitro sliding speed and on the myosin
concentration dependence of this movement, suggesting that non-muscle
tropomyosin isoforms exist, at least in part, to modulate myosin function.
PMID: 11457840 [PubMed - indexed for MEDLINE]
141: J Biol Chem 2001 Sep 14;276(37):34948-57
Saccharomyces cerevisiae protein Pci8p and human protein eIF3e/Int-6 interact
with the eIF3 core complex by binding to cognate eIF3b subunits.
Shalev A, Valasek L, Pise-Masison CA, Radonovich M, Phan L, Clayton J, He H,
Brady JN, Hinnebusch AG, Asano K.
Laboratory of Gene Regulation and Development, NICHD, and the Basic Research
Laboratory, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mammalian, plant, and Schizosaccharomyces pombe eukaryotic initiation factor-3
(eIF3) contains a protein homologous to the product of int-6 (eIF3e), a frequent
integration site of mouse mammary tumor viruses. By contrast, Saccharomyces
cerevisiae does not encode a protein closely related to eIF3e/Int-6. Here, we
characterize a novel S. cerevisiae protein (Pci8p, Yil071cp) that contains a PCI
(proteasome-COP9 signalosome-eIF3) domain conserved in eIF3e/Int-6. We show that
both Pci8p and human eIF3e/Int-6 expressed in budding yeast interact with the
yeast eIF3 complex in vivo and in vitro by binding to a discrete segment of its
eIF3b subunit Prt1p and that human eIF3e/Int-6 interacts with the human eIF3b
segment homologous to the Pci8p-binding site of yeast Prt1p. These results
refine our understanding of subunit interactions in the eIF3 complex and suggest
structural similarity between human eIF3e/Int-6 and yeast Pci8p. However,
deletion of PCI8 had no discernible effect on cell growth or translation
initiation as judged by polysome analysis, suggesting that Pci8p is not required
for the essential function of eIF3 in translation initiation. Motivated by the
involvement of Int-6 in transcriptional control, we investigated the effects of
deleting PCI8 on the total mRNA expression profile by oligonucleotide microarray
analysis and found reduced mRNA levels for a subset of heat shock proteins in
the pci8Delta mutant. We discuss possible dual functions of Pci8p and Int-6 in
transcriptional and translational control.
PMID: 11457827 [PubMed - indexed for MEDLINE]
142: Genetics 2001 Jul;158(3):989-97
The defect in transcription-coupled repair displayed by a Saccharomyces
cerevisiae rad26 mutant is dependent on carbon source and is not associated with
a lack of transcription.
Bucheli M, Lommel L, Sweder K.
Laboratory for Cancer Research, Rutgers, The State University of New Jersey,
Piscataway, New Jersey 08854-8020, USA.
Nucleotide excision repair (NER) is an evolutionarily conserved pathway that
removes DNA damage induced by ultraviolet irradiation and various chemical
agents that cause bulky adducts. Two subpathways within NER remove damage from
the genome overall or the transcribed strands of transcribing genes (TCR). TCR
is a faster repair process than overall genomic repair and has been thought to
require the RAD26 gene in Saccharomyces cerevisiae. Rad26 is a member of the
SWI/SNF family of proteins that either disrupt chromatin or facilitate
interactions between the RNA Pol II and transcription activators. SWI/SNF
proteins are required for the expression or repression of a diverse set of
genes, many of which are differentially transcribed in response to particular
carbon sources. The remodeling of chromatin by Rad26 could affect transcription
and/or TCR following formation of DNA damage and other stress-inducing
conditions. We speculate that another factor(s) can substitute for Rad26 under
particular growth conditions. We therefore measured the level of repair and
transcription in two different carbon sources and found that the defect in the
rad26 mutant for TCR was dependent on the type of carbon source. Furthermore,
TCR did not correlate with transcription rate, suggesting that disruption of
RAD26 leads to a specific defect in DNA repair and not transcription.
PMID: 11454749 [PubMed - indexed for MEDLINE]
143: Curr Opin Cell Biol 2001 Aug;13(4):399-404
Ion homeostasis during salt stress in plants.
Serrano R, Rodriguez-Navarro A.
Instituto de Biologia Molecular y Celular de Plantas Universidad Politecnica de
Valencia-C.S.I.C., Camino de Vera, 46022, Valencia, Spain. serrano@ibmcp.upv.es
Recent progress has been made in the characterization of cation transporters
that maintain ion homeostasis during salt stress in plants. Sodium-proton
antiporters at the vacuolar (NHX1) and plasma membrane (SOS1) have been
identified in Arabidopsis. SOS1 is regulated by the calcium-activated protein
kinase complex SOS2-SOS3. In yeast, a transcription repressor, Sko1, mediates
regulation of the sodium-pump ENA1 gene by the Hog1 MAP kinase. The recent
visualization at the atomic level of the inhibitory site of sodium in the known
target Hal2 has helped identify the interactions determining Na(+) toxicity.
Publication Types:
Review
Review Literature
PMID: 11454443 [PubMed - indexed for MEDLINE]
144: Curr Genet 2001 Jun;39(4):205-9
The Schizosaccharomyces pombe Cdc42p GTPase signals through Pak2p and the
Mkh1p-Pek1p-Spm1p MAP kinase pathway.
Merla A, Johnson DI.
Department of Microbiology and Molecular Genetics, University of Vermont,
Burlington 05405, USA.
The Cdc42p GTPase is involved in many aspects of growth and cell-cycle
regulation, including actin cytoskeletal rearrangements and activation of signal
transduction pathways. To further investigate these functions, genetic
interactions were examined between Schizosaccharomyces pombe Cdc42p, its
effectors Pak1p and Pak2p, and the Mkh1p-Pek1p-Spm1p signal transduction
pathway, which functions in cytokinesis and cell division. Expression of a
truncated version of Pak2p lacking its N-terminal autoinhibitory domain led to a
growth defect that was suppressed by deltamkh1 and deltaspm1 null mutations and
an elongated cell phenotype indicative of a cell division defect that was
suppressed by the deltamkh1 mutation. In addition, expression of the
constitutively activated cdc42G12V mutant allele led to a growth defect that was
rescued by the deltapak2 and deltamkh1 mutations. The deltapak2 mutation did not
suppress the growth defect conferred by plasmid expression of Mkh1p, suggesting
that Pak2p functions upstream of Mkh1p in this pathway. A two-hybrid protein
interaction was observed between Pak2p and Mkh1p, but not between Pak1p and
Mkh1p. These results are consistent with Cdc42p interacting with Pak2p to signal
through the Mkh1p-Pek1p-Spm1p pathway.
PMID: 11453249 [PubMed - indexed for MEDLINE]
145: Nucleic Acids Res 2001 Jul 15;29(14):3069-79
Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease.
Bae SH, Kim JA, Choi E, Lee KH, Kang HY, Kim HD, Kim JH, Bae KH, Cho Y, Park C,
Seo YS.
National Creative Research Initiative Center for Cell Cycle Control, Samsung
Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300
Chunchun-Dong, Changan-Ku, Suwon, Kyunggi-Do 440-746, Korea.
In order to gain insights into the structural basis of the multifunctional Dna2
enzyme involved in Okazaki fragment processing, we performed biochemical,
biophysical and genetic studies to dissect the domain structure of Dna2.
Proteolytic digestion of Dna2 using subtilisin produced a 127 kDa polypeptide
that lacked the 45 kDa N-terminal region of Dna2. Further digestion generated
two subtilisin-resistant core fragments of approximately equal size, 58 and 60
kDa. Surprisingly, digestion resulted in a significant (3- to 8-fold) increase
in both ATPase and endonuclease activities compared to the intact enzyme.
However, cells with a mutant DNA2 allele lacking the corresponding N-terminal
region were severely impaired in growth, being unable to grow at 37 degrees C,
indicating that the N-terminal region contains a domain critical for a cellular
function(s) of Dna2. Analyses of the hydrodynamic properties of and in vivo
complex formation by wild-type and/or mutant Dna2 lacking the N-terminal 45 kDa
domain revealed that Dna2 is active as the monomer and thus the defect in the
mutant Dna2 protein is not due to its inability to multimerize. In addition, we
found that the N-terminal 45 kDa domain interacts physically with a central
region located between the two catalytic domains. Our results suggest that the
N-terminal 45 kDa domain of Dna2 plays a critical role in regulation of the
enzymatic activities of Dna2 by serving as a site for intra- and intermolecular
interactions essential for optimal function of Dna2 in Okazaki fragment
processing. The possible mode of regulation of Dna2 is discussed based upon our
recent finding that replication protein A interacts functionally and physically
with Dna2 during Okazaki fragment processing.
PMID: 11452032 [PubMed - indexed for MEDLINE]
146: Bioinformatics 2001 Jul;17(7):669-71
A Java applet for visualizing protein-protein interaction.
Mrowka R.
Johannes-Muller-Institut fur Physiologie, Charite, Humboldt-Universitat zu
Berlin, Tucholsky Str. 2, D-10117 Berlin, Germany. Mrowka@rz.hu-berlin.de
A web applet for browsing protein-protein interactions was implemented. It
enables the display of interaction relationships, based upon neighboring
distance and biological function. AVAILABILITY: The Java applet is available at
http://www.charite.de/bioinformatics
PMID: 11448890 [PubMed - indexed for MEDLINE]
147: Bioinformatics 2001 Jul;17(7):608-21
Identifying target sites for cooperatively binding factors.
GuhaThakurta D, Stormo GD.
Department of Genetics, Washington University School of Medicine, 4566 Scott
Avenue, Campus Box: 8232, St Louis, MO 63110, USA. dg@genetics.wustl.edu
MOTIVATION: Transcriptional activation in eukaryotic organisms normally requires
combinatorial interactions of multiple transcription factors. Though several
methods exist for identification of individual protein binding site patterns in
DNA sequences, there are few methods for discovery of binding site patterns for
cooperatively acting factors. Here we present an algorithm, Co-Bind (for
COperative BINDing), for discovering DNA target sites for cooperatively acting
transcription factors. The method utilizes a Gibbs sampling strategy to model
the cooperativity between two transcription factors and defines position weight
matrices for the binding sites. Sequences from both the training set and the
entire genome are taken into account, in order to discriminate against commonly
occurring patterns in the genome, and produce patterns which are significant
only in the training set. RESULTS: We have tested Co-Bind on semi-synthetic and
real data sets to show it can efficiently identify DNA target site patterns for
cooperatively binding transcription factors. In cases where binding site
patterns are weak and cannot be identified by other available methods, Co-Bind,
by virtue of modeling the cooperativity between factors, can identify those
sites efficiently. Though developed to model protein-DNA interactions, the scope
of Co-Bind may be extended to combinatorial, sequence specific, interactions in
other macromolecules. AVAILABILITY: The program is available upon request from
the authors or may be downloaded from http://ural.wustl.edu.
PMID: 11448879 [PubMed - indexed for MEDLINE]
148: Virology 2001 Jul 20;286(1):216-24
Ty1 retrotransposition and programmed +1 ribosomal frameshifting require the
integrity of the protein synthetic translocation step.
Harger JW, Meskauskas A, Nielsen J, Justice MC, Dinman JD.
Department of Molecular Genetics and Microbiology, Graduate Program in Molecular
Biosciences at UMDNJ/Rutgers Universities, The Cancer Institute of New Jersey,
Piscataway, New Jersey 08854, USA.
Programmed ribosomal frameshifting is utilized by a number of RNA viruses to
ensure the correct ratio of viral structural to enzymatic proteins for viral
particle assembly. Altering frameshifting efficiencies upsets this ratio,
inhibiting virus propagation. Two yeast viruses that induce host cell ribosomes
to shift translational reading frame were used as tools to explore the
interactions between viruses and host cellular protein synthetic machinery.
Previous studies showed that the ribosome-inactivating protein pokeweed
antiviral protein specifically inhibited propagation of the Ty1
retrotransposable element of yeast as a consequence of inhibition of programmed
+1 ribosomal frameshifting. Here, complementary genetic and pharmacological
approaches were employed to test whether inhibition of Ty1 retrotransposition is
a general feature of alterations in the translocation step of elongation and +1
frameshifting. The results demonstrate that cells harboring a variety of mutant
alleles of two host-encoded proteins that are involved in translocation,
eukaryotic elongation factor-2 and the ribosome-associated protein RPP0, have
Ty1 propagation defects. We also show that sordarin, a fungus-specific inhibitor
of eEF-2 function, specifically inhibits programmed +1 ribosomal frameshifting
and Ty1 retrotransposition. These findings serve to link inhibition of Ty1
retrotransposition and +1 frameshifting to changes in the translocation step of
elongation. Copyright 2001 Academic Press.
PMID: 11448174 [PubMed - indexed for MEDLINE]
149: Yeast 2001 Jul;18(10):943-51
Yeast Lrg1p acts as a specialized RhoGAP regulating 1,3-beta-glucan synthesis.
Watanabe D, Abe M, Ohya Y.
Department of Integrated Biosciences, Graduate School of Frontier Science,
University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Selection of an extragenic suppressor of fks1-1154 Deltafks2, mutations in the
catalytic subunits of yeast 1,3-beta-glucan synthase (GS) conferring
temperature-sensitivity, led to the LRG1 gene, which was originally identified
as a LIM-RhoGAP homologous gene. Mutations in the LRG1 gene restore impaired
1,3-beta-glucan synthesis in the fks1-1154 Deltafks2 mutant as well as that in
rho1-2, a temperature-sensitive mutant of Rho-type GTPase that functions as a
regulatory subunit of GS. Two-hybrid analyses of Lrg1p, which contains a
sequence conserved among Rho GTPase-activating proteins (GAPs), revealed its
specific interactions with the active form of Rho1p. Among eight potential yeast
RhoGAPs, Lrg1p is the only member that negatively regulates GS activity:
mutations in the rest of GAPs, including bem2, Deltabem3, Deltasac7, Deltabag7,
Deltarga1, Deltarga2 and Deltargd1, do not suppress impairment of
1,3-beta-glucan synthesis. Analyses of Mpk1p phosphorylation revealed the
inability of Lrg1p to regulate the Pkc1p-MAP kinase cascade, a distinct
Rho1p-regulating signalling pathway known to be affected by the GAPs, Bem2p and
Sac7p. Thus, different groups of Rho1p GAPs control the activity of different
Rho1p-effector proteins. Copyright 2001 John Wiley & Sons, Ltd.
PMID: 11447600 [PubMed - indexed for MEDLINE]
150: J Biol Chem 2001 Sep 14;276(37):34537-44
Genetic interactions with the yeast Q-SNARE VTI1 reveal novel functions for the
R-SNARE YKT6.
Dilcher M, Kohler B, von Mollard GF.
Zentrum Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II,
Universitat Gottingen, 37073 Gottingen, Germany.
SNARE proteins are required for fusion of transport vesicles with target
membranes. Previously, we found that the yeast Q-SNARE Vti1p is involved in
transport to the cis-Golgi, to the prevacuole/late endosome, and to the vacuole.
Here we identified a previously uncharacterized gene, VTS1, and the R-SNARE YKT6
both as multicopy and as low copy suppressors of the growth and vacuolar
transport defect in vti1-2 cells. Ykt6p was known to function in retrograde
traffic to the cis-Golgi and homotypic vacuolar fusion. We found that VTI1 and
YKT6 also interacted in traffic to the prevacuole and vacuole, indicating that
these SNARE complexes contain Ykt6p, Vti1p, plus Pep12p and Ykt6p, Vti1p, Vam3p,
plus Vam7p, respectively. As Ykt6p was required for several transport steps,
R-SNAREs cannot be the sole determinants of specificity. To study the role of
the 0 layer in the SNARE motif, we introduced the mutations vti1-Q158R and
ykt6-R165Q. SNARE complexes to which Ykt6p contributed a fourth glutamine
residue in the 0 layer were nonfunctional, suggesting an essential function for
arginine in the 0 layer of these complexes. vti1-Q158R cells had severe defects
in several transport steps, indicating that the second arginine in the 0 layer
interfered with function.
PMID: 11445562 [PubMed - indexed for MEDLINE]
151: J Biol Chem 2001 Sep 7;276(36):33689-96
Coordinated ATP hydrolysis by the Hsp90 dimer.
Richter K, Muschler P, Hainzl O, Buchner J.
Institut fur Organische Chemie und Biochemie, Technische Universitat Munchen,
Lichtenbergstr. 4, Garching 85747, Germany.
The Hsp90 dimer is a molecular chaperone with an unusual N-terminal ATP binding
site. The structure of the ATP binding site makes it a member of a new class of
ATP-hydrolyzing enzymes, known as the GHKL family. While for some of the family
members structural data on conformational changes occurring after ATP binding
are available, these are still lacking for Hsp90. Here we set out to investigate
the correlation between dimerization and ATP hydrolysis by Hsp90. The
dimerization constant of wild type (WT) Hsp90 was determined to be 60 nm.
Heterodimers of WT Hsp90 with fragments lacking the ATP binding domain form
readily and exhibit dimerization constants similar to full-length Hsp90.
However, the ATPase activity of these heterodimers was significantly lower than
that of the wild type protein, indicating cooperative interactions in the
N-terminal part of the protein that lead to the activation of the ATPase
activity. To further address the contribution of the N-terminal domains to the
ATPase activity, we used an Hsp90 point mutant that is unable to bind ATP. Since
heterodimers between the WT protein and this mutant showed WT ATPase activity,
this mutant, although unable to bind ATP, still has the ability to stimulate the
activity in its WT partner domain. Thus, contact formation between the
N-terminal domains might not depend on ATP bound to both domains. Together,
these results suggest a mechanism for coupling the hydrolysis of ATP to the
opening-closing movement of the Hsp90 molecular chaperone.
PMID: 11441008 [PubMed - indexed for MEDLINE]
152: Mol Cell Biol 2001 Aug;21(15):5142-55
exo1-Dependent mutator mutations: model system for studying functional
interactions in mismatch repair.
Amin NS, Nguyen MN, Oh S, Kolodner RD.
Ludwig Institute for Cancer Research, University of California, San Diego School
of Medicine, La Jolla, California 92093-0660, USA.
EXO1 interacts with MSH2 and MLH1 and has been proposed to be a redundant
exonuclease that functions in mismatch repair (MMR). To better understand the
role of EXO1 in mismatch repair, a genetic screen was performed to identify
mutations that increase the mutation rates caused by weak mutator mutations such
as exo1Delta and pms1-A130V mutations. In a screen starting with an exo1
mutation, exo1-dependent mutator mutations were obtained in MLH1, PMS1, MSH2,
MSH3, POL30 (PCNA), POL32, and RNR1, whereas starting with the weak pms1 allele
pms1-A130V, pms1-dependent mutator mutations were identified in MLH1, MSH2,
MSH3, MSH6, and EXO1. These mutations only cause weak MMR defects as single
mutants but cause strong MMR defects when combined with each other. Most of the
mutations obtained caused amino acid substitutions in MLH1 or PMS1, and these
clustered in either the ATP-binding region or the MLH1-PMS1 interaction regions
of these proteins. The mutations showed two other types of interactions:
specific pairs of mutations showed unlinked noncomplementation in diploid
strains, and the defect caused by pairs of mutations could be suppressed by
high-copy-number expression of a third gene, an effect that showed allele and
overexpressed gene specificity. These results support a model in which EXO1
plays a structural role in MMR and stabilizes multiprotein complexes containing
a number of MMR proteins. A similar role is proposed for PCNA based on the data
presented.
PMID: 11438669 [PubMed - indexed for MEDLINE]
153: J Clin Lab Anal 2001;15(4):223-9
Direct measurement of HDL cholesterol: method eliminating apolipoprotein E-rich
particles.
Okada M, Matsui H, Ito Y, Fujiwara A.
Department of Laboratory Medicine, Niigata University School of Medicine,
Niigata City, Japan. okadar@med.niigata-u.ac.jp
It has been reported that the existing direct method of high density lipoprotein
(HDL) cholesterol measures particles enriched with apolipoprotein E (apoE). The
aim of our study was to investigate a new analytical protocol to directly
measure HDL cholesterol that eliminates apoE-rich particles. The interactions of
four lipoproteins (HDL(3), HDL(2), LDL, and VLDL + chylomicron) with
surfactants, divalent cations, sugars, and lectins were investigated. By
analyzing sera, HDL(3), and HDL(2), we examined the relationships among the
measurements obtained by our protocol, a precipitation method using
heparin-MnCl(2), and a commercially available kit for this direct method. A
significant difference was found between the direct method and the
heparin-MnCl(2) method, but not between our protocol and the heparin-MnCl(2)
method. Multiple regression analysis showed that the difference between the
direct method and the heparin MnCl(2) method is dependent on sources of
apoE-rich HDL. In conclusion, our protocol enables a direct measurement of HDL
cholesterol that eliminates apoE-rich particles. Copyright 2001 Wiley-Liss, Inc.
PMID: 11436206 [PubMed - indexed for MEDLINE]
154: J Biol Chem 2001 Aug 31;276(35):33093-100
Schwannomin isoform-1 interacts with syntenin via PDZ domains.
Jannatipour M, Dion P, Khan S, Jindal H, Fan X, Laganiere J, Chishti AH, Rouleau
GA.
Center for Research in Neuroscience, McGill University and the Montreal General
Hospital, 1650 Cedar Avenue, Montreal, Quebec, Canada.
The neurofibromatosis type 2 gene (NF2schwannomin isoform-1 (sch-1). Syntenin is an
adapter protein that couples transmembrane proteoglycans to cytoskeletal
components and is involved in intracellular vesicle transport. The C terminus 25
amino acids of sch-1 and the two PDZ domains of syntenin mediate their binding,
and mutations introduced within the VAFFEEL region of sch-1 defined a sequence
crucial for syntenin recognition. We have showed that the two proteins
interacted in vitro and in vivo and localized underneath the plasma membrane.
Fibroblast cells expressing heterologous antisense syntenin display alterations
in the subcellular distribution of sch-1. Together, these results provide the
first functional clue to the existence of schwannomin isoforms and could unravel
novel pathways for the transport and subcellular localization of schwannomin in
vivo.
PMID: 11432873 [PubMed - indexed for MEDLINE]
155: EMBO J 2001 Jul 2;20(13):3506-17
Spt16-Pob3 and the HMG protein Nhp6 combine to form the nucleosome-binding
factor SPN.
Formosa T, Eriksson P, Wittmeyer J, Ginn J, Yu Y, Stillman DJ.
Department of Biochemistry, University of Utah School of Medicine, 50 N. Medical
Drive Rm 211, Salt Lake City, UT 84132, USA. Tim.Formosa@hsc.utah.edu
Yeast Spt16/Cdc68 and Pob3 form a heterodimer that acts in both DNA replication
and transcription. This is supported by studies of new alleles of SPT16
described here. We show that Spt16-Pob3 enhances HO transcription through a
mechanism that is affected by chromatin modification, since some of the defects
caused by mutations can be suppressed by deleting the histone deacetylase Rpd3.
While otherwise conserved among many eukaryotes, Pob3 lacks the HMG1 DNA-binding
motif found in similar proteins such as the SSRP1 subunit of human FACT. SPT16
and POB3 display strong genetic interactions with NHP6A/B, which encodes an HMG1
motif, suggesting that these gene products function coordinately in vivo. While
Spt16-Pob3 and Nhp6 do not appear to form stable heterotrimers, Nhp6 binds to
nucleosomes and these Nhp6-nucleosomes can recruit Spt16-Pob3 to form
SPN-nucleosomes. These complexes have altered electrophoretic mobility and a
distinct pattern of enhanced sensitivity to DNase I. These results suggest that
Spt16-Pob3 and Nhp6 cooperate to function as a novel nucleosome reorganizing
factor.
PMID: 11432837 [PubMed - indexed for MEDLINE]
156: Eur J Biochem 2001 Jul;268(13):3674-84
Mechanism of activation of the double-stranded-RNA-dependent protein kinase,
PKR: role of dimerization and cellular localization in the stimulation of PKR
phosphorylation of eukaryotic initiation factor-2 (eIF2).
Vattem KM, Staschke KA, Wek RC.
Department of Biochemistry and Molecular Biology, Indiana University School of
Medicine, Indianapolis, USA.
An important defense against viral infection involves inhibition of translation
by PKR phosphorylation of the alpha subunit of eIF2. Binding of viral dsRNAs to
two dsRNA-binding domains (dsRBDs) in PKR leads to relief of an inhibitory
region and activation of eIF2 kinase activity. Interestingly, while deletion of
the regulatory region of PKR significantly induces activity in vitro, the
truncated kinase does not inhibit translation in vivo, suggesting that these
sequences carry out additional functions required for PKR control. To delineate
these functions and determine the order of events leading to activation of PKR,
we fused truncated PKR to domains of known function and assayed the chimeras for
in vivo activity. We found that fusion of a heterologous dimerization domain
with the PKR catalytic domain enhanced autophosphorylation and eIF2 kinase
function in vivo. The dsRBDs also mediate ribosome association and we proposed
that such targeting increases the localized concentration of PKR, enhancing
interaction between PKR molecules. We addressed this premise by linking the
truncated PKR to RAS sequences mediating farnesylation and membrane localization
and found that the fusion protein was functional in vivo. These results indicate
that cellular localization along with oligomerization enhances interaction
between PKR molecules. Alanine substitution for the phosphorylation site,
threonine 446, impeded in vivo and in vitro activity of the PKR fusion proteins,
while aspartate or glutamate substitutions partially restored the function of
the truncated kinase. These results indicate that both dimerization and cellular
localization play a role in transient protein-protein interactions and that
trans-autophosphorylation is the final step in the mechanism of activation of
PKR.
PMID: 11432733 [PubMed - indexed for MEDLINE]
157: J Biol Chem 2001 Aug 31;276(35):32474-9
Asymmetric interactions between the acidic P1 and P2 proteins in the
Saccharomyces cerevisiae ribosomal stalk.
Guarinos E, Remacha M, Ballesta JP.
Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas
and Universidad Autonoma de Madrid, Canto Blanco, 28049 Madrid, Spain.
The Saccharomyces cerevisiae ribosomal stalk is made of five components, the
32-kDa P0 and four 12-kDa acidic proteins, P1alpha, P1beta, P2alpha, and P2beta.
The P0 carboxyl-terminal domain is involved in the interaction with the acidic
proteins and resembles their structure. Protein chimeras were constructed in
which the last 112 amino acids of P0 were replaced by the sequence of each
acidic protein, yielding four fusion proteins, P0-1alpha, P0-1beta, P0-2alpha,
and P0-2beta. The chimeras were expressed in P0 conditional null mutant strains
in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally
deprived of acidic proteins, the four fusion proteins can replace the wild-type
P0 with little effect on cell growth. In other genetic backgrounds, the chimeras
either reduce or increase cell growth because of their effect on the ribosomal
stalk composition. An analysis of the stalk proteins showed that each P0 chimera
is able to strongly interact with only one acidic protein. The following
associations were found: P0-1alpha.P2beta, P0-1beta.P2alpha, P0-2alpha.P1beta,
and P0-2beta.P1alpha. These results indicate that the four acidic proteins do
not form dimers in the yeast ribosomal stalk but interact with each other
forming two specific associations, P1alpha.P2beta and P1beta.P2alpha, which have
different structural and functional roles.
PMID: 11431471 [PubMed - indexed for MEDLINE]
158: Nature 2001 Jun 28;411(6841):1073-6
Multiple pathways cooperate in the suppression of genome instability in
Saccharomyces cerevisiae.
Myung K, Chen C, Kolodner RD.
Ludwig Institute for Cancer Research, University of California San Diego, 92093,
USA.
Gross chromosome rearrangements (GCRs), such as translocations, deletion of a
chromosome arm, interstitial deletions and inversions, are often observed in
cancer cells. Spontaneous GCRs are rare in Saccharomyces cerevisiae; however,
the existence of mutator mutants with increased genome instability suggests that
GCRs are actively suppressed. Here we show by genetic analysis that these genome
rearrangements probably result from DNA replication errors and are suppressed by
at least three interacting pathways or groups of proteins: S-phase checkpoint
functions, recombination proteins and proteins that prevent de novo addition of
telomeres at double-strand breaks (DSBs). Mutations that inactivate these
pathways cause high rates of GCRs and show synergistic interactions, indicating
that the pathways that suppress GCRs all compete for the same DNA substrates.
PMID: 11429610 [PubMed - indexed for MEDLINE]
159: Mutat Res 2001 Jul 12;486(2):137-46
Deletion of the SRS2 gene suppresses elevated recombination and DNA damage
sensitivity in rad5 and rad18 mutants of Saccharomyces cerevisiae.
Friedl AA, Liefshitz B, Steinlauf R, Kupiec M.
Institute of Radiation Biology, GSF-National Research Center for Environment and
Health, P.O. Box 1149, 85758, Oberschleissheim, Germany.
anna.friedl@lrz.uni-muenchen.de
The Saccharomyces cerevisiae genes RAD5, RAD18, and SRS2 are proposed to act in
post-replicational repair of DNA damage. We have investigated the genetic
interactions between mutations in these genes with respect to cell survival and
ectopic gene conversion following treatment of logarithmic and early stationary
cells with UV- and gamma-rays. We find that the genetic interaction between the
rad5 and rad18 mutations depends on DNA damage type and position in the cell
cycle at the time of treatment. Inactivation of SRS2 suppresses damage
sensitivity both in rad5 and rad18 mutants, but only when treated in logarithmic
phase. When irradiated in stationary phase, the srs2 mutation enhances the
sensitivity of rad5 mutants, whereas it has no effect on rad18 mutants.
Irrespective of the growth phase, the srs2 mutation reduces the frequency of
damage-induced ectopic gene conversion in rad5 and rad18 mutants. In addition,
we find that srs2 mutants exhibit reduced spontaneous and UV-induced sister
chromatid recombination (SCR), whereas rad5 and rad18 mutants are proficient for
SCR. We propose a model in which the Srs2 protein has pro-recombinogenic or
anti-recombinogenic activity, depending on the context of the DNA damage.
PMID: 11425518 [PubMed - indexed for MEDLINE]
160: RNA 2001 Jun;7(6):833-45
A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change
in branch-site architecture.
Newby MI, Greenbaum NL.
Institute of Molecular Biophysics, Florida State University, Tallahassee
32306-4380, USA.
The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is
catalyzed by the spliceosome, a dynamic assembly involving specific and
sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing
between the U2 small nuclear (sn)RNA and a complementary consensus sequence of
the intron, called the branch site, results in positioning of the 2'OH of an
unpaired intron adenosine residue to initiate nucleophilic attack in the first
step of splicing. To understand the structural features that facilitate
recognition and chemical activity of the branch site, duplexes representing the
paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined
by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine
(psi) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide
on the intron strand, one of which forms the branch site) and with uridine, the
unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated
A-form helical backbone geometry and continuous base stacking throughout the
unmodified molecule. Incorporation of psi at the conserved position, however,
was accompanied by marked deviation from helical parameters and an extrahelical
orientation for the unpaired adenosine. Incorporation of psi also stabilized the
branch-site interaction, contributing -0.7 kcal/mol to duplex deltaG degrees 37.
These findings suggest that the presence of this conserved U2 snRNA
pseudouridine induces a change in the structure and stability of the branch-site
sequence, and imply that the extrahelical orientation of the branch-site
adenosine may facilitate recognition of this base during splicing.
PMID: 11424937 [PubMed - indexed for MEDLINE]
161: Traffic 2001 Jul;2(7):476-86
The class C Vps complex functions at multiple stages of the vacuolar transport
pathway.
Peterson MR, Emr SD.
Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute,
University of California, San Diego, La Jolla, California 92093-0668, USA.
The Class C Vps complex, consisting of Vps11, Vps16, Vps18, and Vps33, is
required for SNARE-mediated membrane fusion at the lysosome-like yeast vacuole.
However, Class C vps mutants display more severe and pleiotropic phenotypes than
mutants specifically defective in endosome-to-vacuole transport, suggesting that
there are additional functions for the Class C Vps complex. A SNARE double
mutant which is defective for both Golgi-to-endosome and endosome-to-vacuole
trafficking replicates many of the phenotypes observed in Class C vps mutants.
We show that genetic interactions exist between Class C vps alleles and alleles
of the Class D vps group, which are defective in the docking and fusion of
Golgi-derived vesicles at the endosome. Moreover, the Class D protein Vac1 was
found to physically bind to the Class C Vps complex through a direct association
with Vps11. Finally, using a random mutagenic screen, a temperature-conditional
allele which shares many of the phenotypes of mutants which are selectively
defective in Golgi-to-endosome trafficking was isolated (vps11-3ts).
Collectively, these results indicate that the Class C Vps complex plays
essential roles in the processes of membrane docking and fusion at both the
Golgi-to-endosome and endosome-to-vacuole stages of transport.
PMID: 11422941 [PubMed - indexed for MEDLINE]
162: Mol Biol Evol 2001 Jul;18(7):1283-92
The yeast protein interaction network evolves rapidly and contains few redundant
duplicate genes.
Wagner A.
Department of Biology, University of New Mexico, Albequerque, New Mexico
87131-1091, USA. wagnera@unm.edu
In this paper, the structure and evolution of the protein interaction network of
the yeast Saccharomyces cerevisiae is analyzed. The network is viewed as a graph
whose nodes correspond to proteins. Two proteins are connected by an edge if
they interact. The network resembles a random graph in that it consists of many
small subnets (groups of proteins that interact with each other but do not
interact with any other protein) and one large connected subnet comprising more
than half of all interacting proteins. The number of interactions per protein
appears to follow a power law distribution. Within approximately 200 Myr after a
duplication, the products of duplicate genes become almost equally likely to (1)
have common protein interaction partners and (2) be part of the same subnetwork
as two proteins chosen at random from within the network. This indicates that
the persistence of redundant interaction partners is the exception rather than
the rule. After gene duplication, the likelihood that an interaction gets lost
exceeds 2.2 x 10(-3)/Myr. New interactions are estimated to evolve at a rate
that is approximately three orders of magnitude smaller. Every 300 Myr, as many
as half of all interactions may be replaced by new interactions.
PMID: 11420367 [PubMed - indexed for MEDLINE]
163: Mol Cell Biol 2001 Jul;21(14):4568-78
GCN5 dependence of chromatin remodeling and transcriptional activation by the
GAL4 and VP16 activation domains in budding yeast.
Stafford GA, Morse RH.
Department of Biomedical Sciences, State University of New York at Albany School
of Public Health, Albany, New York 12201-2002, USA.
Chromatin-modifying enzymes such as the histone acetyltransferase GCN5 can
contribute to transcriptional activation at steps subsequent to the initial
binding of transcriptional activators. However, few studies have directly
examined dependence of chromatin remodeling in vivo on GCN5 or other
acetyltransferases, and none have examined remodeling via nucleosomal activator
binding sites. In this study, we have monitored chromatin perturbation via
nucleosomal binding sites in the yeast episome TALS by GAL4 derivatives in
GCN5(+) and gcn5Delta yeast cells. The strong activator GAL4 shows no dependence
on GCN5 for remodeling TALS chromatin, whereas GAL4-estrogen receptor-VP16 shows
substantial, albeit not complete, GCN5 dependence. Mini-GAL4 derivatives having
weakened interactions with TATA-binding protein and TFIIB exhibit a strong
dependence on GCN5 for both transcriptional activation and TALS remodeling not
seen for native GAL4. These results indicate that GCN5 can contribute to
chromatin remodeling at activator binding sites and that dependence on
coactivator function for a given activator can vary according to the type and
strength of contacts that it makes with other factors. We also found a weaker
dependence for chromatin remodeling on SPT7 than on GCN5, indicating that GCN5
can function via pathways independent of the SAGA complex. Finally, we examine
dependence on GCN5 and SWI-SNF at two model promoters and find that although
these two chromatin-remodeling and/or modification activities may sometimes work
together, in other instances they act in complementary fashion.
PMID: 11416135 [PubMed - indexed for MEDLINE]
164: Biochem J 2001 Jul 1;357(Pt 1):83-95
Functional roles and efficiencies of the thioredoxin boxes of calcium-binding
proteins 1 and 2 in protein folding.
Kramer B, Ferrari DM, Klappa P, Pohlmann N, Soling HD.
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am
Fassberg 11, D37077 Gottingen, Germany.
The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1
and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI)
family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys)
respectively and, like PDI, may be involved in the folding of nascent proteins.
We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the
lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided
that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by
His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo
pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin
(trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be
functionally equivalent, the first trx box of CaBP1 is significantly more active
than the second trx box. Furthermore, only about 65% re-activation of denatured
reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and
the yield of PDI-catalysed reactions was significantly reduced in the presence
of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could
catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), which is a redox-independent process, and neither protein had any
effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can
bind peptides via its b' domain, a property it shares with PDIp, the
pancreas-specific PDI homologue, and although PDI can bind malfolded proteins
such as 'scrambled' ribonuclease, no such interactions could be detected for
CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity
for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2
lacks the general peptide-binding activity attributed to the b' domain of PDI;
(3) interaction of CaBP2 with substrate (RNase AIII) is different from that of
PDI and substrate; and (4) both CaBP2 and CaBP1 may promote oxidative folding by
different kinetic pathways.
PMID: 11415439 [PubMed - indexed for MEDLINE]
165: Biochem Biophys Res Commun 2001 Jun 22;284(4):1083-9
Similar subunit interactions contribute to assembly of clathrin adaptor
complexes and COPI complex: analysis using yeast three-hybrid system.
Takatsu H, Futatsumori M, Yoshino K, Yoshida Y, Shin HW, Nakayama K.
Institute of Biological Sciences and Gene Experiment Center, University of
Tsukuba, Ibaraki, Tsukuba Science City, 305-8572, Japan.
Clathrin adaptor protein (AP) complexes are heterotetramers composed of two
large, one medium, and one small subunits. By exploiting the yeast three-hybrid
system, we have found that an interaction between the two large subunits of the
AP-1 complex, gamma-adaptin and beta1-adaptin, is markedly enhanced in the
presence of the small subunit, sigma1. Similarly, two large subunits of the AP-4
complex, epsilon-adaptin and beta4-adaptin, are found to interact with each
other only in the presence of the small subunit, sigma4. Furthermore, we have
found that an interaction between two large subunits of the COPI F subcomplex,
gamma-COP and beta-COP, is detectable only in the presence of zeta-COP. Because
these COPI subunits have common ancestral origins to the corresponding AP
subunits, these three-hybrid data, taken together with the previous two-hybrid
data, suggest that the AP complexes and the COPI F subcomplex assemble by virtue
of similar subunit interactions. Copyright 2001 Academic Press.
PMID: 11409905 [PubMed - indexed for MEDLINE]
166: FEBS Lett 2001 Jun 8;498(2-3):150-6
Nuclear export of mRNA.
Zenklusen D, Stutz F.
Institute of Microbiology, Centre Hospitalier Universitaire Vaudois, 44, rue du
Bugnon, 1011, Lausanne, Switzerland.
Export of mRNA through nuclear pore complexes (NPC) is preceded by multiple and
well coordinated processing steps, resulting in the formation of an export
competent ribonucleoprotein complex (mRNP). Numerous factors involved in the
translocation of the mRNP through the NPC and its release into the cytoplasm
have been isolated mainly through genetic approaches in yeast, and putative
functional homologues have been identified in metazoan systems. Understanding
the mechanism of mRNA export relies, in part, on the functional characterization
of these factors and the establishment of a complete network of molecular
interactions. Here we summarize recent progress in the characterization of yeast
and mammalian components implicated in the export of an mRNA from the nucleus to
the cytoplasm.
Publication Types:
Review
Review, Tutorial
PMID: 11412847 [PubMed - indexed for MEDLINE]
167: Genes Dev 2001 Jun 15;15(12):1528-39
The 19S complex of the proteasome regulates nucleotide excision repair in yeast.
Gillette TG, Huang W, Russell SJ, Reed SH, Johnston SA, Friedberg EC.
Laboratory of Molecular Pathology, Department of Pathology, University of Texas
Southwestern Medical Center, Dallas, TX 75390-9072, USA.
Previous studies suggest that the amino-terminal ubiquitin-like (ubl) domain of
Rad23 protein can recruit the proteasome for a stimulatory role during
nucleotide excision repair in the yeast Saccharomyces cerevisiae. In this
report, we show that the 19S regulatory complex of the yeast proteasome can
affect nucleotide excision repair independently of Rad23 protein. Strains with
mutations in 19S regulatory subunits (but not 20S subunits) of the proteasome
promote partial recovery of nucleotide excision repair in vivo in rad23 deletion
mutants, but not in other nucleotide excision repair-defective strains tested.
In addition, a strain that expresses a temperature-degradable ATPase subunit of
the 19S regulatory complex manifests a dramatically increased rate of nucleotide
excision repair in vivo. These data indicate that the 19S regulatory complex of
the 26S proteasome can negatively regulate the rate of nucleotide excision
repair in yeast and suggest that Rad23 protein not only recruits the 19S
regulatory complex, but also can mediate functional interactions between the 19S
regulatory complex and the nucleotide excision repair machinery. The 19S
regulatory complex of the yeast proteasome functions in nucleotide excision
repair independent of proteolysis.
PMID: 11410533 [PubMed - indexed for MEDLINE]
168: Proc Natl Acad Sci U S A 2001 Jun 19;98(13):7325-30
Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex.
Mah AS, Jang J, Deshaies RJ.
Division of Biology and Howard Hughes Medical Institute, California Institute of
Technology, Pasadena, CA 91125, USA.
Exit from mitosis in budding yeast requires inactivation of cyclin-dependent
kinases through mechanisms triggered by the protein phosphatase Cdc14. Cdc14
activity, in turn, is regulated by a group of proteins, the mitotic exit network
(MEN), which includes Lte1, Tem1, Cdc5, Cdc15, Dbf2/Dbf20, and Mob1. The direct
biochemical interactions between the components of the MEN remain largely
unresolved. Here, we investigate the mechanisms that underlie activation of the
protein kinase Dbf2. Dbf2 kinase activity depended on Tem1, Cdc15, and Mob1 in
vivo. In vitro, recombinant protein kinase Cdc15 activated recombinant Dbf2, but
only when Dbf2 was bound to Mob1. Conserved phosphorylation sites Ser-374 and
Thr-544 (present in the human, Caenorhabditis elegans, and Drosophila
melanogaster relatives of Dbf2) were required for DBF2 function in vivo, and
activation of Dbf2-Mob1 by Cdc15 in vitro. Although Cdc15 phosphorylated Dbf2,
Dbf2-Mob1, and Dbf2(S374A/T544A)-Mob1, the pattern of phosphate incorporation
into Dbf2 was substantially altered by either the S374A T544A mutations or
omission of Mob1. Thus, Cdc15 promotes the exit from mitosis by directly
switching on the kinase activity of Dbf2. We propose that Mob1 promotes this
activation process by enabling Cdc15 to phosphorylate the critical Ser-374 and
Thr-544 phosphoacceptor sites of Dbf2.
PMID: 11404483 [PubMed - indexed for MEDLINE]
169: Methods 2001 Jul;24(3):297-306
High-throughput yeast two-hybrid assays for large-scale protein interaction
mapping.
Walhout AJ, Vidal M.
Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School,
Boston, Massachusetts 02115, USA.
Protein-protein interactions play fundamental roles in many biological
processes. Hence, protein interaction mapping is becoming a well-established
functional genomics approach to generate functional annotations for predicted
proteins that so far have remained uncharacterized. The yeast two-hybrid system
is currently one of the most standardized protein interaction mapping
techniques. Here, we describe the protocols for a semiautomated,
high-throughput, Gal4-based yeast two-hybrid system. Copyright 2001 Academic
Press.
PMID: 11403578 [PubMed - indexed for MEDLINE]
170: Methods 2001 Jul;24(3):201-17
Using the yeast interaction trap and other two-hybrid-based approaches to study
protein-protein interactions.
Toby GG, Golemis EA.
Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania
19111, USA.
The detection of physical interaction between two or more molecules of interest
can be facilitated if the act of association between the interactive partners
leads to the production of a readily observed biological or physical readout.
Many interacting molecule pairs (X, Y) can be made to induce such a readout if X
and Y are each fused to defined protein elements with desired properties. For
example, in the yeast forward two-hybrid system, X is synthesized as a
translational fusion to a DNA-binding domain (DBD), Y is synthesized as a fusion
to a transcriptional activation domain (AD), and coexpression of DBD-X and AD-Y
induces transcription of easily scored responsive reporters. Other approaches
use paradigms based on the artificial production of two, hybrid, molecules, but
substitute a variety of readouts including the repression of transcription,
activation of signal transduction pathways, or reconstitution of a disrupted
enzymatic activity. In this article, we summarize a number of two-hybrid-based
approaches, and detail the use of the forward yeast two-hybrid system in a
screen to identify novel interacting partners for a protein of interest.
Copyright 2001 Academic Press.
Publication Types:
Review
Review, Tutorial
PMID: 11403570 [PubMed - indexed for MEDLINE]
171: J Mol Biol 2001 Jun 22;309(5):1007-15
Sfl1 functions via the co-repressor Ssn6-Tup1 and the cAMP-dependent protein
kinase Tpk2.
Conlan RS, Tzamarias D.
Institute of Molecular Biology & Biotechnology-Foundation of Research &
Technology, Vassilika Vouton, Heraklion, Crete, GR-711 10, Greece.
r.s.conlan@swan.ac.uk
Ssn6 (Cyc8) is a component of the yeast general corepressor Ssn6-Tup1 that
inhibits the transcription of many diversely regulated genes. The corepressor
does not interact directly with DNA but is recruited to different promoters
through interactions with distinct pathway-specific, DNA-binding repressor
proteins. Using yeast two-hybrid and GST chromatography interaction experiments,
we have determined that Sfl1, a novel repressor protein, interacts directly with
Ssn6, and in vivo repression data suggest that Sfl1 inhibits transcription by
recruiting Ssn6-Tup1 via a specific domain in the Sfl1 protein. Sin4 and Srb10,
components of specific RNA polymerase II sub-complexes that are required for
Ssn6-Tup1 repression activity, are found to be required for Sfl1 repression
function. These results indicate a possible mechanism for Sfl1-mediated
repression via Ssn6-Tup1 and specific subunits of the RNA polymerase II
holoenzyme. Electrophoretic mobility shift and chromatin immuno-precipitation
assays demonstrate that Sfl1 is present at the promoters of three
Ssn6-Tup1-repressible genes; namely, FLO11, HSP26, and SUC2. Sfl1 is known to
interact with Tpk2, a cAMP-dependent protein kinase that negatively regulates
Sfl1 function. Consistently, we show that phosphorylation by protein kinase A
inhibits Sfl1 DNA binding in vitro, and that a tpk2Delta mutation increases the
levels of Sfl1 protein associated with specific promoter elements in vivo. These
data indicate a possible mechanism for regulating Sfl1-mediated repression
through modulation of DNA binding by cAMP-dependent protein kinase-dependent
phosphorylation. Taken together with previous data, these new observations
suggest a link between cAMP signaling and Ssn6-Tup1-mediated transcriptional
repression. Copyright 2001 Academic Press.
PMID: 11399075 [PubMed - indexed for MEDLINE]
172: Mol Membr Biol 2001 Jan-Mar;18(1):105-12
Multiplicity and regulation of genes encoding peptide transporters in
Saccharomyces cerevisiae.
Hauser M, Narita V, Donhardt AM, Naider F, Becker JM.
Department of Microbiology and Biochemistry, University of Tennessee, Knoxville,
37996-0845, USA.
The model eukaryote Saccharomyces cerevisiae has two distinct peptide transport
mechanisms, one for di-/tripeptides (the PTR system) and another for
tetra-/pentapeptides (the OPT system). The PTR system consists of three genes,
PTR1, PTR2 and PTR3. The transporter (Ptr2p), encoded by the gene PTR2, is a 12
transmembrane domain (TMD) integral membrane protein that translocates
di-/tripeptides. Homologues to Ptr2p have been identified in virtually all
organisms examined to date and comprise the PTR family of transport proteins. In
S. cerevisiae, the expression of PTR2 is highly regulated at the cellular level
by complex interactions of many genes, including PTR1, PTR3, CUP9 and SSY1.
Oligopeptides, consisting of four to five amino acids, are transported by the
12-14 TMD integral membrane protein Opt1p. Unlike Ptr2p, distribution of this
protein appears limited to fungi and plants, and there appears to be three
paralogues in S. cerevisiae. This transporter has an affinity for enkephalin, an
endogenous mammalian pentapeptide, as well as for glutathione. Although it is
known that OPT1 is normally expressed only during sporulation, to date little is
known about the genes and proteins involved in the regulation of OPT1
expression.
Publication Types:
Review
Review, Tutorial
PMID: 11396605 [PubMed - indexed for MEDLINE]
173: Mol Cell Biol 2001 Jul;21(13):4233-45
New function of CDC13 in positive telomere length regulation.
Meier B, Driller L, Jaklin S, Feldmann HM.
Institute for Biochemistry, University of Munich (LMU), D-81377 Munich, Germany.
Two roles for the Saccharomyces cerevisiae Cdc13 protein at the telomere have
previously been characterized: it recruits telomerase to the telomere and
protects chromosome ends from degradation. In a synthetic lethality screen with
YKU70, the 70-kDa subunit of the telomere-associated Yku heterodimer, we
identified a new mutation in CDC13, cdc13-4, that points toward an additional
regulatory function of CDC13. Although CDC13 is an essential telomerase
component in vivo, no replicative senescence can be observed in cdc13-4 cells.
Telomeres of cdc13-4 mutants shorten for about 150 generations until they reach
a stable level. Thus, in cdc13-4 mutants, telomerase seems to be inhibited at
normal telomere length but fully active at short telomeres. Furthermore,
chromosome end structure remains protected in cdc13-4 mutants. Progressive
telomere shortening to a steady-state level has also been described for mutants
of the positive telomere length regulator TEL1. Strikingly, cdc13-4/tel1Delta
double mutants display shorter telomeres than either single mutant after 125
generations and a significant amplification of Y' elements after 225
generations. Therefore CDC13, TEL1, and the Yku heterodimer seem to represent
distinct pathways in telomere length maintenance. Whereas several CDC13 mutants
have been reported to display elongated telomeres indicating that Cdc13p
functions in negative telomere length control, we report a new mutation leading
to shortened and eventually stable telomeres. Therefore we discuss a key role of
CDC13 not only in telomerase recruitment but also in regulating telomerase
access, which might be modulated by protein-protein interactions acting as
inhibitors or activators of telomerase activity.
PMID: 11390652 [PubMed - indexed for MEDLINE]
174: Mol Cell Biol 2001 Jul;21(13):4089-96
Phosphorylation of the RNA polymerase II carboxy-terminal domain by the Bur1
cyclin-dependent kinase.
Murray S, Udupa R, Yao S, Hartzog G, Prelich G.
Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx,
New York 10461, USA.
BUR1, which was previously identified by a selection for mutations that have
general effects on transcription in Saccharomyces cerevisiae, encodes a
cyclin-dependent kinase that is essential for viability, but none of its
substrates have been identified to date. Using an unbiased biochemical approach,
we have identified the carboxy-terminal domain (CTD) of Rpb1, the largest
subunit of RNA polymerase II, as a Bur1 substrate. Phosphorylation of Rpb1 by
Bur1 is likely to be physiologically relevant, since bur1 mutations interact
genetically with rpb1 CTD truncations and with mutations in other genes involved
in CTD function. Several genetic interactions are presented, implying a role for
Bur1 during transcriptional elongation. These results identify Bur1 as a fourth
S. cerevisiae CTD kinase and provide striking functional similarities between
Bur1 and metazoan P-TEFb.
PMID: 11390638 [PubMed - indexed for MEDLINE]
175: FEBS Lett 2001 Jun 1;498(1):46-51
Shy1p occurs in a high molecular weight complex and is required for efficient
assembly of cytochrome c oxidase in yeast.
Nijtmans LG, Artal Sanz M, Bucko M, Farhoud MH, Feenstra M, Hakkaart GA, Zeviani
M, Grivell LA.
Section for Molecular Biology, Swammerdam Institute of Life Sciences, University
of Amsterdam, The Netherlands. nijtmans@science.uva.nl
Surf1p is a protein involved in the assembly of mitochondrial respiratory chain
complexes. However its exact role in this process remains to be elucidated. We
studied SHY1, the yeast homologue of SURF1, with an aim to obtain a better
understanding of the molecular pathogenesis of cytochrome c oxidase (COX)
deficiency in SURF1 mutant cells from Leigh syndrome patients. Assembly of COX
was analysed in a shy1 null mutant strain by two-dimensional polyacrylamide gel
electrophoresis (2D-PAGE). Steady-state levels of the enzyme were found to be
strongly reduced, the total amount of assembled complex being approximately 30%
of control. The presence of a significant amount of holo-COX in the
SHY1-disruptant strain suggests that Shy1p may either facilitate assembly of the
enzyme, or increase its stability. However, our observations, based on 2D-PAGE
analysis of mitochondria labelled in vitro, now provide the first direct
evidence that COX assembly is impaired in a Deltashy1 strain. COX enzyme
assembled in the absence of Shy1p appears to be structurally and enzymically
normal. The in vitro labelling studies additionally indicate that mitochondrial
translation is significantly increased in the shy1 null mutant strain, possibly
reflecting a compensatory mechanism for reduced respiratory capacity. Protein
interactions of both Shy1p and Surf1p are implied by their appearance in a high
molecular weight complex of about 250 kDa, as shown by 2D-PAGE.
PMID: 11389896 [PubMed - indexed for MEDLINE]
176: Mol Cell 2001 May;7(5):1013-23
Evolutionarily conserved interaction between CstF-64 and PC4 links
transcription, polyadenylation, and termination.
Calvo O, Manley JL.
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Tight connections exist between transcription and subsequent processing of mRNA
precursors, and interactions between the transcription and polyadenylation
machineries seem especially extensive. Using a yeast two-hybrid screen to
identify factors that interact with the polyadenylation factor CstF-64, we
uncovered an interaction with the transcriptional coactivator PC4. Both human
proteins have yeast homologs, Rna15p and Sub1p, respectively, and we show that
these two proteins also interact. Given evidence that certain polyadenylation
factors, including Rna15p, are necessary for termination in yeast, we show that
deletion or overexpression of SUB1 suppresses or enhances, respectively, both
growth and termination defects detected in an rna15 mutant strain. Our findings
provide an additional, unexpected connection between transcription and
polyadenylation and suggest that PC4/Sub1p, via its interaction with
CstF-64/Rna15p, possesses an evolutionarily conserved antitermination activity.
PMID: 11389848 [PubMed - indexed for MEDLINE]
177: J Biol Chem 2001 Aug 10;276(32):29782-91
Marked stepwise differences within a common kinetic mechanism characterize
TATA-binding protein interactions with two consensus promoters.
Powell RM, Parkhurst KM, Brenowitz M, Parkhurst LJ.
Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304,
USA.
Binding of the TATA-binding protein (TBP) to promoter DNA bearing the TATA
sequence is an obligatory initial step in RNA polymerase II transcription
initiation. The interactions of Saccharomyces cerevisiae TBP with the E4
(TATATATA) and adenovirus major late (TATAAAAG) promoters have been modeled via
global analysis of kinetic and thermodynamic data obtained using fluorescence
resonance energy transfer. A linear two-intermediate kinetic mechanism describes
the reaction of both of these consensus strong promoters with TBP. Qualitative
features common to both interactions include tightly bound TBP-DNA complexes
with similar solution geometries, simultaneous DNA binding and bending, and the
presence of intermediate TBP-DNA conformers at high mole fraction throughout
most of the reaction and at equilibrium. Despite very similar energetic changes
overall, the stepwise entropic and enthalpic compensations along the two
pathways differ markedly following the initial binding/bending event.
Furthermore, TBP-E4 dissociation ensues from both replacement and displacement
processes, in contrast to replacement alone for TBP-adenovirus major late
promoter. A model is proposed that explicitly correlates these similarities and
differences with the sequence-specific structural properties inherent to each
promoter. This detailed mechanistic comparison of two strong promoters
interacting with TBP provides a foundation for subsequent comparison between
consensus and variant promoter sequences reacting with TBP.
PMID: 11387341 [PubMed - indexed for MEDLINE]
178: J Biol Chem 2001 Aug 3;276(31):29268-74
Proteomic analysis of nucleoporin interacting proteins.
Allen NP, Huang L, Burlingame A, Rexach M.
Department of Biological Sciences, Stanford University, Stanford, California
94305-5020, USA.
The Saccharomyces cerevisiae nuclear pore complex is a supramolecular assembly
of 30 nucleoporins that cooperatively facilitate nucleocytoplasmic transport.
Thirteen nucleoporins that contain FG peptide repeats (FG Nups) are proposed to
function as stepping stones in karyopherin-mediated transport pathways. Here,
protein interactions that occur at individual FG Nups were sampled using
immobilized nucleoporins and yeast extracts. We find that many proteins bind to
FG Nups in highly reproducible patterns. Among 135 proteins identified by mass
spectrometry, most were karyopherins and nucleoporins. The PSFG nucleoporin
Nup42p and the GLFG nucleoporins Nup49p, Nup57p, Nup100p, and Nup116p exhibited
generic interactions with karyopherins; each bound 6--10 different karyopherin
betas, including importins as well as exportins. Unexpectedly, the same Nups
also captured the hexameric Nup84p complex and Nup2p. In contrast, the FXFG
nucleoporins Nup1p, Nup2p, and Nup60p were more selective and captured mostly
the Kap95p.Kap60p heterodimer. When the concentration of Gsp1p-GTP was elevated
in the extracts to mimic the nucleoplasmic environment, the patterns of
interacting proteins changed; exportins exhibited enhanced binding to FG Nups,
and importins exhibited reduced binding. The results demonstrate a global role
for Gsp1p-GTP on karyopherin-nucleoporin interactions and provide a rudimentary
map of the routes that karyopherins take as they cross the nuclear pore complex.
PMID: 11387327 [PubMed - indexed for MEDLINE]
179: EMBO J 2001 Jun 1;20(11):2742-56
SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF
ubiquitin ligase.
Farras R, Ferrando A, Jasik J, Kleinow T, Okresz L, Tiburcio A, Salchert K, del
Pozo C, Schell J, Koncz C.
Max-Planck Institut fur Zuchtungsforschung, Carl-von-Linne-Weg 10, D-50829
Cologne, Germany.
Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic
regulation of metabolic, hormonal and stress responses through their interaction
with the kinase inhibitor PRL1 WD-protein. Here we show that SKP1/ASK1, a
conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the
skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal
domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1-binding proteasomal
protein, alpha4/PAD1, which enhances the formation of a trimeric SnRK complex
with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1
with SnRKs. SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit
(AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts.
SKP1/ASK1, cullin and proteasomal alpha-subunits show nuclear co-localization in
differentiated Arabidopsis cells, and are observed in association with mitotic
spindles and phragmoplasts during cell division. Detection of SnRK in purified
26S proteasomes and co-purification of epitope- tagged SKP1/ASK1 with SnRK,
cullin and proteasomal alpha-subunits indicate that the observed protein
interactions between SnRK, SKP1/ASK1 and alpha4/PAD1 are involved in proteasomal
binding of an SCF ubiquitin ligase in Arabidopsis.
PMID: 11387208 [PubMed - indexed for MEDLINE]
180: J Biol Chem 2001 Aug 3;276(31):29393-402
Molecular characterization of mammalian homologues of class C Vps proteins that
interact with syntaxin-7.
Kim BY, Kramer H, Yamamoto A, Kominami E, Kohsaka S, Akazawa C.
Department of Neurochemistry, National Institute of Neuroscience, NCNP, Kodaira,
Tokyo 187-8502, Japan.
Vesicle-mediated protein sorting plays an important role in segregation of
intracellular molecules into distinct organelles. Extensive genetic studies
using yeast have identified more than 40 vacuolar protein sorting (VPS) genes
involved in vesicle transport to vacuoles. However, their mammalian counterparts
are not fully elucidated. In this study, we identified two human homologues of
yeast Class C VPS genes, human VPS11 (hVPS11) and human VPS18 (hVPS18). We also
characterized the subcellular localization and interactions of the protein
products not only from these genes but also from the other mammalian Class C VPS
homologue genes, hVPS16 and rVPS33a. The protein products of hVPS11 (hVps11) and
hVPS18 (hVps18) were ubiquitously expressed in peripheral tissues, suggesting
that they have a fundamental role in cellular function. Indirect
immunofluorescence microscopy revealed that the mammalian Class C Vps proteins
are predominantly associated with late endosomes/lysosomes. Immunoprecipitation
and gel filtration studies showed that the mammalian Class C Vps proteins
constitute a large hetero-oligomeric complex that interacts with syntaxin-7.
These results indicate that like their yeast counterparts, mammalian Class C Vps
proteins mediate vesicle trafficking steps in the endosome/lysosome pathway.
PMID: 11382755 [PubMed - indexed for MEDLINE]
181: Trends Genet 2001 Jun;17(6):346-52
Protein--protein interaction maps: a lead towards cellular functions.
Legrain P, Wojcik J, Gauthier JM.
Hybrigenics, 180 Avenue Daumesnil, Paris 75012, France. plegrain@hybrigenics.fr
The availability of complete genome sequences now permits the development of
tools for functional biology on a proteomic scale. Several experimental
approaches or in silico algorithms aim at clustering proteins into networks with
biological significance. Among those, the yeast two-hybrid system is the
technology of choice to detect protein-protein interactions. Recently, optimized
versions were applied at a genomic scale, leading to databases on the web.
However, as with any other 'genetic' assay, yeast two-hybrid assays are prone to
false positives and false negatives. Here we discuss these various technologies,
their general limitations and the potential advances they make possible,
especially when in combination with other functional genomics or bioinformatics
analyses.
Publication Types:
Review
Review, Tutorial
PMID: 11377797 [PubMed - indexed for MEDLINE]
182: J Biol Chem 2001 Aug 3;276(31):29382-92
Single amino acid substitutions and deletions that alter the G protein coupling
properties of the V2 vasopressin receptor identified in yeast by receptor random
mutagenesis.
Erlenbach I, Kostenis E, Schmidt C, Serradeil-Le Gal C, Raufaste D, Dumont ME,
Pausch MH, Wess J.
Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health,
Bethesda, Maryland 20892, USA.
To facilitate structure-function relationship studies of the V2 vasopressin
receptor, a prototypical G(s)-coupled receptor, we generated V2
receptor-expressing yeast strains (Saccharomyces cerevisiae) that required
arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2
receptors heterologously expressed in yeast were unable to productively interact
with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p
subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2
receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the
C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained
proper G protein coupling selectivity in yeast. To gain insight into the
molecular basis underlying the selectivity of V2 receptor/G protein
interactions, we used receptor saturation random mutagenesis to generate a yeast
library expressing mutant V2 receptors containing mutations within the second
intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant
receptors yielded four mutant receptors that, in contrast to the wild-type
receptor, showed substantial coupling to Gq5. Functional analysis of these
mutant receptors, followed by more detailed site-directed mutagenesis studies,
indicated that single amino acid substitutions at position Met(145) in the
central portion of the second intracellular loop of the V2 receptor had
pronounced effects on receptor/G protein coupling selectivity. We also observed
that deletion of single amino acids N-terminal of Met(145) led to misfolded
receptor proteins, whereas single amino acid deletions C-terminal of Met(145)
had no effect on V2 receptor function. These findings highlight the usefulness
of combining receptor random mutagenesis and yeast expression technology to
study mechanisms governing receptor/G protein coupling selectivity and receptor
folding.
PMID: 11375990 [PubMed - indexed for MEDLINE]
183: Biotechnol Bioeng 2001 Jul 20;74(2):96-107
Prediction of the pilot-scale recovery of a recombinant yeast enzyme using
integrated models.
Varga EG, Titchener-Hooker NJ, Dunnill P.
The Advanced Centre for Biochemical Engineering, Department of Biochemical
Engineering, University College London, Torrington Place, London WC1E 7JE, UK.
This article describes the rapid prediction of recovery process performance for
a new recombinant enzyme product on the basis of a broad portfolio of computer
models and highly targeted experimentation. A process model for the recombinant
system was generated by linking unit operation models in an integrated fashion,
with required parameter estimation and physical property determination
accomplished using data from scale-down studies. This enabled the generic
modeling framework established for processing of a natural enzyme from bakers'
yeast to be applied. An experimental study of the same operations at the pilot
scale showed that the process model gave a conservative prediction of
recombinant enzyme recovery. The model successfully captured interactions
leading to a low overall product yield and indicated the need for further study
of precipitate breakage in the feed zone of a disc stack centrifuge in order to
improve performance. The utility of scale-down units as an aid to fast model
generation and the advantage of integrating computer modeling and scale-down
studies to accelerate bioprocess development are highlighted. Copyright 2001
John Wiley & Sons, Inc.
Publication Types:
Evaluation Studies
PMID: 11369998 [PubMed - indexed for MEDLINE]
184: Protein Sci 2001 Jun;10(6):1113-23
Environmentally induced reversible conformational switching in the yeast cell
adhesion protein alpha-agglutinin.
Zhao H, Chen MH, Shen ZM, Kahn PC, Lipke PN.
Department of Biological Sciences and the Institute for Biomolecular Structure
and Function, Hunter College of the City University of New York, New York
10021,USA.
The yeast cell adhesion protein alpha-agglutinin is expressed on the surface of
a free-living organism and is subjected to a variety of environmental
conditions. Circular dichroism (CD) spectroscopy shows that the binding region
of alpha-agglutinin has a beta-sheet-rich structure, with only approximately 2%
alpha-helix under native conditions (15-40 degrees C at pH 5.5). This region is
predicted to fold into three immunoglobulin-like domains, and models are
consistent with the CD spectra as well as with peptide mapping and site-specific
mutagenesis. However, secondary structure prediction algorithms show that
segments comprising approximately 17% of the residues have high alpha-helical
and low beta-sheet potential. Two model peptides of such segments had helical
tendencies, and one of these peptides showed pH-dependent conformational
switching. Similarly, CD spectroscopy of the binding region of alpha-agglutinin
showed reversible conversion from beta-rich to mixed alpha/beta structure at
elevated temperatures or when the pH was changed. The reversibility of these
changes implied that there is a small energy difference between the all-beta and
the alpha/beta states. Similar changes followed cleavage of peptide or disulfide
bonds. Together, these observations imply that short sequences of high helical
propensity are constrained to a beta-rich state by covalent and local charge
interactions under native conditions, but form helices under non-native
conditions.
PMID: 11369849 [PubMed - indexed for MEDLINE]
185: Arch Biochem Biophys 2001 Jan 15;385(2):301-10
Photoaffinity labeling and photoaffinity cross-linking of phosphofructokinase-1
from Saccharomyces cerevisiae by 8-azidoadeninenucleotides.
Knoche M, Monnich K, Schafer HJ, Kopperschlager G.
Institut fur Biochemie, Fachbereich Chemie und Pharmazie, Johannes
Gutenberg-Universitat Mainz, Germany.
Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four alpha-
and four beta-subunits, each of them carrying catalytic and regulatory bindings
sites for MgATP. In this paper, various photoaffinity labels, such as
8-azidoadenosine 5'-triphosphate, 8-azido-1,N6-ethenoadenosine 5'-triphosphate,
and 8-N3-3'(2')-O-biotinyl-8-azidoadenosine 5'-triphosphate have been used to
study their interaction with the enzyme in the dark and during irradiation. All
nucleotidetriphosphates function as phosphate donor forming fructose
1,6-bisphosphate from fructose 6-phosphate. However, the kinetic analysis
revealed distinctly differences between them. Photolabeling causes a decrease in
enzyme activity to a similar extent, and ATP acts as competitive effector to
inactivation. Three bifunctional diazidodiadeninedinucleotides (8-diN3AP4A,
monoepsilon-8-diN3AP4A, and diepsilon-8-diN3AP4A) were applied for studying the
spatial arrangement of the nucleotide binding sites. No cross-linking of the
subunits was obtained by irradiation of the enzyme with 8-diN3AP4A.
Photolabeling with diepsilon-8-diN3AP4A resulted in the formation of two
alpha-beta cross-links with different mobilities in the SDS-polyacrylamide gel
electrophoresis, while monoepsilon-8-diN3AP4A yielded only one alpha-beta
cross-link. Because an interfacial location of the catalytic sites between two
subunits is less likely, we suggest that the formation of cross-linked subunits
may be the result of specific interactions of the bifunctional photolabels with
regulatory sites at the interface of both subunits.
PMID: 11368011 [PubMed - indexed for MEDLINE]
186: Mol Biol Cell 2001 May;12(5):1381-92
Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export.
Azad AK, Stanford DR, Sarkar S, Hopper AK.
Department of Biochemistry and Molecular Biology, Pennsylvania State University
College of Medicine, Hershey, Pennsylvania 17033, USA.
Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund
and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to
the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases.
We report that in budding yeast there are nuclear pools of tyrosyl-tRNA
synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear
localization sequence and showed it to be sufficient for nuclear location of a
passenger protein. Mutations of this nuclear localization sequence in endogenous
Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important
for nucleus location. The mutations do not significantly affect catalytic
activity, but they do cause defects in export of tRNAs to the cytosol. Despite
export defects, the cells are viable, indicating that nuclear tRNA
aminoacylation is not required for all tRNA nuclear export paths. Because the
tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA
aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No
genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected,
indicating that tRNA nuclear aminoacylation and Los1p operate in the same export
pathway or there are more than two pathways for tRNA nuclear export.
PMID: 11359929 [PubMed - indexed for MEDLINE]
187: Methods Mol Biol 2001;148:431-49
Genetic analysis of DNA-protein interactions using a reporter gene assay in
yeast.
Setzer DR, Schulman DB, Bumbulis MJ.
Department of Molecular Biology and Microbiology, School of Medicine, Case
Western Reserve University, Cleveland, OH, USA.
PMID: 11357604 [PubMed - indexed for MEDLINE]
188: J Biol Chem 2001 Jul 13;276(28):26715-23
Characterization of DNA damage-stimulated self-interaction of Saccharomyces
cerevisiae checkpoint protein Rad17p.
Zhang H, Zhu Z, Vidanes G, Mbangkollo D, Liu Y, Siede W.
Department of Radiation Oncology and the Winship Cancer Institute, Emory
University School of Medicine, Atlanta, Georgia 30322, USA.
Saccharomyces cerevisiae Rad17p is necessary for cell cycle checkpoint arrests
in response to DNA damage. Its known interactions with the checkpoint proteins
Mec3p and Ddc1p in a PCNA-like complex indicate a sensor role in damage
recognition. In a novel application of the yeast two-hybrid system and by
immunoprecipitation, we show here that Rad17p is capable of increased
self-interaction following DNA damage introduced by 4-nitroquinoline-N-oxide,
camptothecin or partial inactivation of DNA ligase I. Despite overlap of regions
required for Rad17p interactions with Rad17p or Mec3p, single amino acid
substitutions revealed that Rad17p x Rad17p complex formation is independent of
Mec3p. E128K (rad17-1) was found to inhibit Rad17p interaction with Mec3p but
not with Rad17p. On the other hand, Phe-121 is essential for Rad17p
self-interaction, and its function in checkpoint arrest but not for Mec3p
interaction. These differential effects indicate that Rad17p-Rad17p interaction
plays a role that is independent of the Rad17p x Mec3p x Ddc1p complex, although
our results are also compatible with Rad17p-mediated supercomplex formation of
the Rad17p x Mec3p x Ddc1p heterotrimer in response to DNA damage.
PMID: 11356855 [PubMed - indexed for MEDLINE]
189: Cancer Radiother 2001 Apr;5(2):109-29
[Molecular mechanisms controlling the cell cycle: fundamental aspects and
implications for oncology]
[Article in French]
Viallard JF, Lacombe F, Belloc F, Pellegrin JL, Reiffers J.
Service de medecine interne et maladies infectieuses, centre Francois-Magendie,
hopital du Haut-Leveque, 5, avenue Magellan, 33604 Pessac, France.
jean-francois.viallard@chu-bordeaux.fr
INTRODUCTION: Comprehension of cell cycle regulation mechanisms has progressed
very quickly these past few years and regulators of the cell cycle have gained
widespread importance in cancer. This review first summarizes major advances in
the understanding of the control of cell cycle mechanisms. Examples of how this
control is altered in tumoral cells are then described. CURRENT KNOWLEDGE AND
KEY POINTS: The typical mammalian cell cycle consists of four distinct phases
occurring in a well-defined order, each of which should be completed
successfully before the next begins. Progression of eukaryotic cells through
major cell cycle transitions is mediated by sequential assembly and activation
of a family of serine-threonine protein kinases, the cyclin dependent kinases
(CDK). The timing of their activation is determined by their post-translational
modifications (phosphorylations/dephosphorylations), and by the association of a
protein called cyclin, which is the regulatory subunit of the kinase complex.
The cyclin family is divided into two main classes. The 'G1 cyclins' include
cyclins C, D1-3, and E, and their accumulation is rate-limiting for progression
from the G1 to S phase. The 'mitotic or G2 cyclins', which include cyclin A and
cyclin B, are involved in the control of G2/M transition and mitosis. The
cyclins bind to and activate the CDK, which leads to phosphorylation (and then
inhibition) of the tumor suppressor protein, pRb. pRb controls commitment to
progress from the G1 to S phase, at least in part by repressing the activity of
the E2F transcription factors known to promote cell proliferation. Both the
D-type cyclins and their partner kinases CDK4/6 have proto-oncogenic properties,
and their activity is carefully regulated at multiple levels including negative
control by two families of CDK inhibitors. While members of the INK4 family
(p16INK4A, p15INK4B, p18INK4C, p19INK4D) interact specifically with CDK4 and
CDK6, the CIP/KIP inhibitors p21CIP1/WAF1, p27KIP1 and p57KIP2 inhibit a broader
spectrum of CDK. The interplay between p16INK4A, cyclin D/CDK, and pRb/E2F
together constitute a functional unit collectively known as the 'pRb pathway'.
Each of the major components of this mechanism may become deregulated in cancer,
and accumulating evidence points to the 'pRb pathway' as a candidate obligatory
target in multistep oncogenesis of possibly all human tumor types. FUTURE
PROSPECTS AND PROJECTS: Major advances in the understanding of cell cycle
regulation mechanisms provided a better knowledge of the molecular interactions
involved in human cancer. This progress has led to the promotion of new
therapeutic agents presently in clinical trials or under development. Moreover,
the components of the cell cycle are probably involved in other non-cancerous
diseases and their role must be defined.
Publication Types:
Review
Review, Academic
PMID: 11355576 [PubMed - indexed for MEDLINE]
190: Biotechniques 2001 May;30(5):984-8
Erratum in:
Biotechniques 2001 Sep;31(3):488
Rapid selection against truncation mutants in yeast reverse two-hybrid screens.
Puthalakath H, Strasser A, Huang DC.
Walter and Eliza Hall Institute, Melbourne, Australia.
The yeast reverse two-hybrid system is a powerful technique for isolating
mutations in a protein that abolish its interaction with a known partner.
Selection is based on abrogation of growth suppression imposed when wild-type
interactions confer 5-fluoroorotic acid (5-FOA) sensitivity to yeast cells. A
laborious component of this system is to eliminate those mutations that cause
protein truncation. By fusing the green fluorescent protein (GFP) to the
C-terminus of a protein of interest, dynein light chain (LC8), we were able to
rapidly isolate mutations that did not result in protein truncation.
Publication Types:
Technical Report
PMID: 11355361 [PubMed - indexed for MEDLINE]
191: J Biol Chem 2001 Jul 13;276(28):26526-33
Interaction of gamma 1-syntrophin with diacylglycerol kinase-zeta. Regulation of
nuclear localization by PDZ interactions.
Hogan A, Shepherd L, Chabot J, Quenneville S, Prescott SM, Topham MK, Gee SH.
Department of Cellular and Molecular Medicine, Center for Neuromuscular Disease,
University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
Syntrophins are modular adapter proteins that link ion channels and signaling
proteins to dystrophin and its homologues. A yeast two-hybrid screen of a human
brain cDNA library using the PDZ domain of gamma 1- syntrophin, a recently
identified brain-specific isoform, yielded overlapping clones encoding the C
terminus of diacylglycerol kinase-zeta (DGK-zeta), an enzyme that converts
diacylglycerol into phosphatidic acid. In biochemical assays, the C terminus of
DGK-zeta, which contains a consensus PDZ-binding motif, was found to be
necessary and sufficient for association with gamma 1-syntrophin. When
coexpressed in HeLa cells, DGK-zeta and gamma 1-syntrophin formed a stable
complex that partitioned between the cytoplasm and nucleus. DGK-zeta
translocates from the cytosol to the nucleus, a process negatively regulated by
protein kinase C phosphorylation. We found that DGK-zeta recruits gamma
1-syntrophin into the nucleus and that the PDZ-binding motif is required.
Disrupting the interaction altered the intracellular localization of both
proteins; DGK-zeta accumulated in the nucleus, whereas gamma 1-syntrophin
remained in the cytoplasm. The level of endogenous syntrophins in the nucleus of
HeLa cells also reflected the amount of nuclear DGK-zeta. In the brain, DGK-zeta
and gamma 1-syntrophin were colocalized in cell bodies and dendrites of
cerebellar Purkinjie neurons and other neuronal cell types, suggesting that
their interaction is physiologically relevant. Moreover, coimmunoprecipitation
and pull-down experiments from brain extracts and cells suggest that DGK-zeta,
gamma 1-syntrophin, and dystrophin form a ternary complex. Collectively, our
results suggest that gamma 1-syntrophin participates in regulating the
subcellular localization of DGK-zeta to ensure correct termination of
diacylglycerol signaling.
PMID: 11352924 [PubMed - indexed for MEDLINE]
192: RNA 2001 Apr;7(4):565-75
An essential protein-binding domain of nuclear RNase P RNA.
Ziehler WA, Morris J, Scott FH, Millikin C, Engelke DR.
Department of Biological Chemistry, University of Michigan, Ann Arbor
48109-0606, USA.
Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and
protein subunits. The RNA subunits of each enzyme share substantial secondary
structural features, and most of the protein subunits are shared between the
two. One of the conserved RNA subdomains, designated P3, has previously been
shown to be required for nucleolar localization. Phylogenetic sequence analysis
suggests that the P3 domain interacts with one of the proteins common to RNase P
and RNase MRP, a conclusion strengthened by an earlier observation that the
essential domain can be interchanged between the two enzymes. To examine
possible functions of the P3 domain, four conserved nucleotides in the P3 domain
of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a
library of all possible sequence combinations at those positions. Selection of
functional genes in vivo identified permissible variations, and viable clones
that caused yeast to exhibit conditional growth phenotypes were tested for
defects in RNase P RNA and tRNA biosynthesis. Under nonpermissive conditions,
the mutants had reduced maturation of the RPR1 RNA precursor, an expected
phenotype in cases where RNase P holoenzyme assembly is defective. This loss of
RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation
characteristic of RNase P defects. To test whether mutations at the conserved
positions inhibited interactions with a particular protein, specific binding of
the individual protein subunits to the RNA subunit was tested in yeast using the
three-hybrid system. Pop1p, the largest subunit shared by RNases P and MRP,
bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was
eliminated by mutations at the conserved P3 residues. These results indicate
that Pop1p interacts with the P3 domain common to RNases P and MRP, and that
this interaction is critical in the maturation of RNase P holoenzyme.
PMID: 11345435 [PubMed - indexed for MEDLINE]
193: Proc Natl Acad Sci U S A 2001 May 22;98(11):6080-5
Five subunits are required for reconstitution of the cleavage and
polyadenylation activities of Saccharomyces cerevisiae cleavage factor I.
Gross S, Moore C.
Department of Molecular Biology and Microbiology, Sackler School of Graduate
Biomedical Sciences, Tufts University, Stearns 509, 136 Harrison Avenue, Boston,
MA 02111, USA.
Cleavage and polyadenylation of mRNA 3' ends in Saccharomyces cerevisiae
requires several factors, one of which is cleavage factor I (CF I). Purification
of CF I activity from yeast extract has implicated numerous proteins as
functioning in both cleavage and/or polyadenylation. Through reconstitution of
active CF I from separately expressed and purified proteins, we show that CF I
contains five subunits, Rna14, Rna15, Pcf11, Clp1, and Hrp1. These five are
necessary and sufficient for reconstitution of cleavage activity in vitro when
mixed with CF II, and for specific polyadenylation when mixed with
polyadenylation factor I, purified poly(A) polymerase, and poly(A) binding
protein. Analysis of the individual protein-protein interactions supports an
architectural model for CF I in which Pcf11 simultaneously interacts with Rna14,
Rna15, and Clp1, whereas Rna14 bridges Rna15 and Hrp1.
PMID: 11344258 [PubMed - indexed for MEDLINE]
194: Science 2001 May 4;292(5518):929-34
Integrated genomic and proteomic analyses of a systematically perturbed
metabolic network.
Ideker T, Thorsson V, Ranish JA, Christmas R, Buhler J, Eng JK, Bumgarner R,
Goodlett DR, Aebersold R, Hood L.
The Institute for Systems Biology, 4225 Roosevelt Way NE, Suite 200, Seattle, WA
98105, USA. tideker@systemsbiology.org
We demonstrate an integrated approach to build, test, and refine a model of a
cellular pathway, in which perturbations to critical pathway components are
analyzed using DNA microarrays, quantitative proteomics, and databases of known
physical interactions. Using this approach, we identify 997 messenger RNAs
responding to 20 systematic perturbations of the yeast galactose-utilization
pathway, provide evidence that approximately 15 of 289 detected proteins are
regulated posttranscriptionally, and identify explicit physical interactions
governing the cellular response to each perturbation. We refine the model
through further iterations of perturbation and global measurements, suggesting
hypotheses about the regulation of galactose utilization and physical
interactions between this and a variety of other metabolic pathways.
PMID: 11340206 [PubMed - indexed for MEDLINE]
195: Mol Cell Biol 2001 Jun;21(11):3725-37
Rfc4 interacts with Rpa1 and is required for both DNA replication and DNA damage
checkpoints in Saccharomyces cerevisiae.
Kim HS, Brill SJ.
Department of Molecular Biology and Biochemistry, Center for Advanced
Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854,
USA.
The large subunit of replication protein A (Rpa1) consists of three
single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown
function. To determine the essential role of this domain we searched for
mutations that require wild-type Rpa1N for viability in yeast. A mutation in
RFC4, encoding a small subunit of replication factor C (RFC), was found to
display allele-specific interactions with mutations in the gene encoding Rpa1
(RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis
inhibitor hydroxyurea, such as rfa1-t11, are lethal in combination with rfc4-2.
The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5
strains, it exhibits defects in the DNA replication block and intra-S
checkpoints. RFC4 and the DNA damage checkpoint gene RAD24 were found to be
epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant
is defective in the G(1)/S DNA damage checkpoint response and that both the
rfc4-2 and rfa1-t11 strains are defective in the G(2)/M DNA damage checkpoint.
Thus, in addition to its essential role as part of the clamp loader in DNA
replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways.
Our results suggest that a physical interaction between Rfc4 and Rpa1N is
required for both roles.
PMID: 11340166 [PubMed - indexed for MEDLINE]
196: Biochem J 2001 May 15;356(Pt 1):207-15
Self-association and precursor protein binding of Saccharomyces cerevisiae
Tom40p, the core component of the protein translocation channel of the
mitochondrial outer membrane.
Gordon DM, Wang J, Amutha B, Pain D.
Department of Physiology, University of Pennsylvania School of Medicine, 3700
Hamilton Walk, D403 Richards Building, Philadelphia, PA 19104-6085, USA.
The precursor protein translocase of the mitochondrial outer membrane (Tom) is a
multi-subunit complex containing receptors and a general import channel, of
which the core component is Tom40p. Nuclear-encoded mitochondrial precursor
proteins are first recognized by surface receptors and then pass through the
import channel. The Tom complex has been purified; however, the protein-protein
interactions that drive its assembly and maintain its stability have been
difficult to study. Here we show that Saccharomyces cerevisiae Tom40p expressed
in bacteria and purified to homogeneity associates efficiently with itself. The
self-association is very strong and can withstand up to 4 M urea or 1 M salt.
The tight self-association does not require the N-terminal segment of Tom40p.
Furthermore, purified Tom40p preferentially recognizes the targeting sequence of
mitochondrial precursor proteins. Although the binding of the targeting sequence
to Tom40p is inhibited by urea concentrations in excess of 1 M, it is moderately
resistant to 1 M salt. Simultaneous self-assembly and precursor protein binding
suggest that Tom40p contains at least two different domains mediating these
processes. The experimental approach described here should be useful for
analysing protein-protein interactions involving individual or groups of
components of the mitochondrial import machinery.
PMID: 11336653 [PubMed - indexed for MEDLINE]
197: FEBS Lett 2001 Apr 27;495(3):148-53
A yeast two-hybrid study of human p97/Gab2 interactions with its SH2
domain-containing binding partners.
Crouin C, Arnaud M, Gesbert F, Camonis J, Bertoglio J.
Inserm Unit 461, Faculte de Pharmacie Paris-XI, Chatenay-Malabry, france.
p97/Gab2 is a recently characterized member of a large family of scaffold
proteins that play essential roles in signal transduction. Gab2 becomes
tyrosine-phosphorylated in response to a variety of growth factors and forms
multimolecular complexes with SH2 domain-containing signaling molecules such as
the p85-regulatory subunit of the phosphoinositide-3-kinase (p85-PI3K), the
tyrosine phosphatase SHP-2 and the adapter protein CrkL. To characterize the
interactions between Gab2 and its SH2-containing binding partners, we designed a
modified yeast two-hybrid system in which the Lyn tyrosine kinase is expressed
in a regulated manner in yeast. Using this assay, we demonstrated that p97/Gab2
specifically interacts with the SH2 domains of PI3K, SHP-2 and CrkL. Interaction
with p85-PI3K is mediated by tyrosine residues Y452, Y476 and Y584 of Gab2,
while interaction with SHP-2 depends exclusively on tyrosine Y614. CrkL
interaction is mediated by its SH2 domain recognizing Y266 and Y293, despite the
latter being in a non-consensus (YTFK) environment.
PMID: 11334882 [PubMed - indexed for MEDLINE]
198: Genetics 2001 May;158(1):187-96
Multiple functional interactions between components of the Lsm2-Lsm8 complex, U6
snRNA, and the yeast La protein.
Pannone BK, Kim SD, Noe DA, Wolin SL.
Department of Cell Biology, Howard Hughes Medical Institute, Yale University
School of Medicine, New Haven, CT 06536, USA.
The U6 small nuclear ribonucleoprotein is a critical component of the eukaryotic
spliceosome. The first protein that binds the U6 snRNA is the La protein, an
abundant phosphoprotein that binds the 3' end of many nascent small RNAs. A
complex of seven Sm-like proteins, Lsm2-Lsm8, also binds the 3' end of U6 snRNA.
A mutation within the Sm motif of Lsm8p causes Saccharomyces cerevisiae cells to
require the La protein Lhp1p to stabilize nascent U6 snRNA. Here we describe
functional interactions between Lhp1p, the Lsm proteins, and U6 snRNA. LSM2 and
LSM4, but not other LSM genes, act as allele-specific, low-copy suppressors of
mutations in Lsm8p. Overexpression of LSM2 in the lsm8 mutant strain increases
the levels of both Lsm8p and U6 snRNPs. In the presence of extra U6 snRNA genes,
LSM8 becomes dispensable for growth, suggesting that the only essential function
of LSM8 is in U6 RNA biogenesis or function. Furthermore, deletions of LSM5,
LSM6, or LSM7 cause LHP1 to become required for growth. Our experiments are
consistent with a model in which Lsm2p and Lsm4p contact Lsm8p in the Lsm2-Lsm8
ring and suggest that Lhp1p acts redundantly with the entire Lsm2-Lsm8 complex
to stabilize nascent U6 snRNA.
PMID: 11333229 [PubMed - indexed for MEDLINE]
199: Genetics 2001 May;158(1):87-93
Multiple functions of the nonconserved N-terminal domain of yeast TATA-binding
protein.
Lee M, Struhl K.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, MA 02115, USA.
The TATA-binding protein (TBP) is composed of a highly conserved core domain
sufficient for TATA-element binding and preinitiation complex formation as well
as a highly divergent N-terminal region that is dispensable for yeast cell
viability. In vitro, removal of the N-terminal region domain enhances TBP-TATA
association and TBP dimerization. Here, we examine the effects of truncation of
the N-terminal region in the context of yeast TBP mutants with specific defects
in DNA binding and in interactions with various proteins. For a subset of
mutations that disrupt DNA binding and the response to transcriptional
activators, removal of the N-terminal domain rescues their transcriptional
defects. By contrast, deletion of the N-terminal region is lethal in combination
with mutations on a limited surface of TBP. Although this surface is important
for interactions with TFIIA and Brf1, TBP interactions with these two factors do
not appear to be responsible for this dependence on the N-terminal region. Our
results suggest that the N-terminal region of TBP has at least two distinct
functions in vivo. It inhibits the interaction of TBP with TATA elements, and it
acts positively in combination with a specific region of the TBP core domain
that presumably interacts with another protein(s).
PMID: 11333220 [PubMed - indexed for MEDLINE]
200: EMBO J 2001 May 1;20(9):2326-37
Multiple roles for the C-terminal domain of eIF5 in translation initiation
complex assembly and GTPase activation.
Asano K, Shalev A, Phan L, Nielsen K, Clayton J, Valasek L, Donahue TF,
Hinnebusch AG.
Laboratory of Gene Regulation and Development, National Institute of Child
Health and Human Development/NIH, Bethesda, MD 20892, USA.
eIF5 stimulates the GTPase activity of eIF2 bound to Met-tRNA(i)(Met), and its
C-terminal domain (eIF5-CTD) bridges interaction between eIF2 and eIF3/eIF1 in a
multifactor complex containing Met-tRNA(i)(Met). The tif5-7A mutation in
eIF5-CTD, which destabilizes the multifactor complex in vivo, reduced the
binding of Met-tRNA(i)(Met) and mRNA to 40S subunits in vitro. Interestingly,
eIF5-CTD bound simultaneously to the eIF4G subunit of the cap-binding complex
and the NIP1 subunit of eIF3. These interactions may enhance association of
eIF4G with eIF3 to promote mRNA binding to the ribosome. In vivo, tif5-7A
eliminated eIF5 as a stable component of the pre-initiation complex and led to
accumulation of 48S complexes containing eIF2; thus, conversion of 48S to 80S
complexes is the rate-limiting defect in this mutant. We propose that eIF5-CTD
stimulates binding of Met-tRNA(i)(Met) and mRNA to 40S subunits through
interactions with eIF2, eIF3 and eIF4G; however, its most important function is
to anchor eIF5 to other components of the 48S complex in a manner required to
couple GTP hydrolysis to AUG recognition during the scanning phase of
initiation.
PMID: 11331597 [PubMed - indexed for MEDLINE]
201: EMBO J 2001 May 1;20(9):2111-9
Oligopeptide repeats in the yeast protein Sup35p stabilize intermolecular prion
interactions.
Parham SN, Resende CG, Tuite MF.
Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ,
UK.
The nuclear-encoded Sup35p protein is responsible for the prion-like [PSI(+)]
determinant of yeast, with Sup35p existing largely as a high molecular weight
aggregate in [PSI(+)] strains. Here we show that the five oligopeptide repeats
present at the N-terminus of Sup35p are responsible for stabilizing aggregation
of Sup35p in vivo. Sequential deletion of the oligopeptide repeats prevented the
maintenance of [PSI(+)] by the truncated Sup35p, although deletants containing
only two repeats could be incorporated into pre-existing aggregates of wild-type
Sup35p. The mammalian prion protein PrP also contains similar oligopeptide
repeats and we show here that a human PrP repeat (PHGGGWGQ) is able functionally
to replace a Sup35p oligopeptide repeat to allow stable [PSI(+)] propagation in
vivo. Our data suggest a model in which the oligopeptide repeats in Sup35p
stabilize intermolecular interactions between Sup35p proteins that initiate
establishment of the aggregated state. Modulating repeat number therefore alters
the rate of yeast prion conversion in vivo. Furthermore, there appears to be
evolutionary conservation of function of the N-terminally located oligopeptide
repeats in prion propagation.
PMID: 11331577 [PubMed - indexed for MEDLINE]
202: Biochemistry 2001 Feb 20;40(7):2080-6
Mitochondrial phosphate transport protein. Reversions of inhibitory conservative
mutations identify four helices and a nonhelix protein segment with
transmembrane interactions and Asp39, Glu137, and Ser158 as nonessential for
transport.
Phelps A, Briggs C, Haefele A, Mincone L, Ligeti E, Wohlrab H.
Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
The mitochondrial phosphate transport protein (PTP) has six (A--F) transmembrane
(TM) helices per subunit of functional homodimer with all mutations referring to
the subunit of the homodimer. In earlier studies, conservative replacements of
several residues located either at the matrix end (Asp39/helix A, Glu137/helix
C, Asp236/helix E) or at the membrane center (His32/helix A, Glu136/helix C) of
TM helices yielded inactive single mutation PTPs. Some of these residues were
suggested to act as phosphate ligands or as part of the proton cotransport path.
We now show that the mutation Ser158Thr, not part of a TM helix but located near
the center of the matrix loop (Ile141--Ser171) between TM helices C and D,
inactivates PTP and is thus also functionally relevant. On the other side of the
membrane, the single mutation Glu192Asp at the intermembrane space end of TM
helix D yields a PTP with 33% wild-type activity. We constructed double mutants
by adding this mutation to the six transport-inactivating mutations. Transport
was detected only in those with Asp39Asn, Glu137Gln, or Ser158Thr. We conclude
that TM helix D can interact with TM helices A and C and matrix loop
Ile141--Ser171 and that Asp39, Glu137, and Ser158 are not essential for
phosphate transport. Since our results are consistent with residues present in
all 12 functionally identified members of the mitochondrial transport protein
(MTP) family, they lead to a general rule that specifies MTP residue types at 7
separate locations. The conformations of all the double mutation PTPs (except
that with the matrix loop Ser158Thr) are significantly different from those of
the single mutation PTPs, as indicated by their very low liposome incorporation
efficiency and their requirement for less detergent (Triton X-100) to stay in
solution. These dramatic conformational differences also suggest an interaction
between TM helices D and E. The results are discussed in terms of TM helix
movements and changes in the PTP monomer/dimer ratio.
PMID: 11329276 [PubMed - indexed for MEDLINE]
203: Biochemistry 2001 Feb 20;40(7):1930-6
Covariation of a specificity-determining structural motif in an aminoacyl-tRNA
synthetase and a tRNA identity element.
Hawko SA, Francklyn CS.
Department of Biochemistry, University of Vermont, Health Sciences Complex,
Burlington, Vermont 05405, USA.
Transfer RNA (tRNA) identity determinants help preserve the specificity of
aminoacylation in vivo, and prevent cross-species interactions. Here, we
investigate covariation between the discriminator base (N73) element in
histidine tRNAs and residues in the histidyl-tRNA synthetase (HisRS) motif 2
loop. A model of the Escherichia coli HisRS--tRNA(His) complex predicts an
interaction between the prokaryotic conserved glutamine 118 of the motif 2 loop
and cytosine 73. The substitution of Gln 118 in motif 2 with glutamate decreased
discrimination between cytosine and uracil some 50-fold, but left overall rates
of adenylation and aminoacylation unaffected. By contrast, substitutions at
neighboring Glu 115 and Arg 121 affected both adenylation and aminoacylation,
consistent with their predicted involvement in both half-reactions. Additional
evidence for the involvement of the motif 2 loop was provided by functional
analysis of a hybrid Saccharomyces cerevisiae-- E. coli HisRS possessing the 11
amino acid motif 2 loop of the yeast enzyme. Despite an overall decreased
activity of nearly 1000-fold relative to the E. coli enzyme, the chimera
nevertheless exhibited a modest preference for the yeast tRNA(His) over the E.
coli tRNA, and preferred wild-type yeast tRNA(His) to a variant with C at the
discriminator position. These experiments suggest that part of, but not all of,
the specificity is provided by the motif 2 loop. The close interaction between
enzyme loop and RNA sequence elements suggested by these experiments reflects a
covariation between enzyme and tRNA that may have acted to preserve
aminoacylation fidelity over evolutionary time.
PMID: 11329259 [PubMed - indexed for MEDLINE]
204: Nat Med 2001 May;7(5):625-9
Whole recombinant yeast vaccine activates dendritic cells and elicits protective
cell-mediated immunity.
Stubbs AC, Martin KS, Coeshott C, Skaates SV, Kuritzkes DR, Bellgrau D,
Franzusoff A, Duke RC, Wilson CC.
Department of Medicine, University of Colorado Health Sciences Center, Denver,
Colorado, USA.
There is currently a need for vaccines that stimulate cell-mediated
immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes
(CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated
immunity requires the presentation of antigens by specialized cells of the
immune system called dendritic cells (DCs). DCs are unique in their ability to
process exogenous antigens via the major histocompatibility complex (MHC) class
I pathway as well as in their ability to activate naive, antigen-specific CD8+
and CD4+ T cells. Vaccine strategies that target or activate DCs in order to
elicit potent CTL-mediated immunity are the subject of intense research. We
report here that whole recombinant Saccharomyces cerevisiae yeast expressing
tumor or HIV-1 antigens potently induced antigen-specific, CTL responses,
including those mediating tumor protection, in vaccinated animals. Interactions
between yeast and DCs led to DC maturation, IL-12 production and the efficient
priming of MHC class I- and class II-restricted, antigen-specific T-cell
responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of
exogenous whole-protein antigen to MHC class I- and class II-restricted T cells.
Recombinant yeast represent a novel vaccine strategy for the induction of
broad-based cellular immune responses.
PMID: 11329066 [PubMed - indexed for MEDLINE]
205: Methods 2001 May;24(1):29-34
Protein recruitment systems for the analysis of protein +/- protein
interactions.
Aronheim A.
Department of Molecular Genetics, Rappaport Family Institute for Research in the
Medical Sciences, Bat-Galim, Haifa 31096, Israel. aronheim@tx.technion.ac.il
The yeast Saccharomyces cerevisiae serves as an excellent genetic tool for the
analysis of protein +/- protein interactions. The most common system, used to
date, is the two-hybrid system. Although proven very powerful, the two-hybrid
system exhibits several inherent problems and limitations. Recently, two
alternative systems have been described that take advantage of the fact that
localization of signal transduction effectors to the inner leaflet of the plasma
membrane is absolutely necessary for yeast viability. These effectors can either
be the Ras guanyl nucleotide exchange factor or Ras itself. The yeast strain
used in both systems is a temperature-sensitive mutant in the yeast Ras guanyl
nucleotide exchange factor, CDC25. Membrane localization of these effectors is
achieved via protein +/- protein interaction. Each system can be used to test
interaction between known protein pairs, as well as for isolation of novel
protein interactions. Described here are the scientific and technical steps to
be considered for both protein recruitment systems. Copyright 2001 Academic
Press.
PMID: 11327799 [PubMed - indexed for MEDLINE]
206: Nat Struct Biol 2001 May;8(5):417-22
UBA domains of DNA damage-inducible proteins interact with ubiquitin.
Bertolaet BL, Clarke DJ, Wolff M, Watson MH, Henze M, Divita G, Reed SI.
Department of Molecular Biology, The Scripps Research Institute, La Jolla,
California 92037, USA.
Rad23 is a highly conserved protein involved in nucleotide excision repair (NER)
that associates with the proteasome via its N-terminus. Its C-terminal
ubiquitin-associated (UBA) domain is evolutionarily conserved from yeast to
humans. However, the cellular function of UBA domains is not completely
understood. Recently, RAD23 and DDI1, both DNA damage-inducible genes encoding
proteins with UBA domains, were implicated genetically in Pds1-dependent mitotic
control in yeast. The UBA domains of RAD23 and DDI1 are required for these
interactions. Timely degradation of Pds1 via the ubiquitin/proteasome pathway
allows anaphase onset and is crucial for chromosome maintenance. Here, we show
that Rad23 and Ddi1 interact directly with ubiquitin and that this interaction
is dependent on their UBA domains, providing a possible mechanism for
UBA-dependent cell cycle control. Moreover, we show that a hydrophobic surface
on the UBA domain, which from structural work had been predicted to be a
protein-protein interaction interface, is indeed required for ubiquitin binding.
By demonstrating that UBA domains interact with ubiquitin, we have provided the
first indication of a cellular function for the UBA domain.
PMID: 11323716 [PubMed - indexed for MEDLINE]
207: J Biol Chem 2001 Jul 13;276(28):26666-73
Assembly of the human origin recognition complex.
Vashee S, Simancek P, Challberg MD, Kelly TJ.
Department of Molecular Biology and Genetics, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205, USA.
The six-subunit origin recognition complex (ORC) was originally identified in
the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of
replication and serves as a platform for the assembly of additional initiation
factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC
subunits have been identified by sequence similarity to their yeast
counterparts, but little is known about the biochemical characteristics of human
ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its
subunit composition using specific antibodies. The endogenous HsORC, identified
in these experiments, contained homologues of Orc1-Orc5 but lacked a putative
homologue of Orc6. By expressing HsORC subunits in insect cells using the
baculovirus system, we were able to identify a complex containing all six
subunits. To explore the subunit-subunit interactions that are required for the
assembly of HsORC, we carried out extensive co-immunoprecipitation experiments
with recombinant ORC subunits expressed in different combinations. These studies
revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4,
HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable
binary complexes with any other HsORC subunit but interacted with sub-complexes
containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by
HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits.
These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core
upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The
characterization of HsORC should facilitate the identification of human origins
of DNA replication.
PMID: 11323433 [PubMed - indexed for MEDLINE]
208: Genes Dev 2001 Apr 15;15(8):1007-20
Recruitment of the transcriptional machinery through GAL11P: structure and
interactions of the GAL4 dimerization domain.
Hidalgo P, Ansari AZ, Schmidt P, Hare B, Simkovich N, Farrell S, Shin EJ,
Ptashne M, Wagner G.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts 02115, USA.
The GAL4 dimerization domain (GAL4-dd) is a powerful transcriptional activator
when tethered to DNA in a cell bearing a mutant of the GAL11 protein, named
GAL11P. GAL11P (like GAL11) is a component of the RNA-polymerase II holoenzyme.
Nuclear magnetic resonance (NMR) studies of GAL4-dd revealed an elongated dimer
structure with C(2) symmetry containing three helices that mediate dimerization
via coiled-coil contacts. The two loops between the three coiled coils form
mobile bulges causing a variation of twist angles between the helix pairs.
Chemical shift perturbation analysis mapped the GAL11P-binding site to the
C-terminal helix alpha3 and the loop between alpha1 and alpha2. One GAL11P
monomer binds to one GAL4-dd dimer rendering the dimer asymmetric and implying
an extreme negative cooperativity mechanism. Alanine-scanning mutagenesis of
GAL4-dd showed that the NMR-derived GAL11P-binding face is crucial for the novel
transcriptional activating function of the GAL4-dd on GAL11P interaction. The
binding of GAL4 to GAL11P, although an artificial interaction, represents a
unique structural motif for an activating region capable of binding to a single
target to effect gene expression.
PMID: 11316794 [PubMed - indexed for MEDLINE]
209: Oncogene 2001 Jan 25;20(4):501-13
p53 mutants exhibiting enhanced transcriptional activation and altered promoter
selectivity are revealed using a sensitive, yeast-based functional assay.
Inga A, Monti P, Fronza G, Darden T, Resnick MA.
Laboratory of Molecular Genetics, National Institute of Environmental Health
Sciences (NIEHS), PO Box 12233, Research Triangle Park, North Carolina, NC
27709, USA.
Changes in promoter specificity and binding affinity that may be associated with
p53 mutations or post-translational modifications are useful in understanding
p53 structure/function relationships and categorizing tumor mutations. We have
exploited variable expression of human p53 in yeast to identify mutants with
novel phenotypes that would correspond to altered promoter selectivity and
affinity. The p53 cDNA regions coding for the DNA binding and tetramerization
domains were subjected to random PCR mutagenesis and were cloned directly by
recombination in yeast into a vector with a GAL1 promoter whose level of
expression could be easily varied. p53 variants exhibiting higher than wild type
levels of transactivation (supertrans) for the RGC responsive element were
identified at low level of p53 protein expression. All the p53 mutants obtained
with this screen were located in the DNA binding domain. Two out of 17
supertrans mutants have been found in tumors. Six mutations were in the L1 loop
region between amino acids 115 and 124. The transactivation potential of a panel
of supertrans p53 mutants on different promoters was evaluated using the p53
responsive elements, RGC, PIG3, p21 and bax. Although all mutants retained some
activity with all promoters, we found different patterns of induction based on
strength and promoter specificity. In particular none of the mutants was
supertrans for the p21 responsive element. Interestingly, further analysis in
yeast showed that the transactivation function could be retained even in the
presence of dominant-negative p53 tumor mutations that could inhibit wild type
p53. Five mutants were also characterized in human cells in terms of growth
suppression and transactivation of various promoters. These novel supertrans p53
mutants may be useful in studies aimed at dissecting p53 downstream pathways,
understanding specific interactions between p53 and the DNA, and could replace
wild type p53 in cancer gene therapy protocols. The approach may also prove
useful in identifying p53 tumor mutations.
PMID: 11313981 [PubMed - indexed for MEDLINE]
210: Trends Cell Biol 2001 Mar;11(3):102-6
Towards an understanding of complex protein networks.
Tucker CL, Gera JF, Uetz P.
Dept of Genetics, University of Washington Box 357360, Seattle, WA 98195, USA.
ctucker@u.washington.edu
Large-scale two-hybrid screens have generated a wealth of information describing
potential protein--protein interactions. When compiled with data from systematic
localizations of proteins, mutant screens and other functional tests, a network
of interactions among proteins and between proteins and other components of
eukaryotic cells can be deduced. These networks can be viewed as maps of the
cell, depicting potential signaling pathways and interactive complexes. Most
importantly, they provide potential clues to the function of previously
uncharacterized proteins. Focusing on recent experiments, we explore these
protein-interaction studies and the maps derived from such efforts.
Publication Types:
Review
Review, Tutorial
PMID: 11306254 [PubMed - indexed for MEDLINE]
211: Genome Biol 2001;2(4):REVIEWS1013
Genome-wide analysis of protein-DNA interactions in living cells.
Pugh BF, Gilmour DS.
Center for Gene Regulation, Department of Biochemistry and Molecular Biology,
Pennsylvania State University, University Park, PA 16802, USA.
Understanding the regulation of gene expression requires an analysis of
gene-specific transcription factors. This review highlights recent work that
uses protein-DNA crosslinking, immunoprecipitation and DNA microarrays to
determine the binding sites for specific transcription factors throughout the
yeast genome.
Publication Types:
Review
Review, Tutorial
PMID: 11305945 [PubMed - indexed for MEDLINE]
212: Methods Enzymol 2001;332:277-300
Two-hybrid dual bait system to discriminate specificity of protein interactions
in small GTPases.
Serebriiskii IG, Mitina OV, Chernoff J, Golemis EA.
Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania
19111, USA.
PMID: 11305104 [PubMed - indexed for MEDLINE]
213: Methods Enzymol 2001;332:260-70
Ras signaling pathway for analysis of protein-protein interactions.
Aronheim A.
Department of Molecular Genetics, B. Rappaport Faculty of Medicine, Israel
Institute of Technology, Haifa 31096, Israel.
PMID: 11305102 [PubMed - indexed for MEDLINE]
214: J Biol Chem 2001 Apr 20;276(16):12636-44
The interactions of yeast SWI/SNF and RSC with the nucleosome before and after
chromatin remodeling.
Sengupta SM, VanKanegan M, Persinger J, Logie C, Cairns BR, Peterson CL,
Bartholomew B.
Program in Molecular Biology, Microbiology, and Molecular Biology and Department
of Biochemistry and Molecular Biology, Southern Illinois University School of
Medicine, Carbondale, Illinois 62901-4413, USA.
Interactions of the yeast chromatin-remodeling complexes SWI/SNF and RSC with
nucleosomes were probed using site-specific DNA photoaffinity labeling. 5 S rDNA
was engineered with photoreactive nucleotides incorporated at different sites in
DNA to scan for the subunits of SWI/SNF in close proximity to DNA when SWI/SNF
is bound to the 5 S nucleosome or to the free 5 S rDNA. The Swi2/Snf2 and Snf6
subunits of SWI/SNF were efficiently cross-linked at several positions in the
nucleosome, whereas only Snf6 was efficiently cross-linked when SWI/SNF was
bound to free DNA. DNA photoaffinity labeling of RSC showed that the Rsc4
subunit is in close proximity to nucleosomal DNA and not when RSC is bound to
free DNA. After remodeling, the Swi2/Snf2 and Rsc4 subunits are no longer
detected near the nucleosomal DNA and are evidently displaced from the surface
of the nucleosome, indicating significant changes in SWI/SNF and RSC contacts
with DNA after remodeling.
PMID: 11304548 [PubMed - indexed for MEDLINE]
215: Biochem Biophys Res Commun 2001 Apr 20;282(5):1211-9
The role of heat shock protein 70 in vitamin D receptor function.
Lutz W, Kohno K, Kumar R.
Department of Internal Medicine, Mayo Clinic and Foundation, Rochester,
Minnesota 55905, USA.
We previously demonstrated that the 1alpha,25-dihydroxyvitamin D(3) receptor
(VDR) interacts with the constitutive heat shock protein, hsc70 in vitro, and
with DnaK (Biochem. Biophys. Res. Commun. 260, 446-452, 1999). The biological
significance of VDR-heat shock protein interactions, however, is unknown. To
examine the role of such interactions in eukaryotic cells, we heterologously
expressed VDR and RXRalpha together with a vitamin D-responsive reporter system
in Saccharomyces cerevisiae and examined the consequences of heat shock protein
70 gene (SSA) deletion in these cells. We show that heterologously expressed VDR
associates with the yeast cytosolic hsp70 protein, Ssa1p. Deletion of the SSA2,
SSA3, and SSA4 genes and reduction of Ssa1p activity, reduces the intracellular
concentrations of the VDR and its heterodimeric partner, RXRalpha and reduces
the activity of a vitamin D-dependent gene. Hsp70-like chaperone proteins play a
role in controlling concentrations of the VDR within the cell. Copyright 2001
Academic Press.
PMID: 11302745 [PubMed - indexed for MEDLINE]
216: J Biol Chem 2001 May 11;276(19):16520-7
5-Lipoxygenase interacts with coactosin-like protein.
Provost P, Doucet J, Hammarberg T, Gerisch G, Samuelsson B, Radmark O.
Department of Medical Biochemistry and Biophysics, Division of Physiological
Chemistry II, Karolinska Institute, S-171 77 Stockholm, Sweden.
We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid
screen using 5-lipoxygenase (5LO) as a bait. In this report, we demonstrate a
direct interaction between 5LO and CLP. 5LO associated with CLP, which was
expressed as a glutathione S-transferase fusion protein, in a dose-dependent
manner. Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP
proteins transiently expressed in human embryonic kidney 293 cells revealed the
presence of CLP in 5LO immunoprecipitates. In reciprocal experiments, 5LO was
detected in CLP immunoprecipitates. Non-denaturing polyacrylamide gel
electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1
molar stoichiometry in a Ca(2+)-independent manner. Site-directed mutagenesis
suggested an important role for lysine 131 of CLP in mediating 5LO binding. In
view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we
determined whether CLP could physically link 5LO to actin filaments. However, no
F-actin-CLP.5LO ternary complex was observed. In contrast, 5LO appeared to
compete with F-actin for the binding of CLP. Moreover, 5LO was found to
interfere with actin polymerization. Our results indicate that the 5LO-CLP and
CLP-F-actin interactions are mutually exclusive and suggest a modulatory role
for 5LO in actin dynamics.
PMID: 11297527 [PubMed - indexed for MEDLINE]
217: Appl Biochem Biotechnol 2001 Feb;90(2):155-86
Thermozymes and their applications: a review of recent literature and patents.
Bruins ME, Janssen AE, Boom RM.
Department of Food Technology and Nutritional Sciences, Wageningen University,
The Netherlands. marieke.bruins@algemeen.pk.wau.nl
Enzymes from thermophilic microorganisms, thermozymes, have unique
characteristics such as temperature, chemical, and pH stability. They can be
used in several industrial processes, in which they replace mesophilic enzymes
or chemicals. Thermozymes are often used when the enzymatic process is
compatible with existing (high-temperature) process conditions. The main
advantages of performing processes at higher temperatures are reduced risk of
microbial contamination, lower viscosity, improved transfer rates, and improved
solubility of substrates. However, cofactors, substrates, or products might be
unstable or other side reactions may occur. Recent developments show that
thermophiles are a good source of novel catalysts that are of great industrial
interest. Thermostable polymer-degrading enzymes such as amylases, pullulanases,
xylanases, proteases, and cellulases are expected to play an important role in
food, chemical, pharmaceutical, paper, pulp, and waste-treatment industries.
Considerable research efforts have been made to better understand the stability
of thermozymes. There are no major conformational differences with mesophilic
enzymes, and a small number of extra salt bridges, hydrophobic interactions, or
hydrogen bounds seem to confer the extra degree of stabilization. Currently,
overexpression of thermozymes in standard Escherichia coli allows the production
of much larger quantities of enzymes, which are easy to purify by heat
treatment. With wider availability and lower cost, thermophilic enzymes will see
more application in industry.
Publication Types:
Review
Review, Tutorial
PMID: 11297390 [PubMed - indexed for MEDLINE]
218: Mol Biol Cell 2001 Apr;12(4):1177-88
Cadherin sequences that inhibit beta-catenin signaling: a study in yeast and
mammalian cells.
Simcha I, Kirkpatrick C, Sadot E, Shtutman M, Polevoy G, Geiger B, Peifer M,
Ben-Ze'ev A.
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot,
Israel, 76100.
Drosophila Armadillo and its mammalian homologue beta-catenin are scaffolding
proteins involved in the assembly of multiprotein complexes with diverse
biological roles. They mediate adherens junction assembly, thus determining
tissue architecture, and also transduce Wnt/Wingless intercellular signals,
which regulate embryonic cell fates and, if inappropriately activated,
contribute to tumorigenesis. To learn more about Armadillo/beta-catenin's
scaffolding function, we examined in detail its interaction with one of its
protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid
system to study cadherin binding in the absence of Armadillo/beta-catenin's
other protein partners, and mammalian cells where interactions were assessed in
their presence. We found that segments of the cadherin cytoplasmic tail as small
as 23 amino acids bind Armadillo or beta-catenin in yeast, whereas a slightly
longer region is required for binding in mammalian cells. We used mutagenesis to
identify critical amino acids required for cadherin interaction with
Armadillo/beta-catenin. Expression of such short cadherin sequences in mammalian
cells did not affect adherens junctions but effectively inhibited
beta-catenin-mediated signaling. This suggests that the interaction between
beta-catenin and T cell factor family transcription factors is a sensitive
target for disruption, making the use of analogues of these cadherin derivatives
a potentially useful means to suppress tumor progression.
PMID: 11294915 [PubMed - indexed for MEDLINE]
219: Nucleic Acids Res 2001 Apr 15;29(8):1715-23
The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is
required for full activity.
Cicero MP, Hubl ST, Harrison CJ, Littlefield O, Hardy JA, Nelson HC.
Johnson Research Foundation and Department of Biochemistry and Biophysics,
University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6089, USA.
The yeast heat shock transcription factor (HSF) belongs to the winged helix
family of proteins. HSF binds DNA as a trimer, and additional trimers can bind
DNA co-operatively. Unlike other winged helix-turn-helix proteins, HSF's wing
does not appear to contact DNA, as based on a previously solved crystal
structure. Instead, the structure implies that the wing is involved in
protein-protein interactions, possibly within a trimer or between adjacent
trimers. To understand the function of the wing in the HSF DNA-binding domain, a
Saccharomyces cerevisiae strain was created that expresses a wingless HSF
protein. This strain grows normally at 30 degrees C, but shows a decrease in
reporter gene expression during constitutive and heat-shocked conditions.
Removal of the wing does not affect the stability or trimeric nature of a
protein fragment containing the DNA-binding and trimerization domains. Removal
of the wing does result in a decrease in DNA-binding affinity. This defect was
mainly observed in the ability to form the first trimer-bound complex, as the
formation of larger complexes is unaffected by the deletion. Our results suggest
that the wing is not involved in the highly co-operative nature of HSF binding,
but may be important in stabilizing the first trimer bound to DNA.
PMID: 11292844 [PubMed - indexed for MEDLINE]
220: J Mol Biol 2001 Apr 13;307(5):1207-21
Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA
strand exchange via specific interactions with the presynaptic Rad51
nucleoprotein filament.
Solinger JA, Lutz G, Sugiyama T, Kowalczykowski SC, Heyer WD.
Institute of General Microbiology, University of Bern, Bern, CH-3012,
Switzerland.
RAD54 is an important member of the RAD52 group of genes that carry out
recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae.
Rad54 protein is a member of the Snf2/Swi2 protein family of
DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54
protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in
the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST)
and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the
functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB
repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity.
Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically
increasing the kinetics of joint molecule formation. This stimulation was
accompanied by a concurrent increase in the formation of heteroduplex DNA. Our
results suggest that Rad54 protein interacts specifically with established Rad51
nucleoprotein filaments before homology search on the duplex DNA and
heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange
by increasing presynaptic complex formation. We conclude that Rad54 protein acts
during the synaptic phase of DNA strand exchange and after the formation of
presynaptic Rad51 protein-ssDNA filaments. Copyright 2001 Academic Press.
PMID: 11292336 [PubMed - indexed for MEDLINE]
221: Plant Mol Biol 2001 Feb;45(3):365-76
Further analysis of the interactions between the Brassica S receptor kinase and
three interacting proteins (ARC1, THL1 and THL2) in the yeast two-hybrid system.
Mazzurco M, Sulaman W, Elina H, Cock JM, Goring DR.
Biology Department, York University, Toronto, Ontario, Canada.
The yeast two-hybrid system was used to further characterize the interactions
between the Brassica S receptor kinase (SRK) and three putative substrates, ARC1
and the two thioredoxin h proteins, THL1 and THL2. Interactions were generally
detectable with kinase domains of both Class I and Class II SRKs. Chimeric
constructs were made between the SRK910 kinase domain and the non-interacting
Arabidopsis RLK5 kinase domain. Only one chimeric construct, SRR2, interacted
with THL1 and THL2, while none of the chimeras were able to interact with ARC1.
SRR2 is largely made up of RLK5 kinase domain with the N-terminal end being
derived from the SRK910 kinase domain and was the only chimeric construct that
retained kinase activity. Deletion or substitution of a conserved cysteine at
the N-terminal end of the SRK910 kinase domain resulted in loss of interaction
with THL1 and THL2, while the addition of this cysteine to a related receptor
kinase, SFR1, conferred the ability to interact with the thioredoxin h proteins.
In addition, substitution of the cysteines in the THL1 active site abolished the
interaction. Lastly, the two Arabidopsis thioredoxin h clones most closely
related to THL1 and THL2 were found to interact with the SRK kinase domains.
Thus, the nature of the interaction of the thioredoxin h clones with SRK
involves the reducing activity of these proteins and is restricted to the class
of thioredoxin h proteins which have the variant CPPC active site.
PMID: 11292081 [PubMed - indexed for MEDLINE]
222: Genetics 2001 Apr;157(4):1451-67
Functional contacts with a range of splicing proteins suggest a central role for
Brr2p in the dynamic control of the order of events in spliceosomes of
Saccharomyces cerevisiae.
van Nues RW, Beggs JD.
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9
3JR, United Kingdom.
Mapping of functional protein interactions will help in understanding
conformational rearrangements that occur within large complexes like
spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we
undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5
snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with
five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p,
second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is
required for splicing at low temperatures. Co-immunoprecipitation experiments
confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p
with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p
to the spliceosome. We provide evidence that the prp8-1 allele disrupts an
interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex
unwinding through another interaction with Brr2p. The interactions of Brr2p with
a wide range of proteins suggest a particular function for the C-terminal half,
bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p
promotes other RNA rearrangements, acting synergistically with other
spliceosomal proteins, including the structurally related Prp2p and Prp16p.
Overall, these protein interaction studies shed light on how splicing factors
regulate the order of events in the large spliceosome complex.
PMID: 11290703 [PubMed - indexed for MEDLINE]
223: Development 2001 May;128(9):1697-707
Orc mutants arrest in metaphase with abnormally condensed chromosomes.
Pflumm MF, Botchan MR.
Department of Molecular and Cell Biology, University of California, Berkeley, CA
94720, USA.
The origin recognition complex (ORC) is a six subunit complex required for
eukaryotic DNA replication initiation and for silencing of the heterochromatic
mating type loci in Saccharomyces cerevisiae. Our discovery of the Drosophila
ORC complex concentrated in the centric heterochromatin of mitotic cells in the
early embryo and its interactions with heterochromatin protein 1 (HP-1) lead us
to speculate that ORC may play some general role in chromosomal folding. To
explore the role of ORC in chromosomal condensation, we have identified a mutant
of subunit 5 of the Drosophila melanogaster origin recognition complex (Orc5)
and have characterized the phenotypes of both the Orc5 and the previously
identified Orc2 mutant, k43. Both Orc mutants died at late larval stages and
surprisingly, despite a reduced number of S-phase cells, an increased fraction
of cells were also detected in mitosis. For this latter population of cells, Orc
mutants arrest in a defective metaphase with shorter and thicker chromosomes
that fail to align at the metaphase plate within a poorly assembled mitotic
spindle. In addition, sister chromatid cohesion was frequently lost. PCNA and
MCM4 mutants had similar phenotypes to Orc mutants. We propose that DNA
replication defects trigger the mitotic arrest, due to the fact that frequent
fragmentation was observed. Thus, cells have a mitotic checkpoint that senses
chromosome integrity. These studies also suggest that the density of functional
replication origins and completion of S phase are requirements for proper
chromosomal condensation.
PMID: 11290306 [PubMed - indexed for MEDLINE]
224: Proc Natl Acad Sci U S A 2001 Apr 10;98(8):4391-6
HDA2 and HDA3 are related proteins that interact with and are essential for the
activity of the yeast histone deacetylase HDA1.
Wu J, Carmen AA, Kobayashi R, Suka N, Grunstein M.
Department of Biological Chemistry, University of California School of Medicine
and the Molecular Biology Institute, Boyer Hall, University of California, Los
Angeles, CA 90095, USA.
Histone deacetylase HDA1, the prototype for the class II mammalian deacetylases,
is likely the catalytic subunit of the HDA1-containing complex that is involved
in TUP1-specific repression and global deacetylation in yeast. Although the
class I RPD3-like enzymatic complexes have been well characterized, little is
known about the identity and interactions of the factors that associate to form
the HDA1 complex. In this paper, we identify related HDA2 and HDA3 proteins that
are found in the HDA1 complex and show that HDA1 interacts with itself and with
the HDA2-HDA3 subcomplex to form a likely tetramer. These interactions are
necessary for catalytic activity because mutations in any of the three
components disrupt activity both in vitro and in vivo. In this respect the HDA1
complex differs from yeast RPD3, which has components such as SIN3 that are not
essential for activity in vitro, and yeast HOS3, which has intrinsic in vitro
activity as a homodimer in the absence of other subunits.
PMID: 11287668 [PubMed - indexed for MEDLINE]
225: Mol Cell Biol 2001 May;21(9):3144-58
Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid
cohesion.
Hanna JS, Kroll ES, Lundblad V, Spencer FA.
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School
of Medicine, Baltimore, Maryland 21205, USA.
CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both
exhibit genetic and physical ties to replication fork constituents. We find that
absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and
leads to a preanaphase accumulation of cells that depends on the spindle
assembly checkpoint. The physical and genetic interactions between CTF4, CTF18,
and core components of replication fork complexes observed in this study and
others suggest that both gene products act in association with the replication
fork to facilitate sister chromatid cohesion. We find that Ctf18p, an RFC1-like
protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p
is not a component of biochemically purified proliferating cell nuclear antigen
loading RF-C, suggesting the presence of a discrete complex containing Ctf18p,
Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of
the budding yeast polymerase kappa, encoded by TRF4, strongly supports a
hypothesis that the DNA replication machinery is required for proper sister
chromatid cohesion. Analogous to the polymerase switching role of the bacterial
and human RF-C complexes, we propose that budding yeast RF-C(CTF18) may be
involved in a polymerase switch event that facilities sister chromatid cohesion.
The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for
replication accessory proteins in this process.
PMID: 11287619 [PubMed - indexed for MEDLINE]
226: Am J Physiol Cell Physiol 2001 May;280(5):C1284-92
A store-operated nonselective cation channel in lymphocytes is activated
directly by Ca(2+) influx factor and diacylglycerol.
Su Z, Csutora P, Hunton D, Shoemaker RL, Marchase RB, Blalock JE.
Departments of Physiology and Biophysics, Schools of Medicine and Dentistry,
University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Agonist-receptor interactions at the plasma membrane often lead to activation of
store-operated channels (SOCs) in the plasma membrane, allowing for sustained
Ca(2+) influx. While Ca(2+) influx is important for many biological processes,
little is known about the types of SOCs, the nature of the depletion signal, or
how the SOCs are activated. We recently showed that in addition to the Ca(2+)
release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human
peripheral blood mononuclear cells express novel store-operated nonselective
cation channels that we termed Ca(2+) release-activated nonselective cation
(CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC
channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition,
CRANC channels in inside-out plasma membrane patches are directly activated upon
exposure of their cytoplasmic side to highly purified CIF preparations.
Furthermore, CRANC channels are also directly activated by diacylglycerol. These
results strongly suggest that the Ca(2+) store-depletion signal is a diffusible
molecule and that at least some SOCs may have dual activation mechanisms.
PMID: 11287342 [PubMed - indexed for MEDLINE]
227: J Cell Biol 2001 Apr 2;153(1):159-68
The surveillance mechanism of the spindle position checkpoint in yeast.
Adames NR, Oberle JR, Cooper JA.
Department of Cell Biology and Physiology, Washington University School of
Medicine, St. Louis, Missouri 63110, USA. nadames@cellbio.wustl.edu
The spindle position checkpoint in Saccharomyces cerevisiae delays mitotic exit
until the spindle has moved into the mother-bud neck, ensuring that each
daughter cell inherits a nucleus. The small G protein Tem1p is critical in
promoting mitotic exit and is concentrated at the spindle pole destined for the
bud. The presumed nucleotide exchange factor for Tem1p, Lte1p, is concentrated
in the bud. These findings suggested the hypothesis that movement of the spindle
pole through the neck allows Tem1p to interact with Lte1p, promoting GTP loading
of Tem1p and mitotic exit. However, we report that deletion of LTE1 had little
effect on the timing of mitotic exit. We also examined several mutants in which
some cells inappropriately exit mitosis even though the spindle is within the
mother. In some of these cells, the spindle pole body did not interact with the
bud or the neck before mitotic exit. Thus, some alternative mechanism must exist
to coordinate mitotic exit with spindle position. In both wild-type and mutant
cells, mitotic exit was preceded by loss of cytoplasmic microtubules from the
neck. Thus, the spindle position checkpoint may monitor such interactions.
PMID: 11285282 [PubMed - indexed for MEDLINE]
228: Nat Cell Biol 2001 Apr;3(4):384-91
Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase
assembly.
Seol JH, Shevchenko A, Shevchenko A, Deshaies RJ.
Division of Biology and Howard Hughes Medical Institute, California Institute of
Technology, Pasadena, California 91125, USA.
SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a
large family of substrate receptors known as F-box proteins (FBPs). Here we
report the identification, using sequential rounds of epitope tagging, affinity
purification and mass spectrometry, of 16 Skp1 and Cdc53-associated proteins in
budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202
(Rav2). Rav1, Rav2 and Skp1 form a complex that we have named 'regulator of the
(H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates
with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase). V-ATPases
are conserved throughout eukaryotes, and have been implicated in tumour
metastasis and multidrug resistance, and here we show that RAVE promotes
glucose-triggered assembly of the V-ATPase holoenzyme. Previous systematic
genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and
Cdc53, only 3 of which overlap with those reported here. Thus, our results
provide a distinct view of the interactions that link proteins into a
comprehensive cellular network.
PMID: 11283612 [PubMed - indexed for MEDLINE]
229: Proc Natl Acad Sci U S A 2001 Apr 10;98(8):4569-74
Comment in:
Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4277-8.
A comprehensive two-hybrid analysis to explore the yeast protein interactome.
Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, Sakaki Y.
Division of Genome Biology, Cancer Research Institute, Kanazawa University,
Kanazawa 920-0934, Japan. titolab@kenroku.kanazawa-u.ac.jp
Protein-protein interactions play crucial roles in the execution of various
biological functions. Accordingly, their comprehensive description would
contribute considerably to the functional interpretation of fully sequenced
genomes, which are flooded with novel genes of unpredictable functions. We
previously developed a system to examine two-hybrid interactions in all possible
combinations between the approximately 6,000 proteins of the budding yeast
Saccharomyces cerevisiae. Here we have completed the comprehensive analysis
using this system to identify 4,549 two-hybrid interactions among 3,278
proteins. Unexpectedly, these data do not largely overlap with those obtained by
the other project [Uetz, P., et al. (2000) Nature (London) 403, 623-627] and
hence have substantially expanded our knowledge on the protein interaction space
or interactome of the yeast. Cumulative connection of these binary interactions
generates a single huge network linking the vast majority of the proteins.
Bioinformatics-aided selection of biologically relevant interactions highlights
various intriguing subnetworks. They include, for instance, the one that had
successfully foreseen the involvement of a novel protein in spindle pole body
function as well as the one that may uncover a hitherto unidentified
multiprotein complex potentially participating in the process of vesicular
transport. Our data would thus significantly expand and improve the protein
interaction map for the exploration of genome functions that eventually leads to
thorough understanding of the cell as a molecular system.
PMID: 11283351 [PubMed - indexed for MEDLINE]
230: J Biol Chem 2001 May 18;276(20):17448-54
A-kinase-anchoring protein AKAP95 is targeted to the nuclear matrix and
associates with p68 RNA helicase.
Akileswaran L, Taraska JW, Sayer JA, Gettemy JM, Coghlan VM.
Neurological Sciences Institute, Oregon Health Sciences University, Beaverton,
Oregon 97006, USA.
The cell nucleus is structurally and functionally organized by the nuclear
matrix. We have examined whether the nuclear cAMP-dependent protein
kinase-anchoring protein AKAP95 contains specific signals for targeting to the
subnuclear compartment and for interaction with other proteins. AKAP95 was
expressed in mammalian cells and found to localize exclusively to the nuclear
matrix. Mutational analysis was used to identify determinants for nuclear
localization and nuclear matrix targeting of AKAP95. These sites were found to
be distinct from previously identified DNA and protein kinase A binding domains.
The nuclear matrix-targeting site is unique but conserved among members of the
AKAP95 family. Direct binding of AKAP95 to isolated nuclear matrix was
demonstrated in situ and found to be dependent on the nuclear matrix-targeting
site. Moreover, Far Western blot analysis identified at least three
AKAP95-binding proteins in nuclear matrix isolated from rat brain. Yeast
two-hybrid cloning identified one binding partner as p68 RNA helicase. The
helicase and AKAP95 co-localized in the nuclear matrix of mammalian cells,
associated in vitro, and were precipitated as a complex from solubilized cell
extracts. The results define novel protein-protein interactions among nuclear
matrix proteins and suggest a potential role of AKAP95 as a scaffold for
coordinating assembly of hormonally responsive transcription complexes.
PMID: 11279182 [PubMed - indexed for MEDLINE]
231: J Biol Chem 2001 May 18;276(20):17261-6
Structure-function analysis of the active site tunnel of yeast RNA
triphosphatase.
Bisaillon M, Shuman S.
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021,
USA.
Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus,
catalyzes metal-dependent gamma phosphate hydrolysis within the hydrophilic
interior of a topologically closed 8-strand beta barrel (the "triphosphate
tunnel"). We used structure-guided alanine scanning to identify 6 side chains
within the triphosphate tunnel that are essential for phosphohydrolase activity
in vitro and in vivo: Arg393, Glu433, Arg458, Arg469, Asp471 and Thr473. Alanine
substitutions at two positions, Asp377 and Lys409, resulted in partial catalytic
defects and a thermosensitive growth phenotype. Structure-function relationships
were clarified by introducing conservative substitutions. Five residues were
found to be nonessential: Lys309, Ser395, Asp397, Lys427 Asn431, and Lys474. The
present findings, together with earlier mutational analyses, reveal an unusually
complex active site in which 15 individual side chains in the tunnel cavity are
important for catalysis, and each of the 8 strands of the beta barrel
contributes at least one functional constituent. The active site residues fall
into three classes: (i) those that participate directly in catalysis via
coordination of the gamma phosphate or the metal; (ii) those that make critical
water-mediated contacts with the gamma phosphate or the metal; and (iii) those
that function indirectly via interactions with other essential side chains or by
stabilization of the tunnel structure.
PMID: 11279161 [PubMed - indexed for MEDLINE]
232: J Biol Chem 2001 May 4;276(18):14996-5002
Importance of homodimerization for the in vivo function of yeast RNA
triphosphatase.
Lehman K, Ho CK, Shuman S.
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021,
USA.
Saccharomyces cerevisiae RNA triphosphatase Cet1 is an essential component of
the yeast mRNA capping apparatus. The active site of Cet1 resides within a
topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that is
supported by a globular hydrophobic core. The homodimeric quaternary structure
of Cet1 is formed by a network of contacts between the partner protomers. By
studying the effects of alanine-cluster mutations, we highlight the
contributions of two separate facets of the crystallographic dimer interface to
Cet1 function in vivo. One essential facet of the interface entails hydrophobic
cross-dimer interactions of Cys(330) and Val(331) and a cross-dimer hydrogen
bond of Asp(280) with the backbone amide of Gln(329). The second functionally
relevant dimer interface involves hydrophobic side-chain interactions of
Phe(272) and Leu(273). Ala-cluster mutations involving these residues elicited
lethal or severe temperature-sensitive phenotypes that were suppressed
completely by fusion of the mutated triphosphatases to the guanylyltransferase
domain of mammalian capping enzyme. The recombinant D279A-D280A and F272A-L273A
proteins retained phosphohydrolase activity but sedimented as monomers. These
results indicate that a disruption of the dimer interface is uniquely
deleterious when the yeast RNA triphosphatase must function in concert with the
endogenous yeast guanylyltransferase. We also identify key residue pairs in the
hydrophobic core of the Cet1 protomer that support the active site tunnel and
stabilize the triphosphatase in vivo.
PMID: 11279098 [PubMed - indexed for MEDLINE]
233: J Biol Chem 2001 May 11;276(19):16207-15
DNA binding by the ETS-domain transcription factor PEA3 is regulated by
intramolecular and intermolecular protein.protein interactions.
Greenall A, Willingham N, Cheung E, Boam DS, Sharrocks AD.
School of Biochemistry and Genetics, The Medical School, University of Newcastle
Upon Tyne, Newcastle Upon Tyne NE2 4HH, United Kingdom.
The control of DNA binding by eukaryotic transcription factors represents an
important regulatory mechanism. Many transcription factors are controlled by
cis-acting autoinhibitory modules that are thought to act by blocking
promiscuous DNA binding in the absence of appropriate regulatory cues. Here, we
have investigated the determinants and regulation of the autoinhibitory
mechanism employed by the ETS-domain transcription factor, PEA3. DNA binding is
inhibited by a module composed of a combination of two short motifs located on
either side of the ETS DNA-binding domain. A second type of protein, Ids, can
act in trans to mimic the effect of these cis-acting inhibitory motifs and
reduce DNA binding by PEA3. By using a one-hybrid screen, we identified the
basic helix-loop-helix-leucine zipper transcription factor USF-1 as an
interaction partner for PEA3. PEA3 and USF-1 form DNA complexes in a cooperative
manner. Moreover, the formation of ternary PEA3.USF-1.DNA complexes requires
parts of the same motifs in PEA3 that form the autoinhibitory module. Thus the
binding of USF-1 to PEA3 acts as a switch that modifies the autoinhibitory
motifs in PEA3 to first relieve their inhibitory action, and second, promote
ternary nucleoprotein complex assembly.
PMID: 11278941 [PubMed - indexed for MEDLINE]
234: J Biol Chem 2001 Apr 13;276(15):12135-9
Biochemical characterization of Gyp6p, a Ypt/Rab-specific GTPase-activating
protein from yeast.
Will E, Gallwitz D.
Max Planck Institute for Biophysical Chemistry, Department of Molecular
Genetics, D-37070 Gottingen, Germany.
Gyp6p from yeast belongs to the GYP family of Ypt/Rab-specific GTPase-activating
proteins, and Ypt6p is its preferred substrate (Strom, M., Vollmer, P., Tan, T.
J., and Gallwitz, D. (1993) Nature 361, 736-739). We have investigated the
kinetic parameters of Gyp6p/Ypt6p interactions and find that Gyp6p accelerates
the intrinsic GTPase activity of Ypt6p (0.0002 min(-1)) by a factor of 5 x 10(6)
and that they have a very low affinity for its preferred substrate (K(m) = 592
micrometer). Substitution with alanine of several arginines, which Gyp6p shares
with other GYP family members, resulted in significant inhibition of GAP
activity. Replacement of arginine-155 with either alanine or lysine abolished
its GAP activity, indicating a direct involvement of this strictly conserved
arginine in catalysis. Physical interaction of the catalytically inactive
Gyp6(R155A) mutant GAP with Ypt6 wild-type and Ypt6 mutant proteins could be
demonstrated with the two-hybrid system. Short N-terminal and C-terminal
truncations of Gyp6p resulted in a complete loss of GAP activity and Ypt6p
binding, showing that in contrast to two other Gyp proteins studied previously,
most of the 458 amino acid-long Gyp6p sequence is required to form a
three-dimensional structure that allows substrate binding and catalysis.
PMID: 11278907 [PubMed - indexed for MEDLINE]
235: J Biol Chem 2001 May 11;276(19):16265-70
Helical stalk segments S4 and S5 of the plasma membrane H+-ATPase from
Saccharomyces cerevisiae are optimized to impact catalytic site environment.
Soteropoulos P, Valiakhmetov A, Kashiwazaki R, Perlin DS.
Public Health Research Institute, New York, New York 10016, USA.
The stalk segments of P-type ion-translocating enzymes are presumed to play
important roles in energy coupling. In this work, stalk segments S4 and S5 of
the yeast H(+)-ATPase were examined for helical character, optimal length, and
segment orientation by a combination of proline substitution, insertion/deletion
mutagenesis, and second-site suppressor analyses. The substitution of various
residues for helix-disrupting proline in both S4 (L353P,L353G; A354P; and G371P)
and S5 (D676P and I684P) resulted in highly defective or inactive enzymes
supporting the importance of helical character and/or the maintenance of
essential interactions. The contiguous helical nature of transmembrane segment
M5 and stalk element S5 was explored and found to be favorable, although not
essential. The deletion or addition of one or more amino acids at positions
Ala(354) in S4 and Asp(676) in S5, which were intended to either rotate helical
faces or extend/reduce the length of helical segments, resulted in enzyme
destabilization that abolished most enzyme assembly. Second-site suppressor
mutations were obtained to primary site mutations G371A (S4) and D676G (S5) and
were analyzed with a molecular structure model of the H(+)-ATPase. Primary site
mutations were predicted to alter the site of phosphorylation either directly or
indirectly. The suppressor mutations either directly changed packing around the
primary site or altered the environment of the site of phosphorylation. Overall,
these data support the view that stalk segments S4 and S5 of the H(+)-ATPase are
helical elements that are optimized for length and interactions with other stalk
elements and can influence the phosphorylation domain.
PMID: 11278840 [PubMed - indexed for MEDLINE]
236: J Biol Chem 2001 May 18;276(20):17524-32
J-domain protein, Jac1p, of yeast mitochondria required for iron homeostasis and
activity of Fe-S cluster proteins.
Kim R, Saxena S, Gordon DM, Pain D, Dancis A.
Department of Medicine, Division of Hematology-Oncology, University of
Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J-proteins are molecular chaperones with a characteristic domain predicted to
mediate interaction with Hsp70 proteins. We have previously isolated yeast
mutants of the mitochondrial Hsp70, Ssq1p, in a genetic screen for mutants with
altered iron homeostasis. Here we describe the isolation of mutants of the
J-domain protein, Jac1p, using the same screen. Mutant jac1 alleles predicted to
encode severely truncated proteins (lacking 70 or 152 amino acids) were
associated with phenotypes strikingly similar to the phenotypes of ssq1 mutants.
These phenotypes include activation of the high affinity cellular iron uptake
system and iron accumulation in mitochondria. In contrast to iron accumulation,
Fe-S proteins of mitochondria were specifically deficient. In jac1 mutants, like
in ssq1 mutants, processing of the Yfh1p precursor protein from intermediate to
mature forms was delayed. In the genetic backgrounds used in this study, jac1
null mutants were found to be viable, permitting analysis of genetic
interactions. The Deltajac1 Deltassq1 double mutant was more severely
compromised for growth than either single mutant, suggesting a synthetic or
additive effect of these mutations. Overexpression of Jac1p partially suppressed
ssq1 slow growth and vice versa. Similar mitochondrial localization and similar
mutant phenotypes suggest that Ssq1p and Jac1p are functional partners in iron
homeostasis.
PMID: 11278728 [PubMed - indexed for MEDLINE]
237: J Biol Chem 2001 May 11;276(19):15768-75
Delineation of functional regions within the subunits of the Saccharomyces
cerevisiae cell adhesion molecule a-agglutinin.
Shen ZM, Wang L, Pike J, Jue CK, Zhao H, de Nobel H, Kurjan J, Lipke PN.
Department of Biological Sciences and the Institute for Biomolecular Structure
and Function, Hunter College of the City University of New York, New York 10021,
USA.
a-Agglutinin from Saccharomyces cerevisiae is a cell adhesion glycoprotein
expressed on the surface of cells of a mating type and consists of an anchorage
subunit Aga1p and a receptor binding subunit Aga2p. Cell wall attachment of
Aga2p is mediated through two disulfide bonds to Aga1p (Cappellaro, C.,
Baldermann, C., Rachel, R., and Tanner, W. (1994) EMBO J. 13, 4737-4744). We
report here that purified Aga2p was unstable and had low molar specific activity
relative to its receptor alpha-agglutinin. Aga2p co-expressed with a 149-residue
fragment of Aga1p formed a disulfide-linked complex with specific activity
43-fold higher than Aga2p expressed alone. Circular dichroism of the complex
revealed a mixed alpha/beta structure, whereas Aga2p alone had no periodic
secondary structure. A 30-residue Cys-rich Aga1p fragment was partially active
in stabilization of Aga2p activity. Mutation of either or both Aga2p cysteine
residues eliminated stabilization of Aga2p. Thus the roles of Aga1p include both
cell wall anchorage and cysteine-dependent conformational restriction of the
binding subunit Aga2p. Mutagenesis of AGA2 identified only C-terminal residues
of Aga2p as being essential for binding activity. Aga2p residues 45-72 are
similar to sequences in soybean Nod genes, and include residues implicated in
interactions with both Aga1p (including Cys(68)) and alpha-agglutinin.
PMID: 11278672 [PubMed - indexed for MEDLINE]
238: J Biol Chem 2001 Apr 13;276(15):11980-7
Genetic analysis of the Escherichia coli FtsZ.ZipA interaction in the yeast
two-hybrid system. Characterization of FtsZ residues essential for the
interactions with ZipA and with FtsA.
Haney SA, Glasfeld E, Hale C, Keeney D, He Z, de Boer P.
Department of Infectious Disease, Wyeth-Ayerst Research, Pearl River, New York
10965 and the Department of Molecular Biology and Microbiology, Case Western
Reserve University Medical School, Cleveland, Ohio 44106-4960.
The recruitment of ZipA to the septum by FtsZ is an early, essential step in
cell division in Escherichia coli. We have used polymerase chain
reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze
this interaction and have identified residues within a highly conserved sequence
at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of
a mutation that causes a loss of interaction (ftsZ(D373G)) identified eight
different changes at two residues within this sequence. In vitro, wild type FtsZ
interacted with ZipA with a high affinity in an enzyme-linked immunosorbent
assay, whereas FtsZ(D373G) failed to interact. Two mutant proteins examined
restored this interaction significantly. In vivo, the alleles tested are
significantly more toxic than the wild type ftsZ and cannot complement a
deletion. We have shown that a fusion, which encodes the last 70 residues of
FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and
ZipA. However, when the wild type sequence is compared with one that encodes
FtsZ(D373G), no interaction was seen with either protein. Mutations surrounding
Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA,
indicating that these proteins bind the C terminus of FtsZ differently.
PMID: 11278571 [PubMed - indexed for MEDLINE]
239: J Biol Chem 2001 Apr 20;276(16):13256-63
Trax (translin-associated factor X), a primarily cytoplasmic protein, inhibits
the binding of TB-RBP (translin) to RNA.
Chennathukuzhi VM, Kurihara Y, Bray JD, Hecht NB.
Department of Obstetrics and Gynecology, Center for Research on Reproduction and
Women's Health, School of Medicine, University of Pennsylvania, Philadelphia,
Pennsylvania 19104-6142, USA.
Trax (Translin-associated factor X) has been shown to interact with
TB-RBP/Translin by its coimmunoprecipitation and in yeast two-hybrid assays.
Here we demonstrate that Trax is widely expressed, does not bind to DNA or RNA,
but forms heterodimers with TB-RBP under reducing conditions. The heterodimer of
TB-RBP and Trax inhibits TB-RBP binding to RNA, but enhances TB-RBP binding to
specific single stranded DNA sequences. The in vitro interactions between TB-RBP
and Trax are confirmed by similar interactions in the yeast two-hybrid system.
Cell fractionation and confocal microscope studies reveal that Trax is
predominantly cytoplasmic. In contrast, TB-RBP is present in both the nuclei and
cytoplasm of transfected cells and uses a highly conserved nuclear export signal
to exit nuclei. In addition to a leucine zipper, two basic domains in TB-RBP are
essential for RNA binding, but only one of these domains is needed for DNA
binding. Trax restores DNA binding to TB-RBP containing an altered form of this
domain. These data suggest that Trax-TB.RBP interactions modulate the DNA- and
RNA-binding activity of TB-RBP.
PMID: 11278549 [PubMed - indexed for MEDLINE]
240: J Biol Chem 2001 Jun 29;276(26):24212-22
The dimerization interface of the metastasis-associated protein S100A4 (Mts1):
in vivo and in vitro studies.
Tarabykina S, Scott DJ, Herzyk P, Hill TJ, Tame JR, Kriajevska M, Lafitte D,
Derrick PJ, Dodson GG, Maitland NJ, Lukanidin EM, Bronstein IB.
Department of Molecular Cancer Biology, Danish Cancer Society, Copenhagen
DK-2100, Denmark.
The S100 calcium-binding proteins are implicated in signal transduction,
motility, and cytoskeletal dynamics. The three-dimensional structure of several
S100 proteins revealed that the proteins form non-covalent dimers. However, the
mechanism of the S100 dimerization is still obscure. In this study we
characterized the dimerization of S100A4 (also named Mts1) in vitro and in vivo.
Analytical ultracentrifugation revealed that apoS100A4 was present in solution
as a mixture of monomers and dimers in a rapidly reversible equilibrium (K(d) =
4 +/- 2 microm). The binding of calcium promoted dimerization. Replacement of
Tyr-75 by Phe resulted in the stabilization of the dimer. Helix IV is known to
form the major part of the dimerization interface in homologous S100 proteins.
By using the yeast two-hybrid system we showed that only a few residues of helix
IV, namely Phe-72, Tyr-75, Phe-78, and Leu-79, are essential for dimerization in
vivo. A homology model demonstrated that these residues form a hydrophobic
cluster on helix IV. Their role is to stabilize the structure of individual
subunits rather than provide specific interactions across the dimerization
surface. Our mutation data showed that the specificity at the dimerization
surface is not particularly stringent, which is consistent with recent data
indicating that S100 proteins can form heterodimers.
PMID: 11278510 [PubMed - indexed for MEDLINE]
241: J Biol Chem 2001 Apr 20;276(16):13127-35
Gaf-1, a gamma -SNAP-binding protein associated with the mitochondria.
Chen D, Xu W, He P, Medrano EE, Whiteheart SW.
Department of Biochemistry, University of Kentucky College of Medicine,
Lexington, Kentucky 40536, USA.
The role of alpha/beta-SNAP (Soluble NSF Attachment Protein) in vesicular
trafficking is well established; however, the function of the ubiquitously
expressed gamma-SNAP remains unclear. To further characterize the cellular role
of this enigmatic protein, a two-hybrid screen was used to identify new,
gamma-SNAP-binding proteins and to uncover potentially novel functions for
gamma-SNAP. One such SNAP-binding protein, termed Gaf-1 (gamma-SNAP associate
factor-1) specifically binds gamma- but not alpha-SNAP. The full-length Gaf-1
(75 kDa) is ubiquitously expressed and is found stoichiometrically associated
with gamma-SNAP in cellular extracts. This binding is distinct from other SNAP
interactions since no alpha-SNAP or NSF coprecipitated with Gaf-1. Subcellular
fractionation and immunofluorescence analysis show that Gaf-1 is peripherally
associated with the outer mitochondrial membrane. Only a fraction of gamma-SNAP
was mitochondrial with the balance being either cytosolic or associated with
other membrane fractions. GFP-gamma-SNAP and the C-terminal domain of Gaf-1 both
show a reticular distribution in HEK-293 cells. This reticular structure
colocalizes with Gaf-1 and mitochondria as well as with microtubules but not
with other cytoskeletal elements. These data identify a class of gamma-SNAP
interactions that is distinct from other members of the SNAP family and point to
a potential role for gamma-SNAP in mitochondrial dynamics.
PMID: 11278501 [PubMed - indexed for MEDLINE]
242: Cell Physiol Biochem 2001;11(1):55-60
Determination of protein-protein interactions of ICIn by the yeast two-hybrid
system.
Schmarda A, Fresser F, Gschwentner M, Furst J, Ritter M, Lang F, Baier G,
Paulmichl M.
Department of Physiology, Institute for Medical Biology and Human Genetics,
University of Innsbruck, Austria.
ICln is a ubiquitously expressed eukaryotic protein. Expression of the protein
in Xenopus laevis oocytes, the knocking-down of the protein in fibroblasts, or
the reconstitution of the protein in lipid bilayer led to the assumption that
this protein is an ionic channel or a significant part thereof. However, other
possible roles for ICln in potential regulatory mechanisms have been postulated,
as diverse as regulator of cell morphology by interacting with the Skb1 protein
and/or interaction with core spliceosomal proteins. Here we show that ICln is
able to interact with SnRNP core proteins SmD1, SmD2, SmD3, SmX5 and SmB/B'.
PMID: 11275683 [PubMed - indexed for MEDLINE]
243: J Biol Chem 2001 Jun 15;276(24):21594-600
Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and
selectively with members of the membrane-associated guanylate kinase family of
PDZ (PSD95/Dlg/ZO-1) domain-containing proteins.
DeMarco SJ, Strehler EE.
Program in Molecular Neuroscience, Department of Biochemistry, Mayo Graduate
School, Mayo Clinic, Rochester, Minnesota 55905, USA.
Spatial and temporal regulation of intracellular Ca(2+) signaling depends on
localized Ca(2+) microdomains containing the requisite molecular components for
Ca(2+) influx, efflux, and signal transmission. Plasma membrane Ca(2+)-ATPase
(PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1)
interaction domains. The COOH-terminal tail of PMCA2b isolated the
membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding
partner in a yeast two-hybrid screen. The related MAGUKs SAP90/PSD95,
PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of
PMCA4b, whereas only the first three bound to the tail of PMCA2b.
Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and
SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other
SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and
colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized
Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was
abundant throughout the somatodendritic compartment and often extended into the
neck and head of individual spines where it colocalized with SAP90/PSD95. These
data show that PMCA "b" splice forms interact promiscuously but also with
specificity with different members of the PSD95 family of SAPs. PMCA-SAP
interactions may play a role in the recruitment and maintenance of the PMCA at
specific membrane domains involved in local Ca(2+) regulation.
PMID: 11274188 [PubMed - indexed for MEDLINE]
244: J Mol Biol 2001 Mar 30;307(3):929-38
Mapping protein family interactions: intramolecular and intermolecular protein
family interaction repertoires in the PDB and yeast.
Park J, Lappe M, Teichmann SA.
European Bioinformatics Institute, Hinxton, Cambridgeshire, CB10 1SD, UK.
In the postgenomic era, one of the most interesting and important challenges is
to understand protein interactions on a large scale. The physical interactions
between protein domains are fundamental to the workings of a cell: in
multi-domain polypeptide chains, in multi-subunit proteins and in transient
complexes between proteins that also exist independently. To study the
large-scale patterns and evolution of interactions between protein domains, we
view interactions between protein domains in terms of the interactions between
structural families of evolutionarily related domains. This allows us to
classify 8151 interactions between individual domains in the Protein Data Bank
and the yeast Saccharomyces cerevisiae in terms of 664 types of interactions,
between protein families. At least 51 interactions do not occur in the Protein
Data Bank and can only be derived from the yeast data. The map of interactions
between protein families has the form of a scale-free network, meaning that most
protein families only interact with one or two other families, while a few
families are extremely versatile in their interactions and are connected to many
families. We observe that almost half of all known families engage in
interactions with domains from their own family. We also see that the
repertoires of interactions of domains within and between polypeptide chains
overlap mostly for two specific types of protein families: enzymes and
same-family interactions. This suggests that different types of protein
interaction repertoires exist for structural, functional and regulatory reasons.
Copyright 12001 Academic Press.
PMID: 11273711 [PubMed - indexed for MEDLINE]
245: Protein Sci 2001 Jan;10(1):12-6
Second virial coefficients as a measure of protein--osmolyte interactions.
Weatherly GT, Pielak GJ.
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel
Hill, North Carolina 27599, USA.
The cytoplasm contains high concentrations of cosolutes. These cosolutes include
macromolecules and small organic molecules called osmolytes. However, most
biophysical studies of proteins are conducted in dilute solutions. Two broad
classes of models have been used to describe the interaction between osmolytes
and proteins. One class focuses on excluded volume effects, while the other
focuses on binding between the protein and the osmolyte. To better understand
protein--smolyte interactions, we have conducted sedimentation equilibrium
analytical ultracentrifugation experiments using ferricytochrome c as a model
protein. From these experiments, we determined the second virial coefficients
for a series of osmolytes. We have interpreted the second virial coefficient as
a measure of both excluded volume and protein--osmolyte binding. We conclude
that simple models are not sufficient to understand the interactions between
osmolytes and proteins.
PMID: 11266589 [PubMed - indexed for MEDLINE]
246: Nucleic Acids Res 2001 Apr 1;29(7):1524-33
Transcriptional adaptor and histone acetyltransferase proteins in Arabidopsis
and their interactions with CBF1, a transcriptional activator involved in
cold-regulated gene expression.
Stockinger EJ, Mao Y, Regier MK, Triezenberg SJ, Thomashow MF.
Department of Crop and Soil Sciences and Department of Biochemistry, Michigan
State University, East Lansing, MI 48824, USA.
The ARABIDOPSIS CBF transcriptional activators bind to the CRT/DRE regulatory
element present in the promoters of many cold-regulated genes and stimulate
their transcription. Expression of the CBF1 proteins in yeast activates reporter
genes carrying a minimal promoter with the CRT/DRE as an upstream regulatory
element. Here we report that this ability of CBF1 is dependent upon the
activities of three key components of the yeast Ada and SAGA complexes, namely
the histone acetyltransferase (HAT) Gcn5 and the transcriptional adaptor
proteins Ada2 and Ada3. This result suggested that CBF1 might function through
the action of similar complexes in ARABIDOPSIS In support of this hypothesis we
found that ARABIDOPSIS has a homolog of the GCN5 gene and two homologs of ADA2,
the first report of multiple ADA2 genes in an organism. The ARABIDOPSIS GCN5
protein has intrinsic HAT activity and can physically interact in vitro with
both the ARABIDOPSIS ADA2a and ADA2b proteins. In addition, the CBF1
transcriptional activator can interact with the ARABIDOPSIS GCN5 and ADA2
proteins. We conclude that ARABIDOPSIS encodes HAT-containing adaptor complexes
that are related to the Ada and SAGA complexes of yeast and propose that the
CBF1 transcriptional activator functions through the action of one or more of
these complexes.
PMID: 11266554 [PubMed - indexed for MEDLINE]
247: Nucleic Acids Res 2001 Apr 1;29(7):1410-9
All Tcf HMG box transcription factors interact with Groucho-related
co-repressors.
Brantjes H, Roose J, van De Wetering M, Clevers H.
Department of Immunology, University Medical Center Utrecht, PO Box 85500, 3508
GA, Utrecht, The Netherlands.
Tcf/Lef family transcription factors are the downstream effectors of the
Wingless/Wnt signal transduction pathway. Upon Wingless/Wnt signalling,
beta-catenin translocates to the nucleus, interacts with Tcf (1-3) and thus
activates transcription of target genes (4,5). Tcf factors also interact with
members of the Groucho (Grg/TLE) family of transcriptional co-repressors (6). We
have now tested all known mammalian Groucho family members for their ability to
interact specifically with individual Tcf/Lef family members. Transcriptional
activation by any Tcf could be repressed by Grg-1, Grg-2/TLE-2, Grg-3 and Grg-4
in a reporter assay. Specific interactions between Tcf and Grg proteins may be
achieved in vivo by tissue- or cell type-limited expression. To address this, we
determined the expression of all Tcf and Grg/TLE family members in a panel of
cell lines. Within any cell line, several Tcfs and TLEs are co-expressed. Thus,
redundancy in Tcf/Grg interactions appears to be the rule. The 'long' Groucho
family members containing five domains are repressors of Tcf-mediated
transactivation, whereas Grg-5, which only contains the first two domains, acts
as a de-repressor. As previously shown for DROSOPHILA: Groucho, we show that
long Grg proteins interact with histone deacetylase-1. Although Grg-5 contains
the GP homology domain that mediates HDAC binding in long Grg proteins, Grg-5
fails to bind this co-repressor, explaining how it can de-repress transcription.
PMID: 11266540 [PubMed - indexed for MEDLINE]
248: Pac Symp Biocomput 2001;:115-26
SAMIE: statistical algorithm for modeling interaction energies.
Benos PV, Lapedes AS, Fields DS, Stormo GD.
Dept. of Genetics, Campus Box 8232, Medical School, Washington University, 4566
Scott Ave., St. Louis, MO 63110, USA. benos@genetics.wustl.edu
We are investigating the rules that govern protein-DNA interactions, using a
statistical mechanics based formalism that is related to the Boltzmann Machine
of the neural net literature. Our approach is data-driven, in which
probabilistic algorithms are used to model protein-DNA interactions, given SELEX
and/or phage data as input. In the current report, we trained the network using
SELEX data, under the "one-to-one" model of interactions (i.e. one amino acid
contacts one base). The trained network was able to successfully identify the
wild-type binding sites of EGR and MIG protein families. The predictions using
our method are the same or better than that of methods existing in the
literature. However our methodology offers the potential to capitalise in
quantitative detail, as well as to be used to explore more general model of
interactions, given availability of data.
PMID: 11262933 [PubMed - indexed for MEDLINE]
249: Mol Cell Biol 2001 Apr;21(7):2337-48
Protein import channel of the outer mitochondrial membrane: a highly stable
Tom40-Tom22 core structure differentially interacts with preproteins, small tom
proteins, and import receptors.
Meisinger C, Ryan MT, Hill K, Model K, Lim JH, Sickmann A, Muller H, Meyer HE,
Wagner R, Pfanner N.
Institut fur Biochemie und Molekularbiologie, Universitat Freiburg, Germany.
The preprotein translocase of the yeast mitochondrial outer membrane (TOM)
consists of the initial import receptors Tom70 and Tom20 and a approximately
400-kDa (400 K) general import pore (GIP) complex that includes the central
receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6,
and Tom5. We report that the GIP complex is a highly stable complex with an
unusual resistance to urea and alkaline pH. Under mild conditions for
mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively
associated with the GIP complex, forming a 500K to 600K TOM complex. A
preprotein, stably arrested in the GIP complex, is released by urea but not high
salt, indicating that ionic interactions are not essential for keeping the
preprotein in the GIP complex. Under more stringent detergent conditions,
however, Tom20 and all three small Tom proteins are released, while the
preprotein remains in the GIP complex. Moreover, purified outer membrane
vesicles devoid of translocase components of the intermembrane space and inner
membrane efficiently accumulate the preprotein in the GIP complex. Together,
Tom40 and Tom22 thus represent the functional core unit that stably holds
accumulated preproteins. The GIP complex isolated from outer membranes exhibits
characteristic TOM channel activity with two coupled conductance states, each
corresponding to the activity of purified Tom40, suggesting that the complex
contains two simultaneously active and coupled channel pores.
PMID: 11259583 [PubMed - indexed for MEDLINE]
250: Mol Cell Biol 2001 Apr;21(7):2281-91
Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide
excision repair, without a preassembled repairosome.
Araujo SJ, Nigg EA, Wood RD.
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms,
Hertfordshire EN6 3LD, United Kingdom.
In mammalian cells, the core factors involved in the damage recognition and
incision steps of DNA nucleotide excision repair are XPA, TFIIH complex,
XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF. Many interactions
between these components have been detected, using different physical methods,
in human cells and for the homologous factors in Saccharomyces cerevisiae.
Several human nucleotide excision repair (NER) complexes, including a
high-molecular-mass repairosome complex, have been proposed. However, there have
been no measurements of activity of any mammalian NER protein complex isolated
under native conditions. In order to assess relative strengths of interactions
between NER factors, we captured TFIIH from cell extracts with an anti-cdk7
antibody, retaining TFIIH in active form attached to magnetic beads.
Coimmunoprecipitation of other NER proteins was then monitored functionally in a
reconstituted repair system with purified proteins. We found that all detectable
TFIIH in gently prepared human cell extracts was present in the intact
nine-subunit form. There was no evidence for a repair complex that contained all
of the NER components. At low ionic strength TFIIH could associate with
functional amounts of each NER factor except RPA. At physiological ionic
strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not
other repair factors. The strongest interaction was between TFIIH and XPC-HR23B,
indicating a coupled role of these proteins in early steps of repair. A panel of
antibodies was used to estimate that there are on the order of 10(5) molecules
of each core NER factor per HeLa cell.
PMID: 11259578 [PubMed - indexed for MEDLINE]
251: Infect Immun 2001 Apr;69(4):2037-44
Interactions of surfactant proteins A and D with Saccharomyces cerevisiae and
Aspergillus fumigatus.
Allen MJ, Voelker DR, Mason RJ.
Department of Medicine, National Jewish Medical and Research Center, Denver,
Colorado 80206, USA.
Surfactant proteins A (SP-A) and D (SP-D) are members of the collectin family of
calcium-dependent lectins and are important pulmonary host defense molecules.
Human SP-A and SP-D and rat SP-D bind to Aspergillus fumigatus conidia, but the
ligand remains unidentified. To identify a fungal ligand for SP-A and/or SP-D,
we examined the interactions of the proteins with Saccharomyces cerevisiae. SP-D
but not SP-A bound yeast cells, and EDTA inhibited the binding. SP-D also
aggregated yeast cells and isolated yeast cell walls. Treating yeast cells to
remove cell wall mannoprotein did not reduce SP-D binding, and SP-D failed to
aggregate chitin. However, SP-D aggregated yeast glucan before and after
treatment with a beta(1-->3)-glucanase, suggesting a specific interaction
between the collectin and beta(1-->6)-glucan. In support of this idea,
SP-D-induced yeast aggregation was strongly inhibited by pustulan [a
beta(1-->6)-linked glucose homopolymer] but was not inhibited by laminarin [a
beta(1-->3)-linked glucose homopolymer]. Additionally, pustulan but not
laminarin strongly inhibited SP-D binding to A. fumigatus. The pustulan
concentration for 50% inhibition of SP-D binding to A. fumigatus is 1.0 +/- 0.3
microM glucose equivalents. Finally, SP-D showed reduced binding to the
beta(1-->6)-glucan-deficient kre6 yeast mutant. Taken together, these
observations demonstrate that beta(1-->6)-glucan is an important fungal ligand
for SP-D and that glycosidic bond patterns alone can determine if an extended
carbohydrate polymer is recognized by SP-D.
PMID: 11254556 [PubMed - indexed for MEDLINE]
252: Mol Gen Genet 2001 Feb;264(6):842-51
A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of
global genome nucleotide excision repair.
Wong JM, Ingles CJ.
Banting and Best Department of Medical Research, University of Toronto, Ontario,
Canada.
Nucleotide excision repair is the major pathway responsible for removing
UV-induced DNA damage, and is therefore essential for cell survival following
exposure to UV radiation. In this report, we have assessed the contributions of
some components of the RNA polymerase II (Pol II) transcription machinery to UV
resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II
elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in
the absence of global genome repair dependent on the RAD7 and RAD16 genes, a
result seen previously with deletions of RAD26 and RAD28, yeast homologs of the
human Cockayne syndrome genes CSB and CSA, respectively. A RAD7/16-dependent
reduction in survival after UV irradiation was also seen in the presence of
mutations in RNA Pol II that confer a defect in its response to SII, as well as
with other mutations which reside in regions of the largest subunit of Pol II
not involved in SII interactions. Indeed, an increase in UV sensitivity was
achieved by simply decreasing the steadystate level of RNA Pol II. Truncation of
the C-terminal domain and other RNA Pol II mutations conferred sensitivity to
the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and
RNR2 mRNAs after UV irradiation was attenuated in these mutant cells. That UV
sensitivity can be a consequence of mutations in the RNA Pol II machinery in
yeast cells suggests that alterations in transcriptional programs could underlie
some of the pathophysiological defects seen in the human disease Cockayne
syndrome.
PMID: 11254132 [PubMed - indexed for MEDLINE]
253: Biotechniques 2001 Mar;30(3):634-6, 638, 640 passim
Redefinition of the yeast two-hybrid system in dialogue with changing priorities
in biological research.
Serebriiskii IG, Khazak V, Golemis EA.
Fox Chase Cancer Center, Philadelphia, PA, USA.
Examination of the pattern of reagent creation and application in the two-hybrid
system since 1989 reveals the expansion of a simple core technology to address
increasingly sophisticated problems in protein interaction. As the technology
has matured, its clear suitability for large-scale proteomic projects has made a
major focus of its application the generation of global organismal protein
interaction networks. In an inversion of emphasis, the increasing availability
of such information now provides a master plan with the potential to specify the
most promising directions for biological investigations (i.e., by directing the
physiological validation of predicted critical protein-protein interactions).
Recent derivatives of the two-hybrid system enable the targeting of such key
interactions by facilitating the identification of essential amino acids
conferring protein interaction specificity and of small molecules that
selectively disrupt defined interaction pairs. Finally, the creation of
mammalian expression systems based on two-hybrid principles became a new tool to
create and probe novel biological systems. Taken in sum, this trajectory
emphasizes the point that the creation of tools and the evolution of the idea of
what is an interesting biological problem are in intimate dialogue.
Publication Types:
Review
Review, Tutorial
PMID: 11252799 [PubMed - indexed for MEDLINE]
254: Mol Biol Cell 2001 Mar;12(3):521-38
Selective formation of Sed5p-containing SNARE complexes is mediated by
combinatorial binding interactions.
Tsui MM, Tai WC, Banfield DK.
Department of Biology, The Hong Kong University of Science and Technology,
Clearwater Bay, Kowloon, Hong Kong, China.
Sed5p is the only syntaxin family member required for protein transport through
the yeast Golgi and it is known to bind up to nine other soluble
N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in vivo.
We describe in vitro binding experiments in which we identify ternary and
quaternary Sed5p-containing SNARE complexes. The formation of SNARE complexes
among these endoplasmic reticulum- and Golgi-localized proteins requires Sed5p
and is syntaxin-selective. In addition, Sed5p-containing SNARE complexes form
selectively and this selectivity is mediated by Sed5p-containing intermediates
that discriminate among subsequent binding partners. Although many of these
SNAREs have overlapping distributions in vivo, the SNAREs that form complexes
with Sed5p in vitro reflect their functionally distinct locales. Although
SNARE-SNARE interactions are promiscuous and a single SNARE protein is often
found in more than one complex, both the biochemical as well as genetic analyses
reported here suggest that this is not a result of nonselective direct
substitution of one SNARE for another. Rather our data are consistent with the
existence of multiple (perhaps parallel) trafficking pathways where
Sed5p-containing SNARE complexes play overlapping and/or distinct functional
roles.
PMID: 11251068 [PubMed - indexed for MEDLINE]
255: J Bacteriol 2001 Apr;183(7):2306-15
Functional domains of yeast plasmid-encoded Rep proteins.
Sengupta A, Blomqvist K, Pickett AJ, Zhang Y, Chew JS, Dobson MJ.
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie
University, Halifax, Nova Scotia, Canada B3H 4H7.
Both of the Saccharomyces cerevisiae 2 microm circle-encoded Rep1 and Rep2
proteins are required for efficient distribution of the plasmid to daughter
cells during cellular division. In this study two-hybrid and in vitro protein
interaction assays demonstrate that the first 129 amino acids of Rep1 are
sufficient for self-association and for interaction with Rep2. Deletion of the
first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still
allowed some self-association, suggesting that different but overlapping domains
specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion
proteins were unable to complement defective segregation of a 2 microm-based
stability vector with rep1 deleted, supporting the idea of the requirement of
Rep protein interaction for plasmid segregation but indicating a separate
required function for the carboxy-terminal portion of Rep1. The results of in
vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both
of which are capable of mediating Rep2 self-association. The amino-terminal
domain interacts with Rep1, while the carboxy-terminal domain was shown by
Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and
Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1,
Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2
microm circle plasmid stability locus, forming a structure that mediates plasmid
segregation. In this model, competition between Rep1 and Rep2 for association
with Rep1 determines the formation or disassembly of the segregation complex.
PMID: 11244071 [PubMed - indexed for MEDLINE]
256: Cell 2001 Feb 9;104(3):397-408
Comment in:
Cell. 2001 Feb 9;104(3):329-32.
Suppression of spontaneous chromosomal rearrangements by S phase checkpoint
functions in Saccharomyces cerevisiae.
Myung K, Datta A, Kolodner RD.
Ludwig Institute for Cancer Research, Cancer Center and Department of Medicine,
University of California-San Diego School of Medicine, La Jolla, CA 92093, USA.
Cancer cells show increased genome rearrangements, although it is unclear what
defects cause these rearrangements. Mutations in Saccharomyces cerevisiae RFC5,
DPB11, MEC1, DDC2 MEC3, RAD53, CHK1, PDS1, and DUN1 increased the rate of genome
rearrangements up to 200-fold whereas mutations in RAD9, RAD17, RAD24, BUB3, and
MAD3 had little effect. The rearrangements were primarily deletion of a portion
of a chromosome arm along with TEL1-dependent addition of a new telomere. tel1
mutations increased the proportion of translocations observed, and in some cases
showed synergistic interactions when combined with mutations that increased the
genome rearrangement rate. These data suggest that one role of S phase
checkpoint functions in normal cells is to suppress spontaneous genome
rearrangements resulting from DNA replication errors.
PMID: 11239397 [PubMed - indexed for MEDLINE]
257: Mol Cell Biol 2001 Mar;21(6):2098-106
Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays:
analyses using recombinant yeast histones and immobilized templates.
Gelbart ME, Rechsteiner T, Richmond TJ, Tsukiyama T.
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle,
Washington 98109-1024, USA.
To facilitate the biochemical characterization of chromatin-associated proteins
in the budding yeast Saccharomyces cerevisiae, we have developed a system to
assemble nucleosomal arrays on immobilized templates using recombinant yeast
core histones. This system enabled us to analyze the interaction of Isw2
ATP-dependent chromatin remodeling complex with nucleosomal arrays. We found
that Isw2 complex interacts efficiently with both naked DNA and nucleosomal
arrays in an ATP-independent manner, suggesting that ATP is required at steps
subsequent to this physical interaction. We identified the second subunit of
Isw2 complex, encoded by open reading frame YGL 133w (herein named ITC1), and
found that both subunits of the complex, Isw2p and Itc1p, are essential for
efficient interaction with DNA and nucleosomal arrays. Both subunits are also
required for nucleosome-stimulated ATPase activity and chromatin remodeling
activity of the complex. Finally, we found that ITC1 is essential for function
of Isw2 complex in vivo, since isw2 and itc1 deletion mutants exhibit virtually
identical phenotypes. These results demonstrate the utility of our in vitro
system in studying interactions between chromatin-associated proteins and
nucleosomal arrays.
PMID: 11238944 [PubMed - indexed for MEDLINE]
258: Mol Cell Biol 2001 Mar;21(6):2026-37
Fip1 regulates the activity of Poly(A) polymerase through multiple interactions.
Helmling S, Zhelkovsky A, Moore CL.
Department of Biochemistry, Tufts University, School of Medicine, Boston,
Massachusetts 02111, USA.
Fip1 is an essential component of the Saccharomyces cerevisiae polyadenylation
machinery and the only protein known to interact directly with poly(A)
polymerase (Pap1). Its association with Pap1 inhibits the extension of an
oligo(A) primer by limiting access of the RNA substrate to the C-terminal RNA
binding domain (C-RBD) of Pap1. We present here the identification of separate
functional domains of Fip1. Amino acids 80 to 105 are required for binding to
Pap1 and for the inhibition of Pap1 activity. This region is also essential for
viability, suggesting that Fip1-mediated repression of Pap1 has a crucial
physiological function. Amino acids 206 to 220 of Fip1 are needed for the
interaction with the Yth1 subunit of the complex and for specific
polyadenylation of the cleaved mRNA precursor. A third domain within amino acids
105 to 206 helps to limit RNA binding at the C-RBD of Pap1. Our data demonstrate
that the C terminus of Fip1 is required to relieve the Fip1-mediated repression
of Pap1 in specific polyadenylation. In the absence of this domain, Pap1 remains
in an inhibited state. These findings show that Fip1 has a crucial regulatory
function in the polyadenylation reaction by controlling the activity of poly(A)
tail synthesis through multiple interactions within the polyadenylation complex.
PMID: 11238938 [PubMed - indexed for MEDLINE]
259: J Virol 2001 Apr;75(7):3207-19
Brome mosaic virus Protein 1a recruits viral RNA2 to RNA replication through a
5' proximal RNA2 signal.
Chen J, Noueiry A, Ahlquist P.
Institute for Molecular Virology, University of Wisconsin-Madison, Madison,
Wisconsin 53706, USA.
Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like
superfamily, encodes two RNA replication factors. Membrane-associated 1a protein
contains a helicase-like domain and RNA capping functions. 2a, which is targeted
to membranes by 1a, contains a central polymerase-like domain. In the absence of
2a and RNA replication, 1a acts through an intergenic replication signal in BMV
genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular
membrane. Multiple results imply that 1a-induced RNA3 stabilization reflects
interactions involved in recruiting RNA3 templates into replication. To
determine if 1a had similar effects on another BMV RNA replication template, we
constructed a plasmid expressing BMV genomic RNA2 in vivo. In vivo-expressed
RNA2 templates were replicated upon expression of 1a and 2a. In the absence of
2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane. Deletion
analysis demonstrated that 1a-induced membrane association of RNA2 was mediated
by sequences in the 5'-proximal third of RNA2. The RNA2 5' untranslated region
was sufficient to confer 1a-induced membrane association on a nonviral RNA.
However, sequences in the N-terminal region of the 2a open reading frame
enhanced 1a responsiveness of RNA2 and a chimeric RNA. A 5'-terminal RNA2
stem-loop important for RNA2 replication was essential for 1a-induced membrane
association of RNA2 and, like the 1a-responsive RNA3 intergenic region,
contained a required box B motif corresponding to the TPsiC stem-loop of host
tRNAs. The level of 1a-induced membrane association of various RNA2 mutants
correlated well with their abilities to serve as replication templates. These
results support and expand the conclusion that 1a-induced BMV RNA stabilization
and membrane association reflect early, 1a-mediated steps in viral RNA
replication.
PMID: 11238847 [PubMed - indexed for MEDLINE]
260: Genetics 2001 Mar;157(3):1179-89
The Saccharomyces cerevisiae MUM2 gene interacts with the DNA replication
machinery and is required for meiotic levels of double strand breaks.
Davis L, Barbera M, McDonnell A, McIntyre K, Sternglanz R, Jin Q, Loidl J,
Engebrecht J.
Department of Pharmacological Sciences, Graduate Program in Genetics, State
University of New York, Stony Brook, New York 11794-8651, USA.
The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not
mitotic, DNA replication and thus sporulation. Genetic interactions between MUM2
and a component of the origin recognition complex and polymerase alpha-primase
suggest that MUM2 influences the function of the DNA replication machinery.
Early meiotic gene expression is induced to a much greater extent in mum2 cells
than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea. This
result indicates that the mum2 meiotic arrest is downstream of the arrest
induced by hydroxyurea and suggests that DNA synthesis is initiated in the
mutant. Genetic analyses indicate that the recombination that occurs in mum2
mutants is dependent on the normal recombination machinery and on synaptonemal
complex components and therefore is not a consequence of lesions created by
incompletely replicated DNA. Both meiotic ectopic and allelic recombination are
similarly reduced in the mum2 mutant, and the levels are consistent with the
levels of meiosis-specific DSBs that are generated. Cytological analyses of mum2
mutants show that chromosome pairing and synapsis occur, although at reduced
levels compared to wild type. Given the near-wild-type levels of meiotic gene
expression, pairing, and synapsis, we suggest that the reduction in DNA
replication is directly responsible for the reduced level of DSBs and meiotic
recombination.
PMID: 11238403 [PubMed - indexed for MEDLINE]
261: Genetics 2001 Mar;157(3):1107-16
Genes encoding ribosomal proteins Rps0A/B of Saccharomyces cerevisiae interact
with TOM1 mutants defective in ribosome synthesis.
Tabb AL, Utsugi T, Wooten-Kee CR, Sasaki T, Edling SA, Gump W, Kikuchi Y, Ellis
SR.
Department of Biochemistry and Molecular Biology, University of Louisville,
Louisville, Kentucky 40292, USA.
The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal
subunit that are required for the maturation of 18S rRNA. We show here that the
RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the
hect-domain-containing E3 ubiquitin-protein ligase family that is required for
growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have
synergistic effects on cell growth at temperatures permissive for TOM1 mutants.
Moreover, the growth arrest of TOM1 mutants at elevated temperatures is
partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles
of TOM1 are defective in multiple steps in rRNA processing, and interactions
between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of
ribosomal subunits. Ribosome synthesis is therefore included among the cellular
processes governed by members of the hect-domain-containing E3 ubiquitin-protein
ligase family.
PMID: 11238398 [PubMed - indexed for MEDLINE]
262: Biochem J 2001 Mar 15;354(Pt 3):655-61
Identification of Cdc6 protein domains involved in interaction with Mcm2 protein
and Cdc4 protein in budding yeast cells.
Jang SW, Elsasser S, Campbell JL, Kim J.
Graduate School of Biotechnology, Department of Genetic Engineering, Kyung Hee
University, Yongin, Kyonggi-Do, 449-701, Korea.
The Cdc6 protein (Cdc6p) has essential roles in regulating initiation of DNA
replication. Cdc6p is recruited to origins of replication by the origin
recognition complex (ORC) late in mitosis; Cdc6p in turn recruits minichromosome
maintenance (Mcm) proteins to form the pre-replicative complex. Cdc6p is thought
to interact with one or more Mcm proteins but this point has not yet been
demonstrated. In the present study we observed that Cdc6p interacted
significantly only with Mcm2p out of six Mcm proteins in yeast two-hybrid cells.
Our results indicate that the interaction of Cdc6p with Mcm2p is specific,
although we cannot exclude the possibility that the interaction might not be
direct. In attempts to identify domains of Cdc6p important for interaction with
Mcm2p, we tested interactions of various deleted versions of Cdc6p with Mcm2p
and also with Cdc4p, which was previously known to interact with Cdc6p. The
portion of Cdc6p from amino acid residues 51 to 394 was able to interact with
Mcm2p. During the course of the studies we also discovered a previously
undetected Cdc4p interaction domain between residues 51 and 394. Interestingly,
when all six putative Cdc28 phosphorylation sites in Cdc6p were changed to
alanine, a 6-7-fold increase in binding to Mcm2p was observed. This result
suggests that unphosphorylated Cdc6p has higher affinity than phosphorylated
Cdc6p for Mcm2p; this might partly explain the previous observation that Cdc6p
failed to load Mcm proteins on replication origins during S phase when the
cyclin-dependent protein kinase was active, thus helping to prevent the
reinitiation of activated replicons.
PMID: 11237870 [PubMed - indexed for MEDLINE]
263: J Mol Biol 2001 Mar 9;306(5):903-13
Specific interactions of the telomeric protein Rap1p with nucleosomal binding
sites.
Rossetti L, Cacchione S, De Menna A, Chapman L, Rhodes D, Savino M.
Dipartimento di Genetica e Biologia Molecolare, Fondazione Istituto Pasteur
-Fondazione Cenci Bolognetti, Universita di Roma La Sapienza, Piazzale A Moro
5,00185, Roma, Italy.
The telomeres of Saccharomyces cerevisiae are structurally and functionally well
characterized. Their telomeric DNA is packaged by the protein Rap1p (repressor
activator protein 1). Rap1p is a multifunctional, sequence-specific, DNA-binding
protein which, besides participating in the regulation of telomeres structure
and length, is also involved in transcriptional regulation of genes essential
for cell growth and in silencing. Whereas the long tracts of telomeric DNA
repeats of higher eukaryotes are mostly organized in closely spaced canonical
nucleosomal arrays, it has been proposed that the 300 base-pairs of S.
cerevisiae telomeric DNA are organized in a large non-nucleosomal structure that
has been called the telosome. Recently, nucleosomes have been found also in
Tetrahymena thermophila telomeres, suggesting that, in general, telomere
structural differences between lower and higher eukaryotes could be
quantitative, rather than qualitative. Using an in vitro model system, we have
addressed the question of whether Rap1p can form a stable ternary complex with
nucleosomes containing telomeric binding sites, or competes with nucleosome core
formation. The approach we have taken is to place a single Rap1p-binding site at
different positions within a nucleosome core and then test the binding of Rap1p
and its DNA-binding domain (Rap1p-DBD). We show here that both proteins are able
to specifically recognize their nucleosomal binding site, but that binding is
dependent on the location of the site within the nucleosome core structure.
These results show that a ternary complex between a nucleosome and Rap1p is
stable and could be a possible intermediate between telomeric nucleosomes and
telosomes in the dynamics of S. cerevisiae telomere organization.
PMID: 11237607 [PubMed - indexed for MEDLINE]
264: Biochem Cell Biol 2001;79(1):83-91
A tale of two charges: distinct roles for an acidic and a basic amino acid in
the structure and function of cytochrome c.
Parrish JC, Guillemette JG, Wallace CJ.
Department of Biochemistry, Dalhousie University, Halifax, NS, Canada.
Cytochrome c is a small electron transport protein found in the intermembrane
space of mitochondria. As it interacts with a number of different physiological
partners in a specific fashion, its structure varies little over eukaryotic
evolutionary history. Two highly conserved residues found within its sequence
are those at positions 13 and 90 (numbering is based on the standard horse
cytochrome c); with single exceptions, residue 13 is either Lys or Arg, and
residue 90 is either Glu or Asp. There have been conflicting views on the roles
to be ascribed to these residues, particularly residue 13, so the functional
properties of a number of site-directed mutants of Saccaromyces cerevisiae iso-1
cytochrome c have been examined. Results indicate that the two residues do not
interact specifically with each other; however, residue 13 (Arg) is likely to be
involved in interactions between cytochrome c and other electrostatically
oriented physiological partners (intermolecular), whereas residue 90 (Asp) is
involved in maintaining the intrinsic structure and stability of cytochrome c
(intramolecular). This is supported by molecular dynamics simulations carried
out for these mutants where removal of the negative charge at position 90 leads
to significant shifts in the conformations of neighboring residues, particularly
lysine 86. Both charged residues appear to exert their effects through
electrostatics; however, biological activity is significantly more sensitive to
substitutions of residue 13 than of residue 90.
PMID: 11235919 [PubMed - indexed for MEDLINE]
265: EMBO J 2001 Jan 15;20(1-2):118-27
Identification of a structural motif that confers specific interaction with the
WD40 repeat domain of Arabidopsis COP1.
Holm M, Hardtke CS, Gaudet R, Deng XW.
Department of Molecular, Cellular and Developmental Biology, Yale University,
OML 354, Yale University, PO Box 20-8104, 165 Prospect Street, New Haven, CT
06520-8104, USA.
Arabidopsis COP1 is a photomorphogenesis repressor capable of directly
interacting with the photomorphogenesis-promoting factor HY5. This interaction
between HY5 and COP1 results in targeted deg radation of HY5 by the 26S
proteasome. Here we characterized the WD40 repeat domain-mediated interactions
of COP1 with HY5 and two new proteins. Mutational analysis of those interactive
partners revealed a conserved motif responsible for the interaction with the
WD40 domain. This novel motif, with the core sequence V-P-E/D-φ-G (φ =
hydrophobic residue) in conjunction with an upstream stretch of 4-5 negatively
charged residues, interacts with a defined surface area of the ss-propeller
assembly of the COP1 WD40 repeat domain through both hydrophobic and ionic
interactions. Several residues in the COP1 WD40 domain that are critical for the
interaction with this motif have been revealed. The fact that point mutations
either in the COP1 WD40 domain or in the HY5 motif that abolish the interaction
between COP1 and HY5 in yeast result in a dramatic reduction of HY5 degradation
in transgenic plants validates the biological significance of this defined
interaction.
PMID: 11226162 [PubMed - indexed for MEDLINE]
266: Acta Crystallogr D Biol Crystallogr 2001 Mar;57(Pt 3):459-61
Crystallization and preliminary X-ray diffraction studies of FHA domains of Dun1
and Rad53 protein kinases.
Blanchard H, Fontes MR, Hammet A, Pike BL, Teh T, Gleichmann T, Gooley PR, Kobe
B, Heierhorst J.
Department of Biochemistry, University of Queensland, St Lucia, Brisbane, Qld
4072, Australia. helenb@biosci.uq.edu.au
Forkhead-associated (FHA) domains are modular protein-protein interaction
domains of approximately 130 amino acids present in numerous signalling
proteins. FHA-domain-dependent protein interactions are regulated by
phosphorylation of target proteins and FHA domains may be multifunctional
phosphopeptide-recognition modules. FHA domains of the budding yeast cell-cycle
checkpoint protein kinases Dun1p and Rad53p have been crystallized. Crystals of
the Dun1-FHA domain exhibit the symmetry of the space group P6(1)22 or P6(5)22,
with unit-cell parameters a = b = 127.3, c = 386.3 A; diffraction data have been
collected to 3.1 A resolution on a synchrotron source. Crystals of the
N-terminal FHA domain (FHA1) of Rad53p diffract to 4.0 A resolution on a
laboratory X-ray source and have Laue-group symmetry 4/mmm, with unit-cell
parameters a = b = 61.7, c = 104.3 A.
PMID: 11223532 [PubMed - indexed for MEDLINE]
267: RNA 2001 Jan;7(1):133-42
Yeast U1 snRNP-pre-mRNA complex formation without U1snRNA-pre-mRNA base pairing.
Du H, Rosbash M.
Department of Biology, Howard Hughes Medical Institute, Brandeis University,
Waltham, Massachusetts 02454, USA.
Base pairing between the 5' end of U1 snRNA and the conserved 5' splice site of
pre-mRNA is important for commitment complex formation in vitro. However, the
biochemical mechanisms by which pre-mRNA is initially recognized by the splicing
machinery is not well understood. To evaluate the role of this base pairing
interaction, we truncated U1 snRNA to eliminate the RNA-RNA interaction and
surprisingly found that U1 snRNP can still form a nearly normal RNA-protein
complex and maintain sequence specificity. We propose that some feature of U1
snRNP, perhaps one or more protein factors, is more important than the base
pairing for initial 5' splice site recognition. In addition, at least five sets
of interactions contribute to complex formation or stability. Only one of these
is base pairing between the 5' splice site and the 5' end of U1 snRNA, without
which the U1 snRNP-pre-mRNA complex is less stable and has a somewhat altered
conformation.
PMID: 11214175 [PubMed - indexed for MEDLINE]
268: Nature 2001 Jan 25;409(6819):533-8
Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF.
Iyer VR, Horak CE, Scafe CS, Botstein D, Snyder M, Brown PO.
Department of Biochemistry, Stanford University Medical Center, California
94305, USA.
Proteins interact with genomic DNA to bring the genome to life; and these
interactions also define many functional features of the genome. SBF and MBF are
sequence-specific transcription factors that activate gene expression during the
G1/S transition of the cell cycle in yeast. SBF is a heterodimer of Swi4 and
Swi6, and MBF is a heterodimer of Mbpl and Swi6 (refs 1, 3). The related Swi4
and Mbp1 proteins are the DNA-binding components of the respective factors, and
Swi6 mayhave a regulatory function. A small number of SBF and MBF target genes
have been identified. Here we define the genomic binding sites of the SBF and
MBF transcription factors in vivo, by using DNA microarrays. In addition to the
previously characterized targets, we have identified about 200 new putative
targets. Our results support the hypothesis that SBF activated genes are
predominantly involved in budding, and in membrane and cell-wall biosynthesis,
whereas DNA replication and repair are the dominant functions among MBF
activated genes. The functional specialization of these factors may provide a
mechanism for independent regulation of distinct molecular processes that
normally occur in synchrony during the mitotic cell cycle.
PMID: 11206552 [PubMed - indexed for MEDLINE]
269: Med Mycol 2000;38 Suppl 1:125-37
Candida albicans: adherence, signaling and virulence.
Calderone R, Suzuki S, Cannon R, Cho T, Boyd D, Calera J, Chibana H, Herman D,
Holmes A, Jeng HW, Kaminishi H, Matsumoto T, Mikami T, O'Sullivan JM, Sudoh M,
Suzuki M, Nakashima Y, Tanaka T, Tompkins GR, Watanabe T.
Department of Microbiology and Immunology, Georgetown University Medical Center,
Washington, DC 20007, USA. calderor@gunet.georgetown.edu
The focus of this symposium was to present new information on the morphogenesis
of Candida albicans, particularly how it relates to signal transduction pathways
and other genes involved in the regulation of morphogenesis. In addition, we
discuss the role of adherence and colonization of the oral cavity by the
organism and discuss the role of mannan as an adhesin that recognizes the human
red blood cell. C. albicans utilizes at least two signal pathways to regulate
its conversion from a yeast form to filamentous growth (hyphae). One of these
two pathways is similar to the Saccharomyces cerevisiae pseudohyphal/mating
pathway, which utilizes the regulatory protein, Cphlp. The other pathway is not
totally defined but requires a second regulatory protein, referred to as Efg1p.
Other signal pathways may exist, which include a two-component histidine kinase
and response regulator proteins. The latter pathway(s) may include proteins such
as Chk1p, Ssk1p, Shi1p and Cos1p/Nik1p. Mutations in strains, which specifically
target these proteins, result in morphogenesis defects and avirulence or
attenuation of strains. A growth regulatory gene has also been recently defined
whose expression is associated with growth cessation and which appears to be a
necessary prerequisite in conversion of the organism to a filamentous growth
form. Starvation of yeast cells induces exponentially grown cells (and usually
non-germinative) to germinate. This phenomenon is also observed in cells that
are transiently treated with metabolic inhibitors. During each of these
treatments (starvation, metabolic inhibition), expression of a growth regulatory
gene (CGRI) increases. Adherence of C. albicans to host cells and tissues is
complex; several proteins, which appear to have host recognition functions, have
been defined. In the oral cavity, C. albicans selectively adheres to salivary
proteins, which are absorbed to many oral surfaces. This mechanism enables the
cells to colonize surfaces of the oral cavity. An understanding of these
interactions may lead to strategies to prevent oral disease. Mannan from C.
albicans may provide a host recognition function for C. albicans. Recent
experiments indicate that mannan binds to human red blood cells and causes
hemolysis. Binding of mannan to the band 3 protein of human red blood cells has
been established. This activity may be associated with the ability of the
organism to utilize hemoglobin (and iron).
Publication Types:
Review
Review, Tutorial
PMID: 11204138 [PubMed - indexed for MEDLINE]
270: Plant Mol Biol 2000 Nov;44(4):513-27
Two rice MADS domain proteins interact with OsMADS1.
Lim J, Moon YH, An G, Jang SK.
Department of Life Science, Pohang University of Science and Technology,
Kyunghuk, Korea.
OsMADS1 is a MADS box gene controlling flower development in rice. In order to
learn more about the function of OsMADS1, we searched for cellular proteins
interacting with OsMADS1 employing the yeast two-hybrid system. Two novel
proteins with MADS domains, which were named OsMADS14 and OsMADS15, were
isolated from a rice cDNA library. OsMADS14 and -15 are highly homologous to the
maize MADS box gene ZAP1 which is an orthologue of the floral homeotic gene
APETALA1 (AP1). Interactions among the three MADS domain proteins were confirmed
by in vitro experiments using GST-fused OsMADS1 expressed in Escherichia coli
and in vitro translated proteins of OsMADS14 and -15. We determined which
domains in OsMADS1, -14, and -15 were required for protein-protein interaction
employing the two-hybrid system and pull-down experiments. While the K domain
was essential for protein-protein interaction, a region preceded by the K domain
augmented this interaction. Interestingly, the C-terminal region of OsMADS1
functioned as a transcriptional activation domain in yeast and mammalian cells,
while, on the other hand, the C domains of OsMADS14 and -15 exhibited only very
weak transcriptional activator functionality, if any at all.
PMID: 11197326 [PubMed - indexed for MEDLINE]
271: Biotechniques 2001 Jan;30(1):94-8, 100
cDNA library screening using the SOS recruitment system.
Huang W, Wang SL, Lozano G, de Crombrugghe B.
University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
The SOS recruitment system (SRS), a recently developed method for detecting
protein-protein interactions, provides an attractive alternative to identify
biologically important protein interactions. In SRS, the protein-protein
interactions take place in the cytoplasm instead of the nucleus, as is the case
in the conventional two-hybrid system. Although the SRS has overcome some of the
disadvantages of the conventional two-hybrid system, it still has several
problems and limitations. Here, we describe a new protocol for SRS library
screening. A new combination of growth media to avoid the tedious step of
replica plating greatly increases the number of independent colonies in a single
library screening. Furthermore, we designed a pair of ras-specific primers and a
one-step simple PCR to rule out the most abundant false positive, the mammalian
ras cDNA, in SRS library screening.
Publication Types:
Technical Report
PMID: 11196326 [PubMed - indexed for MEDLINE]
272: Curr Genet 2000 Dec;38(5):248-55
Isolation and molecular characterization of the carboxy-terminal pdr3 mutants in
Saccharomyces cerevisiae.
Simonics T, Kozovska Z, Michalkova-Papajova D, Delahodde A, Jacq C, Subik J.
Department of Microbiology and Virology, Comenius University, Bratislava, Slovak
Republic.
Multidrug resistance in Saccharomyces cerevisiae mainly results from the
overexpression of genes coding for the membrane efflux pumps, the major
facilitators and the ABC binding cassette transporters, under the control of key
transcription regulators encoded by the PDR1 and PDR3 genes. Pdr3p
transcriptional activator contains a weak activation domain near the N-terminal
zinc finger, a central regulatory domain, and a strong activation domain near
the carboxyl terminus. Here we report the results of the mutational analysis of
the C-terminal region of Pdr3p. After in vitro mutagenesis of the PDR3 gene six
single amino acid substitutions were identified and resulted in resistance to
cycloheximide, sulfomethuron methyl, 4-nitroquinoline oxide, fluconazole,
mucidin, chloramphenicol and oligomycin. All the C-terminal pdr3 mutant alleles
also conferred multidrug resistance in the presence of the wild-type PDR3 gene.
The pdr3 mutations resulted in overexpression of both the PDR3 and PDR5 genes as
revealed by transactivation experiments involving the PDR3-lacZ and PDR5-lacZ
fusion genes and Western blot analyses using antibodies against Pdr5p. Most of
the C-terminal pdr3 mutations were found in two sequence stretches exhibiting a
high degree of amino acid identity with Pdr1p indicating that they might play a
significant role in protein-protein interactions during the initiation of
transcription of genes involved in multidrug resistance.
PMID: 11191208 [PubMed - indexed for MEDLINE]
273: Curr Genet 2000 Dec;38(5):233-40
Alterations in the Saccharomyces MAL-activator cause constitutivity but can be
suppressed by intragenic mutations.
Danzi SE, Zhang B, Michels CA.
Department of Biology, Queens College and the Graduate School of CUNY, Flushing,
NY 11367, USA.
The Saccharomyces MAL-activator regulates the maltose-inducible expression of
the MAL structural genes encoding maltose permease and maltase. Constitutive
MAL-activator mutant alleles of two types were identified. The first were
truncation mutations deleting C-terminal residues 283-470 and the second
contained a large number of alterations compared to inducible alleles scattered
throughout the C-terminal 200 residues. We used site-directed in vitro
mutagenesis of the inducible MAL63 and MAL63/23 genes to identify the residues
responsible for the negative regulatory function of the C-terminal domain.
Intragenic suppressors that restored the inducible phenotype to the constitutive
mutants were identified at closely linked and more distant sites within the
MAL-activator protein. MAL63/mal64 fusions of the truncated mutants suggest that
residues in the N-terminal 100 residues containing the DNA-binding domain also
modulate basal expression. Moreover, a transcription activator protein
consisting of LexA(1-87)-Gal4(768-881)-Mal63(200-470) allowed constitutive
reporter gene expression, suggesting that the C-terminal regulatory domain is
not sufficient for maltose-inducible control of this heterologous activation
domain. These results suggest that complex and very specific intramolecular
protein-protein interactions regulate the MAL-activator.
PMID: 11191206 [PubMed - indexed for MEDLINE]
274: IUBMB Life 2000 Aug;50(2):105-13
A CD2-based model of yeast alpha-agglutinin elucidates solution properties and
binding characteristics.
Grigorescu A, Chen MH, Zhao H, Kahn PC, Lipke PN.
Department of Biological Sciences and The Institute for Biomolecular Structure
and Function, Hunter College of the City University of New York, NY 10021, USA.
We have previously shown that the Saccharomyces cerevisiae cell adhesion protein
alpha-agglutinin has sequence characteristics of immunoglobulin-like proteins
and have successfully modeled residues 200-325, based on the structure of
immunoglobulin variable-type domains. Alignments matching residues 20-200 of
alpha-agglutinin with domains I and II of members of the CD2/CD4 subfamily of
the immunoglobulin superfamily showed > 80% conservation of key residues despite
low sequence similarity overall. Three-dimensional models of two
alpha-agglutinin domains constructed on the basis of these alignments were shown
to conform to peptide mapping data and biophysical properties of
alpha-agglutinin. In addition, the residue volume and surface accessibility
characteristics of these models resembled those of the well-packed structures of
related proteins. Residue-by-residue analysis showed that packing and
accessibility anomalies were largely confined to glycosylated and
protease-susceptible loop regions of the domains. Surface accessibility of
hydrophobic residues was typical of proteins with extensive domain interactions,
a finding compatible with the hydrodynamic properties of alpha -agglutinin and
the hydrophobic nature of binding to its peptide ligand alpha-agglutinin. The
procedures used to align the alpha-agglutinin sequence and test the quality of
the model may be applicable to other proteins, especially those that resist
crystallization because of extensive glycosylation.
PMID: 11185954 [PubMed - indexed for MEDLINE]
275: Traffic 2000 Oct;1(10):763-8
The use of yeast two-hybrid screens in studies of protein:protein interactions
involved in trafficking.
Stephens DJ, Banting G.
Department of Cell Biology and Cell Biophysics, EMBL-Heidelberg, Germany.
The yeast two-hybrid system has provided a convenient means to both screen for
proteins that interact with a protein of interest and to characterise the known
interaction between two proteins. Several groups with an interest in the
molecular mechanisms that underlie discrete steps along trafficking pathways
have exploited the yeast two-hybrid system. Here, we provide a brief background
to the technology, attempt to point out some of the pitfalls and benefits of the
different systems that can be employed, and mention some of the areas (within
the trafficking field) where yeast two-hybrid interaction assays have been
particularly informative.
Publication Types:
Review
Review, Tutorial
PMID: 11208066 [PubMed - indexed for MEDLINE]
276: Cell Microbiol 1999 Jul;1(1):7-17
Identification of the intimin-binding domain of Tir of enteropathogenic
Escherichia coli.
de Grado M, Abe A, Gauthier A, Steele-Mortimer O, DeVinney R, Finlay BB.
Biotechnology Laboratory, University of British Columbia, Vancouver, Canada.
Enteropathogenic Escherichia coli (EPEC) attaches intimately to mammalian cells
via a bacterial outer membrane adhesion molecule, intimin, and its receptor in
the host cell membrane, Tir. Tir is a bacterial protein translocated into the
host cell membrane and tyrosine phosphorylated after insertion. Tir-intimin
binding induces organized actin polymerization beneath the adherent bacteria,
resulting in the formation of pedestal-like structures. A series of Tir deletion
derivatives were constructed to analyse which Tir domains are involved in
intimin binding. We have localized the intimin-binding domain (IBD) of Tir using
a yeast two-hybrid system and a gel-overlay approach to a region of 109 amino
acids that is predicted to be exposed on the surface of the plasma membrane. A
truncated Tir protein lacking this domain was translocated to the host cell
membrane and tyrosine phosphorylated, but failed to bind intimin or to induce
either actin polymerization or Tir accumulation beneath the bacteria. These
results indicate that only a small region of Tir is needed to bind intimin and
support the predicted topology for Tir, with both N- and C-terminal regions in
the mammalian cell cytosol. They also confirm that Tir-intimin interactions are
needed for cytoskeletal organization. We have also identified N-terminal regions
involved in Tir stability and Tir secretion to the media.
PMID: 11207537 [PubMed - indexed for MEDLINE]
277: Mol Biol Cell 2001 Feb;12(2):475-85
Vps10p transport from the trans-Golgi network to the endosome is mediated by
clathrin-coated vesicles.
Deloche O, Yeung BG, Payne GS, Schekman R.
Howard Hughes Medical Institute and Department of Molecular and Cell Biology,
University of California, 229 Stanley Hall, Berkeley, California 94720-3206,
USA.
A native immunoisolation procedure has been used to investigate the role of
clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between
the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the
yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the
adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein
receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is
reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport
from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p.
Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic
domain, the portion of the receptor responsible for carboxypeptidase Y sorting,
is also coimmunoprecipitated with clathrin. Our results suggest that CCVs
mediate Vps10p transport from the TGN to the endosome independent of direct
interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain
appears to play a principal role in retrieval of Vps10p from the prevacuolar
compartment rather than in sorting from the TGN.
PMID: 11179429 [PubMed - indexed for MEDLINE]
278: EMBO J 2001 Feb 15;20(4):891-904
Related eIF3 subunits TIF32 and HCR1 interact with an RNA recognition motif in
PRT1 required for eIF3 integrity and ribosome binding.
Valasek L, Phan L, Schoenfeld LW, Valaskova V, Hinnebusch AG.
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and
Human Development, Bethesda, MD 20892, USA.
eIF3 binds to 40S ribosomal subunits and stimulates recruitment of Met-tRNAiMet
and mRNA to the pre-initiation complex. Saccharomyces cerevisiae contains an
ortholog of human eIF3 subunit p35, HCR1, whose interactions with yeast eIF3 are
not well defined. We found that HCR1 has a dual function in translation
initiation: it binds to, and stabilizes, the eIF3-eIF5- eIF1-eIF2 multifactor
complex and is required for the normal level of 40S ribosomes. The RNA
recognition motif (RRM) of eIF3 subunit PRT1 interacted simultaneously with HCR1
and with an internal domain of eIF3 subunit TIF32 that has sequence and
functional similarity to HCR1. PRT1, HCR1 and TIF32 were also functionally
linked by genetic suppressor analysis. We propose that HCR1 stabilizes or
modulates interaction between TIF32 and the PRT1 RRM. Removal of the PRT1 RRM
resulted in dissociation of TIF32, NIP1, HCR1 and eIF5 from eIF3 in vivo, and
destroyed 40S ribosome binding by the residual PRT1-TIF34-TIF35 subcomplex.
Hence, the PRT1 RRM is crucial for the integrity and ribosome-binding activity
of eIF3.
PMID: 11179233 [PubMed - indexed for MEDLINE]
279: EMBO J 2001 Feb 15;20(4):841-51
Gal80-Gal80 interaction on adjacent Gal4p binding sites is required for complete
GAL gene repression.
Melcher K, Xu HE.
Department of Internal Medicine, University of Texas Southwestern Medical
Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-8573, USA.
K.Melcher@em.uni-frankfurt.de
Regulation of the GAL genes of Saccharomyces cerevisiae is determined by the
interplay of the transcriptional activator Gal4p and the repressor Gal80p, which
binds and masks the activation domain of Gal4p under non-inducing conditions.
Here we demonstrate that Gal80p dimerizes with high affinity and that this
dimerization appears to stabilize the Gal4p-Gal80p interaction and also,
indirectly, the Gal4p-DNA interaction in a (Gal4p)2(Gal80p)2DNA complex. In
addition, Gal80 dimers transiently interact with each other to form higher order
multimers. We provide evidence that adjacent Gal4p binding sites, when correctly
spaced, greatly stabilize Gal80p dimer-dimer interactions and that this
stabilization results in the complete repression of GAL genes with multiple
Gal4p binding sites. In contrast, GAL genes under the control of a single Gal4p
binding site do not stabilize Gal80p multimers, resulting in significant and
biologically important transcriptional leakage. Cooperative binding experiments
indicate that Gal80p dimer-dimer interaction probably does not lead to a
stronger Gal4p-Gal80p interaction, but most likely to a more complete shielding
of the Gal4p activation domain.
PMID: 11179228 [PubMed - indexed for MEDLINE]
280: Biochemistry (Mosc) 2000 Dec;65(12):1362-6
Interaction of catalytic domains in cytochrome P450scc--adrenodoxin
reductase--adrenodoxin fusion protein imported into yeast mitochondria.
Novikova LA, Nazarov PA, Saveliev AS, Drutsa VL, Sergeev VN, Miller WL, Luzikov
VN.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State
University, Moscow, 119899 Russia.
We have constructed plasmids for yeast expression of the fusion protein
pre-cytochrome P450scc--adrenodoxin reductase-adrenodoxin (F2) and a variant of
F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from
transformed yeast cells contained the F2 fusion protein at about 0.5% of total
protein and showed cholesterol hydroxylase activity with
22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated
mitochondria were supplemented with an excess of purified P450scc or a mixture
of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These
data suggest that, at least in yeast mitochondria, the interactions of the
catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain
are restricted.
PMID: 11173506 [PubMed - indexed for MEDLINE]
281: Biochem Soc Trans 2000 Dec;28(6):615-6
Protein interactions of fatty acid synthase II.
Honeyman G, Fawcett T.
Department of Biological Sciences, University of Durham, South Road, Durham DH1
3LE, UK.
We have used a yeast two-hybrid approach to detect direct protein interactions
between fatty acid synthase components. Enoyl-acyl carrier protein (ACP)
reductase was found to interact with stearoyl-ACP desaturase and acyl-ACP
thioesterase, but none of these proteins interacted with ACP in the yeast
nucleus.
PMID: 11171144 [PubMed - indexed for MEDLINE]
282: Biochemistry 2001 Jan 23;40(3):712-8
DNA topoisomerase II as the target for the anticancer drug TOP-53: mechanistic
basis for drug action.
Byl JA, Cline SD, Utsugi T, Kobunai T, Yamada Y, Osheroff N.
Department of Biochemistry and Medicine (Hematology/Oncology), Vanderbilt
University School of Medicine, Nashville, Tennessee 37232-0146, USA.
TOP-53 is a promising anticancer agent that displays high activity against
non-small cell lung cancer in animal tumor models [Utsugi, T., et al. (1996)
Cancer Res. 56, 2809-2814]. Compared to its parent compound, etoposide, TOP-53
is considerably more toxic to non-small cell lung cancer cells, is more active
at generating chromosomal breaks, and displays improved cellular uptake and
pharmacokinetics in animal lung tissues. Despite the preclinical success of
TOP-53, several questions remain regarding its cytotoxic mechanism. Therefore,
this study characterized the basis for drug action. Results indicate that
topoisomerase II is the primary cytotoxic target for TOP-53. Furthermore, the
drug kills cells by acting as a topoisomerase II poison. TOP-53 exhibits a DNA
cleavage site specificity that is identical to that of etoposide. Like its
parent compound, the drug increases the number of enzyme-mediated DNA breaks by
interfering with the DNA religation activity of the enzyme. TOP-53 is
considerably more efficient than etoposide at enhancing topoisomerase
II-mediated DNA cleavage and exhibits high activity against human topoisomerase
IIalpha and IIbeta in vitro and in cultured cells. Therefore, at least in part,
the enhanced cytotoxic activity of TOP-53 can be attributed to an enhanced
activity against topoisomerase II. Finally, TOP-53 displays nearly wild-type
activity against a mutant yeast type II enzyme that is highly resistant to
etoposide. This finding suggests that TOP-53 can retain activity against systems
that have developed resistance to etoposide, and indicates that substituents on
the etoposide C-ring are important for topoisomerase II-drug interactions.
PMID: 11170388 [PubMed - indexed for MEDLINE]
283: Genes Cells 2000 Dec;5(12):975-89
Interactions between Mcm10p and other replication factors are required for
proper initiation and elongation of chromosomal DNA replication in Saccharomyces
cerevisiae.
Kawasaki Y, Hiraga S, Sugino A.
Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka,
Suita, Osaka 565-0871, Japan.
BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication
in Saccharomyces cerevisiae. Previous work showed that Mcm10p interacts with the
Mcm2-7 protein complex that may be functioning as the replication-licensing
factor. In addition, Mcm10p is required during origin activation and disassembly
of the prereplicative complex, which allows smooth passage of replication forks.
RESULTS: We show that an mcm10 mutation causes a slow progression of DNA
synthesis and a loss of chromosome integrity during the S phase and prevents
entry into mitosis, despite apparent completion of chromosomal DNA replication
at nonpermissive temperatures. Furthermore, Mcm10p interacts genetically with
the origin recognition complex (ORC) and various replication elongation factors,
including a subunit of DNA polymerases epsilon and delta. Mcm10p is an abundant
protein (approximately 4 x 10(4) copies per haploid cell) that is almost
exclusively localized in the chromatin and/or nuclear matrix fractions during
all phases of the cell cycle. When it is visualized by the chromosome-spreading
method followed by immunostaining, Mcm10p forms punctate foci on chromatin
throughout the cell cycle and these foci mostly overlap with those of Orc1p, a
component of ORC. CONCLUSIONS: These results suggest that Mcm10p, like the
Mcm2-7 proteins, is a critical component of the prereplication chromatin and
acts together with ORC during the initiation of chromosomal DNA replication; in
addition, Mcm10p plays an important role during the elongation of DNA
replication.
PMID: 11168584 [PubMed - indexed for MEDLINE]
284: Biochem Biophys Res Commun 2001 Jan 12;280(1):151-7
Imaging and mapping protein-binding sites on DNA regulatory regions with atomic
force microscopy.
Moreno-Herrero F, Herrero P, Colchero J, Baro AM, Moreno F.
Departamento de Bioquimica y Biologia Molecular, Instituto Universitario de
Biotecnologia de Asturias, 33006 Oviedo, Spain.
Regulation of gene expression is fundamental in biological systems. A systematic
search for protein binding sites in gene promoters has been done in recent
years. Biochemical techniques are easy and reliable when analysing protein
interactions with short pieces of DNA, but are difficult and tedious when long
pieces of DNA have to be analysed. Here we propose AFM as a reliable and easy
technique for identifying protein interaction sites in long DNA molecules like
gene promoters. We support this idea using a well-known model: the interaction
of the Pho4 protein with the PHO5 gene promoter. We have also applied the
technique to demonstrate that Mig1 protein binds to two motifs in the promoter
of HXK2 gene. Our results allow us to define Mig1p as a new factor probably
contributing to the carbon source-dependent transcription regulation of HXK2
gene. Copyright 2001 Academic Press.
PMID: 11162492 [PubMed - indexed for MEDLINE]
285: Nucleic Acids Res 2001 Feb 15;29(4):E18
A novel approach for the identification of protein-protein interaction with
integral membrane proteins.
Hubsman M, Yudkovsky G, Aronheim A.
Department of Molecular Genetics and the Rappaport Family Institute for Research
in the Medical Sciences and the B. Rappaport Faculty of Medicine,
Technion-Israel Institute of Technology, PO Box 9649, Bat-Galim, Haifa 31096,
Israel.
Protein-protein interaction plays a major role in all biological processes. The
currently available genetic methods such as the two-hybrid system and the
protein recruitment system are relatively limited in their ability to identify
interactions with integral membrane proteins. Here we describe the development
of a reverse Ras recruitment system (reverse RRS), in which the bait used
encodes a membrane protein. The bait is expressed in its natural environment,
the membrane, whereas the protein partner (the prey) is fused to a cytoplasmic
Ras mutant. Protein-protein interaction between the proteins encoded by the prey
and the bait results in Ras membrane translocation and activation of a viability
pathway in yeast. We devised the expression of the bait and prey proteins under
the control of dual distinct inducible promoters, thus enabling a rapid
selection of transformants in which growth is attributed solely to specific
protein-protein interaction. The reverse RRS approach greatly extends the
usefulness of the protein recruitment systems and the use of integral membrane
proteins as baits. The system serves as an attractive approach to explore novel
protein-protein interactions with high specificity and selectivity, where other
methods fail.
PMID: 11160938 [PubMed - indexed for MEDLINE]
286: Nucleic Acids Res 2001 Feb 1;29(3):629-37
Leucine zipper motif of chicken histone acetyltransferase-1 is essential for in
vivo and in vitro interactions with the p48 subunit of chicken chromatin
assembly factor-1.
Ahmad A, Nagamatsu N, Kouriki H, Takami Y, Nakayama T.
Department of Biochemistry, Miyazaki Medical College, 5200, Kihara, Kiyotake,
Miyazaki 889-1692, Japan.
We cloned cDNA encoding chicken cytoplasmic histone acetyltransferase-1,
chHAT-1, comprising 408 amino acids including a putative initiation Met. It
exhibits 80.4% identity to the human homolog and possesses a typical leucine
zipper motif. The glutathione S:-transferase (GST) pull-down assay, involving
truncated and missense mutants of the chicken chromatin assembly factor-1
(chCAF-1)p48, revealed not only that a region (comprising amino acids 376-405 of
chCAF-1p48 and containing the seventh WD dipeptide motif) binds to chHAT-1 in
vitro, but also that mutation of the motif has no influence on the in vitro
interaction. The GST pull-down assay, involving truncated and missense chHAT-1
mutants, established that a region, comprising amino acids 380-408 of chHAT-1
and containing the leucine zipper motif, is required for its in vitro
interaction with chCAF-1p48. In addition, mutation of each of four Leu residues
in the leucine zipper motif prevents the in vitro interaction. The yeast
two-hybrid assay revealed that all four Leu residues within the leucine zipper
motif of chHAT-1 are necessary for its in vivo interaction with chCAF-1p48.
These results indicate not only that the proper leucine zipper motif of chHAT-1
is essential for its interaction with chCAF-1p48, but also that the propeller
structure of chCAF-1p48 expected to act as a platform for protein-protein
interactions may not be necessary for this interaction of chHAT-1.
PMID: 11160883 [PubMed - indexed for MEDLINE]
287: Mol Biol Cell 2001 Jan;12(1):37-51
Vps41p function in the alkaline phosphatase pathway requires
homo-oligomerization and interaction with AP-3 through two distinct domains.
Darsow T, Katzmann DJ, Cowles CR, Emr SD.
Department of Cellular and Molecular Medicine and Division of Biology, Howard
Hughes Medical Institute, University of California, San Diego, School of
Medicine, La Jolla, California 92093-0668, USA.
Transport of proteins through the ALP (alkaline phosphatase) pathway to the
vacuole requires the function of the AP-3 adaptor complex and Vps41p. However,
unlike other adaptor protein-dependent pathways, the ALP pathway has not been
shown to require additional accessory proteins or coat proteins, such as
membrane recruitment factors or clathrin. Two independent genetic approaches
have been used to identify new mutants that affect transport through the ALP
pathway. These screens yielded new mutants in both VPS41 and the four AP-3
subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null
mutants of VPS41, which are defective in vacuolar morphology and protein
transport through both the ALP and CPY sorting pathways. The new alleles
displayed severe ALP sorting defects, normal vacuolar morphology, and defects in
ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41
alleles revealed mutations encoding amino acid changes in two distinct domains
of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain
repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required
for binding to AP-3, whereas the C-terminal CHCR domain directs
homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form
of Vps41p is required for the formation of ALP containing vesicles at the Golgi
complex via interactions with AP-3.
PMID: 11160821 [PubMed - indexed for MEDLINE]
288: Biophys J 2001 Jan;80(1):427-34
Tryptophan fluorescence of yeast actin resolved via conserved mutations.
Doyle TC, Hansen JE, Reisler E.
Department of Chemistry and Biochemistry and the Molecular Biology Institute,
University of California, Los Angeles, Los Angeles, California 90095, USA.
Actin contains four tryptophan residues, W79, W86, W340, and W356, all located
in subdomain 1 of the protein. Replacement of each of these residues with either
tyrosine (W79Y and W356Y) or phenylalanine (W86F and W340F) generated viable
proteins in the yeast Saccharomyces cerevisiae, which, when purified, allowed
the analysis of the contribution of these residues to the overall tryptophan
fluorescence of actin. The sum of the relative contributions of these
tryptophans was found to account for the intrinsic fluorescence of wild-type
actin, indicating that energy transfer between the tryptophans is not the main
determinant of their quantum yield, and that these mutations induce little
conformational change to the protein. This was borne out by virtually identical
polymerization rates and similar myosin interactions of each of the mutants and
the wild-type actin. In addition, these mutants allowed the dissection of the
microenvironment of each tryptophan as actin undergoes conformational changes
upon metal cation exchange and polymerization. Based on the relative tryptophan
contributions determined from single mutants, a triple mutant of yeast actin
(W79) was generated that showed small intrinsic fluorescence and should be
useful for studies of actin interactions with actin-binding proteins.
PMID: 11159413 [PubMed - indexed for MEDLINE]
289: Proc Natl Acad Sci U S A 2001 Jan 30;98(3):914-9
Subunit interactions influence the biochemical and biological properties of
Hsp104.
Schirmer EC, Ware DM, Queitsch C, Kowal AS, Lindquist SL.
Department of Molecular Genetics and Cell Biology and Howard Hughes Medical
Institute, University of Chicago, Chicago, IL 60637, USA.
Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104
(NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vitro, NBD1
mutations virtually eliminate ATP hydrolysis with little effect on
hexamerization; analogous NBD2 mutations reduce ATPase activity and severely
impair hexamerization. We report that high protein concentrations overcome the
assembly defects of NBD2 mutants and increase ATP hydrolysis severalfold,
changing V(max) with little effect on K(m). In a complementary fashion, the
detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate inhibits
hexamerization of wild-type (WT) Hsp104, lowering V(max) with little effect on
K(m). ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating
that it is influenced by cooperative subunit interactions. To further analyze
the effects of subunit interactions on Hsp104, we assessed the effects of mutant
Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that hexamerizes but
does not hydrolyze ATP reduces the ATPase activity of WT Hsp104 in vitro. In
vivo, this mutant is not toxic but specifically inhibits the thermotolerance
function of WT Hsp104. Thus, interactions between subunits influence the ATPase
activity of Hsp104, play a vital role in its biological functions, and provide a
mechanism for conditionally inactivating Hsp104 function in vivo.
PMID: 11158570 [PubMed - indexed for MEDLINE]
290: Mol Endocrinol 2001 Feb;15(2):241-54
Two distinct nuclear receptor-interaction domains and CREB-binding
protein-dependent transactivation function of activating signal cointegrator-2.
Lee SK, Jung SY, Kim YS, Na SY, Lee YC, Lee JW.
Center for Ligand and Transcription, Department of Biology, Chonnam National
University, Kwangju 500-757, Korea.
ASC-2 is a recently isolated transcriptional cointegrator molecule, which is
amplified in human cancers and stimulates transactivation by nuclear receptors,
AP-1, nuclear factor kappaB (NFkappaB), serum response factor (SRF), and
numerous other transcription factors. ASC-2 contained two nuclear
receptor-interaction domains, both of which are dependent on the integrity of
their core LXXLL sequences. Surprisingly, the C-terminal LXXLL motif
specifically interacted with oxysterol receptor LXRss, whereas the N-terminal
motif bound a broad range of nuclear receptors. These interactions appeared to
be essential because a specific subregion of ASC-2 including the N- or
C-terminal LXXLL motif acted as a potent dominant negative mutant with
transactivation by appropriate nuclear receptors. In addition, the autonomous
transactivation domain (AD) of ASC-2 was found to consist of three separable
subregions; i.e. AD1, AD2, and AD3. In particular, AD2 and AD3 were binding
sites for CREB binding protein (CBP), and CBP-neutralizing E1A repressed the
autonomous transactivation function of ASC-2. Furthermore, the receptor
transactivation was not enhanced by ASC-2 in the presence of E1A and
significantly impaired by overexpressed AD2. From these results, we concluded
that ASC-2 directly binds to nuclear receptors and recruits CBP to mediate the
nuclear receptor transactivation in vivo.
PMID: 11158331 [PubMed - indexed for MEDLINE]
291: J Cell Biol 2001 Feb 5;152(3):451-69
A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.
Song S, Lee KS.
Laboratory of Metabolism, Division of Basic Sciences, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892, USA.
Coordination of mitotic exit with timely initiation of cytokinesis is critical
to ensure completion of mitotic events before cell division. The Saccharomyces
cerevisiae polo kinase Cdc5 functions in a pathway leading to the degradation of
mitotic cyclin Clb2, thereby permitting mitotic exit. Here we provide evidence
that Cdc5 also plays a role in regulating cytokinesis and that an intact
polo-box, a conserved motif in the noncatalytic COOH-terminal domain of Cdc5, is
required for this event. Depletion of Cdc5 function leads to an arrest in
cytokinesis. Overexpression of the COOH-terminal domain of Cdc5 (cdc5DeltaN),
but not the corresponding polo-box mutant, resulted in connected cells. These
cells shared cytoplasms with incomplete septa, and possessed aberrant septin
ring structures. Provision of additional copies of endogenous CDC5 remedied this
phenotype, suggesting a dominant-negative inhibition of cytokinesis. The
polo-box-dependent interactions between Cdc5 and septins (Cdc11 and Cdc12) and
genetic interactions between the dominant-negative cdc5DeltaN and Cyk2/Hof1 or
Myo1 suggest that direct interactions between cdc5DeltaN and septins resulted in
inhibition of Cyk2/Hof1- and Myo1-mediated cytokinetic pathways. Thus, we
propose that Cdc5 may coordinate mitotic exit with cytokinesis by participating
in both anaphase promoting complex activation and a polo-box-dependent
cytokinetic pathway.
PMID: 11157974 [PubMed - indexed for MEDLINE]
292: Hum Mol Genet 2001 Feb 15;10(4):423-9
Direct interactions of the five known Fanconi anaemia proteins suggest a common
functional pathway.
Medhurst AL, Huber PA, Waisfisz Q, de Winter JP, Mathew CG.
Division of Medical and Molecular Genetics, Guy's, King's and St Thomas' School
of Medicine, 8th Floor, Guy's Tower, Guy's Hospital, London SE1 9RT, UK.
Fanconi anaemia (FA) is an autosomal recessive inherited disorder associated
with a progressive aplastic anaemia, diverse congenital abnormalities and
cancer. The condition is genetically heterogeneous, with at least seven
complementation groups (A-G) described. Cells from individuals who are
homozygous for mutations in FA genes are characterized by chromosomal
instability and hypersensitivity to DNA interstrand crosslinking agents. These
features suggest a possible role for the encoded proteins in the recognition or
repair of these lesions, but neither their function nor whether they operate in
a concerted or discrete functional pathways is known. The recent cloning of the
FANCF and FANCE genes has allowed us to investigate the interaction of the
proteins encoded by five of the seven complementation groups of FA. We used the
yeast two-hybrid system and co-immunoprecipitation analysis to test the 10
possible pairs of proteins for direct interaction. In addition to the previously
described binding of FANCA to FANCG, we now demonstrate direct interaction of
FANCF with FANCG, of FANCC with FANCE and a weaker interaction of FANCE with
both FANCA and FANCG. These findings show that the newly identified FANCE
protein is an integral part of the FA pathway, and support the concept of a
functional link between all known proteins encoded by the genes that are mutated
in this disorder. These proteins may act either as a multimeric complex or by
sequential recruitment of subsets of the proteins in a common pathway that
protects the genomic integrity of mammalian cells.
PMID: 11157805 [PubMed - indexed for MEDLINE]
293: Genes Dev 2001 Jan 15;15(2):147-57
The Sir1 protein's association with a silenced chromosome domain.
Gardner KA, Fox CA.
Department of Biomolecular Chemistry, University of Wisconsin, Madison,
Wisconsin 53706, USA.
Silencing of the cryptic mating-type locus HMR requires recognition of a small
DNA sequence element, the HMR-E silencer, by the Sir1p, one of four Sir proteins
required for the assembly of silenced chromatin domains in Saccharomyces
cerevisiae. The Sir1p recognizes the silencer through interactions with the
origin recognition complex (ORC), a protein complex that binds the silencer DNA
directly. Sir1p was physically associated with HMR in chromatin, and this
association required a Sir1p-ORC interaction, suggesting that it reflected the
Sir1p silencer-recognition function required for silencing. Sir1p was not
associated with nonsilencer replication origins that bind the ORC, indicating
that a Sir1p-ORC interaction is confined to silencers. Significantly, the other
SIR genes were required for Sir1p's association with HMR. Thus, multiple protein
contacts required for and unique to silent chromatin may confine a Sir1p-ORC
interaction to silencers. The Sir1p was present at extremely low concentrations
in yeast cells yet was associated with HMR at all stages of the cell cycle
examined. These data provide insights into the mechanisms that establish and
restrict the assembly of silenced chromatin to only a few discrete chromosomal
domains.
PMID: 11157772 [PubMed - indexed for MEDLINE]
294: J Biol Chem 2000 Apr 14;275(15):11141-6
Interaction in vivo and in vitro of the metastasis-inducing S100 protein, S100A4
(p9Ka) with S100A1.
Wang G, Rudland PS, White MR, Barraclough R.
Cancer and Polio Research Fund Laboratories, School of Biological Sciences,
University of Liverpool, Liverpool L69 7ZB, United Kingdom.
The calcium-binding protein S100A4 (p9Ka) has been shown to cause a metastatic
phenotype in rodent mammary tumor cells and in transgenic mouse model systems.
mRNA for S100A4 (p9Ka) is present at a generally higher level in breast
carcinoma than in benign breast tumor specimens, and the presence of
immunocytochemically detected S100A4 correlates strongly with a poor prognosis
for breast cancer patients. Recombinant S100A4 (p9Ka) has been reported to
interact in vitro with cytoskeletal components and to form oligomers,
particularly homodimers in vitro. Using the yeast two-hybrid system, a strong
interaction between S100A4 (p9Ka) and another S100 protein, S100A1, was
detected. Site-directed mutagenesis of conserved amino acid residues involved in
the dimerization of S100 proteins abolished the interactions. The interaction
between S100A4 and S100A1 was also observed in vitro using affinity column
chromatography and gel overlay techniques. Both S100A1 and S100A4 can occur in
the same cultured mammary cells, suggesting that in cells containing both
proteins, S100A1 might modulate the metastasis-inducing capability of S100A4.
PMID: 10753920 [PubMed - indexed for MEDLINE]
295: J Biol Chem 2001 Mar 30;276(13):9846-54
The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is
essential for its E1 activity and E1-E2 complex formation.
Komatsu M, Tanida I, Ueno T, Ohsumi M, Ohsumi Y, Kominami E.
Department of Biochemistry, Juntendo University School of Medicine, Tokyo
113-8421, Japan.
Apg7p/Cvt2p, a protein-activating enzyme, is essential for both the Apg12p-Apg5p
conjugation system and the Apg8p membrane targeting in autophagy and
cytoplasm-to-vacuole targeting in the yeast Saccharomyces cerevisiae. Similar to
the ubiquitin-conjugating system, both Apg12p and Apg8p are activated by Apg7p,
an E1-like enzyme. Apg12p is then transferred to Apg10p, an E2-like enzyme, and
conjugated with Apg5p, whereas Apg8p is transferred to Apg3p, another E2-like
enzyme, followed by conjugation with phosphatidylethanolamine. Evidence is
presented here that Apg7p forms a homodimer with two active-site cysteine
residues via the C-terminal region. The dimerization of Apg7p is independent of
the other Apg proteins and facilitated by overexpressed Apg12p. The C-terminal
123 amino acids of Apg7p (residues 508 to 630 out of 630 amino acids) are
sufficient for its dimerization, where there is neither an ATP binding domain
nor an active-site cysteine essential for its E1 activity. The deletion of its
carboxyl 40 amino acids (residues 591-630 out of 630 amino acids) results in
several defects of not only Apg7p dimerization but also interactions with two
substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant
Apg7p contains both an ATP binding domain and an active-site cysteine.
Furthermore, the carboxyl 40 amino acids of Apg7p are also essential for the
interaction of Apg7p with Apg3p to form the E1-E2 complex for Apg8p. These
results suggest that Apg7p forms a homodimer via the C-terminal region and that
the C-terminal region is essential for both the activity of the E1 enzyme for
Apg12p and Apg8p as well as the formation of an E1-E2 complex for Apg8p.
PMID: 11139573 [PubMed - indexed for MEDLINE]
296: Mol Cell Biol 2001 Feb;21(3):966-76
Molecular dissection of interactions between Rad51 and members of the
recombination-repair group.
Krejci L, Damborsky J, Thomsen B, Duno M, Bendixen C.
Department of Analysis of Biologically Important Molecular Complexes, Masaryk
University, 612 65 Brno, Czech Republic.
Recombination is important for the repair of DNA damage and for chromosome
segregation during meiosis; it has also been shown to participate in the
regulation of cell proliferation. In the yeast Saccharomyces cerevisiae,
recombination requires products of the RAD52 epistasis group. The Rad51 protein
associates with the Rad51, Rad52, Rad54, and Rad55 proteins to form a dynamic
complex. We describe a new strategy to screen for mutations which cause specific
disruption of the interaction between certain proteins in the complex, leaving
other interactions intact. This approach defines distinct protein interaction
domains and protein relationships within the Rad51 complex. Alignment of the
mutations onto the constructed three-dimensional model of the Rad51 protein
reveal possible partially overlapping interfaces for the Rad51-Rad52 and the
Rad51-Rad54 interactions. Rad51-Rad55 and Rad51-Rad51 interactions are affected
by the same spectrum of mutations, indicating similarity between the two modes
of binding. Finally, the detection of a subset of mutations within Rad51 which
disrupt the interaction with mutant Rad52 protein but activate the interaction
with Rad54 suggests that dynamic changes within the Rad51 protein may contribute
to an ordered reaction process.
PMID: 11154282 [PubMed - indexed for MEDLINE]
297: J Cell Biol 2001 Jan 8;152(1):197-212
Mitotic spindle integrity and kinetochore function linked by the Duo1p/Dam1p
complex.
Cheeseman IM, Enquist-Newman M, Muller-Reichert T, Drubin DG, Barnes G.
Department of Molecular and Cell Biology, University of California at Berkeley,
Berkeley, California 94720, USA.
Duo1p and Dam1p were previously identified as spindle proteins in the budding
yeast, Saccharomyces cerevisiae. Here, analyses of a diverse collection of duo1
and dam1 alleles were used to develop a deeper understanding of the functions
and interactions of Duo1p and Dam1p. Based on the similarity of mutant
phenotypes, genetic interactions between duo1 and dam1 alleles, interdependent
localization to the mitotic spindle, and Duo1p/Dam1p coimmunoprecipitation from
yeast protein extracts, these analyses indicated that Duo1p and Dam1p perform a
shared function in vivo as components of a protein complex. Duo1p and Dam1p are
not required to assemble bipolar spindles, but they are required to maintain
metaphase and anaphase spindle integrity. Immunofluorescence and electron
microscopy of duo1 and dam1 mutant spindles revealed a diverse variety of
spindle defects. Our results also indicate a second, previously unidentified,
role for the Duo1p/Dam1p complex. duo1 and dam1 mutants show high rates of
chromosome missegregation, premature anaphase events while arrested in
metaphase, and genetic interactions with a subset of kinetochore components
consistent with a role in kinetochore function. In addition, Duo1p and Dam1p
localize to kinetochores in chromosome spreads, suggesting that this complex may
serve as a link between the kinetochore and the mitotic spindle.
PMID: 11149931 [PubMed - indexed for MEDLINE]
298: J Cell Biol 2001 Jan 8;152(1):51-64
Membrane recruitment of Aut7p in the autophagy and cytoplasm to vacuole
targeting pathways requires Aut1p, Aut2p, and the autophagy conjugation complex.
Kim J, Huang WP, Klionsky DJ.
Department of Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Autophagy is a degradative pathway by which cells sequester nonessential, bulk
cytosol into double-membrane vesicles (autophagosomes) and deliver them to the
vacuole for recycling. Using this strategy, eukaryotic cells survive periods of
nutritional starvation. Under nutrient-rich conditions, autophagy machinery is
required for the delivery of a resident vacuolar hydrolase, aminopeptidase I, by
the cytoplasm to vacuole targeting (Cvt) pathway. In both pathways, the vesicle
formation process requires the function of the starvation-induced Aut7 protein,
which is recruited from the cytosol to the forming Cvt vesicles and
autophagosomes. The membrane binding of Aut7p represents an early step in
vesicle formation. In this study, we identify several requirements for Aut7p
membrane association. After synthesis in the cytosol, Aut7p is proteolytically
cleaved in an Aut2p-dependent manner. While this novel processing event is
essential for Aut7p membrane binding, Aut7p must undergo additional physical
interactions with Aut1p and the autophagy (Apg) conjugation complex before
recruitment to the membrane. Lack of these interactions results in a cytosolic
distribution of Aut7p rather than localization to forming Cvt vesicles and
autophagosomes. This study assigns a functional role for the Apg conjugation
system as a mediator of Aut7p membrane recruitment. Further, we demonstrate that
Aut1p, which physically interacts with components of the Apg conjugation complex
and Aut7p, constitutes an additional factor required for Aut7p membrane
recruitment. These findings define a series of steps that results in the
modification of Aut7p and its subsequent binding to the sequestering transport
vesicles of the autophagy and cytoplasm to vacuole targeting pathways.
PMID: 11149920 [PubMed - indexed for MEDLINE]
299: Biochemistry 2001 Jan 16;40(2):422-8
Interactions between yeast iso-1-cytochrome c and its peroxidase.
Pielak GJ, Wang X.
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel
Hill, North Carolina 27599-3290, USA. gary_pielak@unc.edu
Isothermal titration calorimetry was used to study the formation of 19 complexes
involving yeast iso-1-ferricytochrome c (Cc) and ferricytochrome c peroxidase
(CcP). The complexes comprised combinations of the wild-type proteins, six CcP
variants, and three Cc variants. Sixteen protein combinations were designed to
probe the crystallographically defined interface between Cc and CcP. The data
show that the high-affinity sites on Cc and CcP coincide with the
crystallographically defined sites. Changing charged residues to alanine
increases the enthalpy of complex formation by a constant amount, but the
decrease in stability depends on the location of the amino acid substitution.
Deleting methyl groups has a small effect on the binding enthalpy and a larger
deleterious effect on the binding free energy, consistent with model studies of
the hydrophobic effect, and showing that nonpolar interactions also stabilize
the complex. Double-mutant cycles were used to determine the coupling energies
for nine Cc-CcP residue pairs. Comparing these energies to the crystal structure
of the complex leads to the conclusion that many of the substitutions induce a
rearrangement of the complex.
PMID: 11148036 [PubMed - indexed for MEDLINE]
300: RNA 2000 Dec;6(12):1882-94
The bI4 group I intron binds directly to both its protein splicing partners, a
tRNA synthetase and maturase, to facilitate RNA splicing activity.
Rho SB, Martinis SA.
Department of Biology and Biochemistry, University of Houston, Texas 77204-5513,
USA.
The imported mitochondrial leucyl-tRNA synthetase (NAM2p) and a
mitochondrial-expressed intron-encoded maturase protein are required for
splicing the fourth intron (bI4) of the yeast cob gene, which expresses an
electron transfer protein that is essential to respiration. However, the role of
the tRNA synthetase, as well as the function of the bI4 maturase, remain
unclear. As a first step towards elucidating the mechanistic role of these
protein splicing factors in this group I intron splicing reaction, we tested the
hypothesis that both leucyl-tRNA synthetase and bI4 maturase interact directly
with the bI4 intron. We developed a yeast three-hybrid system and determined
that both the tRNA synthetase and bI4 maturase can bind directly and
independently via RNA-protein interactions to the large bI4 group I intron. We
also showed, using modified two-hybrid and three-hybrid assays, that the bI4
intron bridges interactions between the two protein splicing partners. In the
presence of either the bI4 maturase or the Leu-tRNA synthetase, bI4 intron
transcribed recombinantly with flanking exons in the yeast nucleus exhibited
splicing activity. These data combined with previous genetic results are
consistent with a novel model for a ternary splicing complex (two protein: one
RNA) in which both protein splicing partners bind directly to the bI4 intron and
facilitate its self-splicing activity.
PMID: 11142386 [PubMed - indexed for MEDLINE]
301: Nucleic Acids Res 2001 Jan 15;29(2):536-44
Cnr interferes with dimerization of the replication protein alpha in
phage-plasmid P4.
Tocchetti A, Serina S, Oliva I, Deho G, Ghisotti D.
Dipartimento di Genetica e di Biologia dei Microrganismi, Universita di Milano,
Via Celoria 26, 20133 Milano, Italy.
DNA replication of phage-plasmid P4 in its host Escherichia coli depends on its
replication protein alpha. In the plasmid state, P4 copy number is controlled by
the regulator protein Cnr (copy number regulation). Mutations in alpha
(alpha(cr)) that prevent regulation by Cnr cause P4 over-replication and cell
death. Using the two-hybrid system in Saccharomyces cerevisiae and a system
based on lambda immunity in E.coli for in vivo detection of protein-protein
interactions, we found that (i) alpha protein interacts with Cnr, whereas
alpha(cr) proteins do not; (ii) both alpha-alpha and alpha(cr)-alpha(cr)
interactions occur and the interaction domain is located within the C-terminal
of alpha; (iii) Cnr-Cnr interaction also occurs. Using an in vivo competition
assay, we found that Cnr interferes with both alpha-alpha and
alpha(cr)-alpha(cr) dimerization. Our data suggest that Cnr and alpha interact
in at least two ways, which may have different functional roles in P4
replication control.
PMID: 11139624 [PubMed - indexed for MEDLINE]
302: Genetics 2001 Jan;157(1):91-101
WW domains of Rsp5p define different functions: determination of roles in fluid
phase and uracil permease endocytosis in Saccharomyces cerevisiae.
Gajewska B, Kaminska J, Jesionowska A, Martin NC, Hopper AK, Zoladek T.
Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy
of Sciences, 02-106 Warsaw, Poland.
Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway,
contains three WW domains that mediate protein-protein interactions. To
determine if these domains adapt Rsp5p to a subset of substrates involved in
numerous cellular processes, we generated mutations in individual or
combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles
complement RSP5 deletions at 30 degrees. Thus, individual WW domains are not
essential. Each rsp5-w mutation caused temperature-sensitive growth. Among
variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the
deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To
determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil
permease (Fur4p) endocytosis was examined. The WW3 domain is important for both
processes. WW2 appears not to be important for fluid phase endocytosis whereas
it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid
phase endocytosis, but it does not appear to function in Fur4p endocytosis.
Thus, various WW domains play different roles in the endocytosis of these two
substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does
not change during the cell cycle. Altering WW domains does not change the
location of Rsp5p.
PMID: 11139494 [PubMed - indexed for MEDLINE]
303: Exp Cell Res 2001 Jan 15;262(2):75-83
The human histone deacetylase family.
Gray SG, Ekstrom TJ.
Laboratory for Molecular Development and Tumor Biology, Centre for Molecular
Medicine (CMM), Stockholm, S-171 76, Sweden. Steven.Gray@vai.org
Since the identification of the first histone deacetylase (Taunton et al.,
Science 272, 408-411), several new members have been isolated. They can loosely
be separated into entities on the basis of their similarity to various yeast
histone deacetylases. The first class is represented by its closeness to the
yeast Rpd3-like proteins, and the second most recently discovered class has
similarities to yeast Hda1-like proteins. However, due to the fact that several
different research groups isolated the Hda1-like histone deacetylases
independently, there have been various different nomenclatures used to describe
the various members, which can lead to confusion in the interpretation of this
family's functions and interactions. With the discovery of another novel murine
histone deacetylase, homologous to yeast Sir2, the number of members of this
family is set to increase, as 7 human homologues of this gene have been
isolated. In the light of these recent discoveries, we have examined the
literature data and conducted a database analysis of the isolated histone
deacetylases and potential candidates. The results obtained suggest that the
number of histone deacetylases within the human genome may be as high as 17 and
are discussed in relation to their homology to the yeast histone deacetylases.
Copyright 2001 Academic Press.
Publication Types:
Review
Review, Tutorial
PMID: 11139331 [PubMed - indexed for MEDLINE]
304: J Biol Chem 2001 Mar 23;276(12):8848-55
Mapping the DNA topoisomerase III binding domain of the Sgs1 DNA helicase.
Fricke WM, Kaliraman V, Brill SJ.
Department of Molecular Biology and Biochemistry, Center for Advanced
Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08855,
USA. brill@mbcl.rutgers.edu
Several members of the RecQ family of DNA helicases are known to interact with
DNA topoisomerase III (Top3). Here we show that the Saccharomyces cerevisiae
Sgs1 and Top3 proteins physically interact in cell extracts and bind directly in
vitro. Sgs1 and Top3 proteins coimmunoprecipitate from cell extracts under
stringent conditions, indicating that Sgs1 and Top3 are present in a stable
complex. The domain of Sgs1 which interacts with Top3 was identified by
expressing Sgs1 truncations in yeast. The results indicate that the
NH(2)-terminal 158 amino acids of Sgs1 are sufficient for the high affinity
interaction between Sgs1 and Top3. In vitro assays using purified Top3 and
NH(2)-terminal Sgs1 fragments demonstrate that at least part of the interaction
is through direct protein-protein interactions with these 158 amino acids.
Consistent with these physical data, we find that mutant phenotypes caused by a
point mutation or small deletions in the Sgs1 NH(2) terminus can be suppressed
by Top3 overexpression. We conclude that Sgs1 and Top3 form a tight complex in
vivo and that the first 158 amino acids of Sgs1 are necessary and sufficient for
this interaction. Thus, a primary role of the Sgs1 amino terminus is to mediate
the Top3 interaction.
PMID: 11124263 [PubMed - indexed for MEDLINE]
305: Mol Gen Genet 2000 Nov;264(4):378-91
Allele-specific interactions between the yeast RFC1 and RFC5 genes suggest a
basis for RFC subunit-subunit interactions.
Beckwith W, McAlear MA.
Department of Molecular Biology and Biochemistry, Wesleyan University,
Middletown, CT 06459-0175, USA.
Replication factor C (RFC) is an essential, multi-subunit ATPase that functions
in DNA replication, DNA repair, and DNA metabolism-related checkpoints. In order
to investigate how the individual RFC subunits contribute to these functions in
vivo, we undertook a genetic analysis of RFC genes from budding yeast. We
isolated and characterized mutations in the RFC5 gene that could suppress the
cold-sensitive phenotype of rfc1-1 mutants. Analysis of the RFC5 suppressors
revealed that they could not suppress the elongated telomere phenotype, the
sensitivity to DNA damaging agents, or the mutator phenotype of rfc1-1 mutants.
Unlike the checkpoint-defective rfc5-1 mutation, the RFC5 suppressor mutations
did not interfere with the methylmethane sulfonate- or hydroxyurea-induced
phosphorylation of Rad53p. The Rfc5p suppressor substitutions mapped to amino
acid positions in the conserved RFC box motifs IV-VII. Comparisons of the
structures of related RFC box-containing proteins suggest that these RFC motifs
may function to coordinate interactions between neighboring subunits of
multi-subunit ATPases.
PMID: 11129041 [PubMed - indexed for MEDLINE]
306: J Mol Biol 2001 Jan 12;305(2):219-30
Isolation of mutations that disrupt cooperative DNA binding by the Drosophila
bicoid protein.
Burz DS, Hanes SD.
Molecular Genetics Program Wadsworth Center, New York State Department of
Health, USA.
Cooperative DNA binding is thought to contribute to the ability of the
Drosophila melanogaster protein, Bicoid, to stimulate transcription of target
genes in precise sub-domains within the embryo. As a first step toward testing
this idea, we devised a genetic screen to isolate mutations in Bicoid that
specifically disrupt cooperative interactions, but do not disrupt DNA
recognition or transcription activation. The screen was carried out in
Saccharomyces cerevisiae and 12 cooperativity mutants were identified. The
mutations map across most of the Bicoid protein, with some located within the
DNA-binding domain (homeodomain). Four homeodomain mutants were characterized in
yeast and shown to activate a single-site reporter gene to levels comparable to
that of wild-type, indicating that DNA binding per se is not affected. However,
these mutants failed to show cooperative coupling between high and low-affinity
sites, and showed reduced activation of a reporter gene carrying a natural
Drosophila enhancer. Homology modeling indicated that none of the four mutations
is in residues that contact DNA. Instead, these residues are likely to interact
with other DNA-bound Bicoid monomers or other parts of the Bicoid protein. In
vitro, the isolated homeodomains did not show strong cooperativity defects,
supporting the idea that other regions of Bicoid are also important for
cooperativity. This study describes the first systematic screen to identify
cooperativity mutations in a eukaryotic DNA-binding protein. Copyright 2001
Academic Press.
PMID: 11124901 [PubMed - indexed for MEDLINE]
307: J Biol Chem 2001 Apr 20;276(16):13034-8
A mutational epitope for cytochrome C binding to the apoptosis protease
activation factor-1.
Yu T, Wang X, Purring-Koch C, Wei Y, McLendon GL.
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Cytochrome c (Cc) binding to apoptosis protease activation factor-1 (Apaf-1) is
a critical activation step in the execution phase of apoptosis. Here we report
studies that help define the Cc:Apaf-1 binding surface. It is shown that a large
number of Cc residues, including residues 7, 25, 39, 62-65, and 72, are involved
in the Cc:Apaf-1 interaction. Mutation of residue 72 eliminated Cc activity
whereas mutations of residues 7, 25, 39, and 62-65 showed reduced activity in an
additive fashion. The implications of this binding model for both recognition
and modulation of protein-protein interactions are briefly discussed.
PMID: 11112785 [PubMed - indexed for MEDLINE]
308: J Mol Biol 2000 Dec 15;304(5):941-51
Structure of the FHA1 domain of yeast Rad53 and identification of binding sites
for both FHA1 and its target protein Rad9.
Liao H, Yuan C, Su MI, Yongkiettrakul S, Qin D, Li H, Byeon IJ, Pei D, Tsai MD.
Departments of Chemistry and Biochemistry, The Ohio State Biochemistry Program,
and Campus Chemical Instrument Center, The Ohio State University, Columbus, OH
43210, USA.
Forkhead-associated (FHA) domains have been shown to recognize both pThr and
pTyr-peptides. The solution structures of the FHA2 domain of Rad53 from
Saccharomyces cerevisiae, and its complex with a pTyr peptide, have been
reported recently. We now report the solution structure of the other FHA domain
of Rad53, FHA1 (residues 14-164), and identification of binding sites of FHA1
and its target protein Rad9. The FHA1 structure consists of 11 beta-strands,
which form two large twisted anti-parallel beta-sheets folding into a
beta-sandwich. Three short alpha-helices were also identified. The beta-strands
are linked by several loops and turns. These structural features of free FHA1
are similar to those of free FHA2, but there are significant differences in the
loops. Screening of a peptide library [XXX(pT)XXX] against FHA1 revealed an
absolute requirement for Asp at the +3 position and a preference for Ala at the
+2 position. These two criteria are met by a pThr motif (192)TEAD(195) in Rad9.
Surface plasmon resonance analysis showed that a pThr peptide containing this
motif, (188)SLEV(pT)EADATFVQ(200) from Rad9, binds to FHA1 with a K(d) value of
0.36 microM. Other peptides containing pTXXD sequences also bound to FHA1, but
less tightly (K(d)=4-70 microM). These results suggest that Thr192 of Rad9 is
the likely phosphorylation site recognized by the FHA1 domain of Rad53. The
tight-binding peptide was then used to identify residues of FHA1 involved in the
interaction with the pThr peptide. The results are compared with the
interactions between the FHA2 domain and a pTyr peptide derived from Rad9
reported previously. Copyright 2000 Academic Press.
PMID: 11124038 [PubMed - indexed for MEDLINE]
309: Curr Opin Microbiol 2000 Dec;3(6):573-81
Pheromone response, mating and cell biology.
Elion EA.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, 240 Longwood Avenue, Boston, MA 02115, USA. eline_elion@hms.harvard.edu
Saccharomyces cerevisiae responds to mating pheromones by activating a
receptor-G-protein-coupled mitogen-activated protein kinase (MAPK) cascade that
is also used by other signaling pathways. The activation of the MAPK cascade may
involve conformational changes through prebound receptor and heterotrimeric
G-protein. G beta may then recruit Cdc42-bound MAPKKKK Ste20 to MAPKKK Ste11
through direct interactions with Ste20 and the Ste5 scaffold. Ste20 activates
Ste11 by derepressing an autoinhibitory domain. An underlying nuclear shuttling
machinery may be required for proper recruitment of Ste5 to G beta. Subsequent
polarized growth is mediated by a similar mechanism involving Far1, which binds
G beta in addition to Cdc24 and Bem1. Far1 and Cdc24 also undergo nuclear
shuttling and the nuclear pool of Far1 may temporally regulate access of Cdc24
to the cell cortex.
Publication Types:
Review
Review, Tutorial
PMID: 11121776 [PubMed - indexed for MEDLINE]
310: Biochim Biophys Acta 2000 Dec 11;1499(1-2):85-100
Mutations in SPC110, encoding the yeast spindle pole body calmodulin-binding
protein, cause defects in cell integrity as well as spindle formation.
Stirling DA, Stark MJ.
Department of Biochemistry, University of Dundee, MSI/WTB Complex, DD1 5EH,
Dundee, UK. d.a.stirling@dundee.ac.uk
The 110 kDa spindle pole body component, Spc110p, is an essential target of
calmodulin in budding yeast. Cells with mutations which reduce calmodulin
binding to Spc110p are unable to form a mitotic spindle and die. Here we show
that these effects can be overcome either directly by increasing extracellular
calcium or calmodulin expression, which reverse the primary spindle defect, or
indirectly through increased extracellular osmolarity or high dosage of MID2 or
SLG1/HCS77/WSC1 which preserve viability. We propose that overcoming a cell
integrity defect associated with the mitotic arrest enables the defective
spindle pole bodies to provide sufficient function for proliferation of a large
proportion of mutant cells. Our findings demonstrate a role for calcium in the
Spc110p-calmodulin interaction in vivo and have important general implications
for the interpretation of genetic interactions involving cell integrity genes.
PMID: 11118641 [PubMed - indexed for MEDLINE]
311: Biochim Biophys Acta 2000 Dec 11;1499(1-2):63-73
Oligomerization properties of the acidic ribosomal P-proteins from Saccharomyces
cerevisiae: effect of P1A protein phosphorylation on the formation of the
P1A-P2B hetero-complex.
Tchorzewski M, Boguszewska A, Dukowski P, Grankowski N.
Maria Curie-Skllodowska University, Institute of Microbiology and Biotechnology,
Department of Molecular Biology, Akademicka Street 19, 20-033, Lublin, Poland.
Acidic ribosomal P-proteins form, in all eukaryotic cells, a lateral
protuberance, the so-called 'stalk', which is directly involved in translational
activity of the ribosomes. In Saccharomyces cerevisiae cells, there are four
distinct P-proteins: P1A, P1B, P2A and P2B. In spite of the high level of their
structural homology, they are not completely equivalent and may perform
different functions. As yet, the protein-protein interactions between yeast
P-proteins have not been fully defined. In this paper, the interplay between
yeast P-proteins has been investigated by means of a two-hybrid system, chemical
cross-linking and gel filtration. The data presented herein show that all
P-proteins are able to form homo-oligomeric complexes. By analyzing
hetero-interactions, we were able to detect strong interactions between P1A and
P2B proteins. Additionally, the pair of P1B and P2A proteins is also able to
form a hetero-complex, though at a very low efficiency. All P-proteins are
phosphorylated by numerous protein kinases. Using the multifunctional protein
kinase CK II, we have shown that incorporation of phosphate into P1A protein can
exert its effect on the hetero-oligomerization process, namely by preventing the
formation of the hetero-oligomer P1A-P/P2B. These findings are the first to show
differences in the oligomerization behavior of the yeast P-proteins; moreover,
they emphasize a significant impact of the phosphorylation on the formations of
P-protein complex.
PMID: 11118639 [PubMed - indexed for MEDLINE]
312: J Biol Chem 2001 Mar 16;276(11):8616-22
Nam1p, a protein involved in RNA processing and translation, is coupled to
transcription through an interaction with yeast mitochondrial RNA polymerase.
Rodeheffer MS, Boone BE, Bryan AC, Shadel GS.
Department of Biochemistry, Emory University School of Medicine, Rollins
Research Center, Atlanta, Georgia 30322, USA.
Alignment of three fungal mtRNA polymerases revealed conserved amino acid
sequences in an amino-terminal region of the Saccharomyces cerevisiae enzyme
implicated previously as harboring an important functional domain. Phenotypic
analysis of deletion and point mutations, in conjunction with a yeast two-hybrid
assay, revealed that Nam1p, a protein involved in RNA processing and translation
in mitochondria, binds specifically to this domain. The significance of this
interaction in vivo was demonstrated by the fact that the temperature-sensitive
phenotype of a deletion mutation (rpo41Delta2), which impinges on this
amino-terminal domain, is suppressed by overproducing Nam1p. In addition,
mutations in the amino-terminal domain result specifically in decreased
steady-state levels of mature mitochondrial CYTB and COXI transcripts, which is
a primary defect observed in NAM1 null mutant yeast strains. Finally, one point
mutation (R129D) did not abolish Nam1p binding, yet displayed an obvious
COX1/CYTB transcript defect. This mutation exhibited the most severe
mitochondrial phenotype, suggesting that mutations in the amino-terminal domain
can perturb other critical interactions, in addition to Nam1p binding, that
contribute to the observed phenotypes. These results implicate the
amino-terminal domain of mtRNA polymerases in coupling additional factors and
activities involved in mitochondrial gene expression directly to the
transcription machinery.
PMID: 11118450 [PubMed - indexed for MEDLINE]
313: Plant Physiol 2000 Dec;124(4):1844-53
Interaction specificity of Arabidopsis calcineurin B-like calcium sensors and
their target kinases.
Kim KN, Cheong YH, Gupta R, Luan S.
Department of Plant and Microbial Biology, University of California, Berkeley,
California 94720, USA.
Calcium is a critical component in a number of plant signal transduction
pathways. A new family of calcium sensors called calcineurin B-like proteins
(AtCBLs) have been recently identified from Arabidopsis. These calcium sensors
have been shown to interact with a family of protein kinases (CIPKs). Here we
report that each individual member of AtCBL family specifically interacts with a
subset of CIPKs and present structural basis for the interaction and for the
specificity underlying these interactions. Although the C-terminal region of
CIPKs is responsible for interaction with AtCBLs, the N-terminal region of CIPKs
is also involved in determining the specificity of such interaction. We have
also shown that all three EF-hand motifs in AtCBL members are required for the
interaction with CIPKs. Several AtCBL members failed to interact with any of the
CIPKs presented in this study, suggesting that these AtCBL members either have
other CIPKs as targets or they target distinct proteins other than CIPKs. These
results may provide structural basis for the functional specificity of CBL
family of calcium sensors and their targets.
PMID: 11115898 [PubMed - indexed for MEDLINE]
314: Curr Biol 2000 Nov 30;10(23):1519-22
Yeast Eap1p, an eIF4E-associated protein, has a separate function involving
genetic stability.
Chial HJ, Stemm-Wolf AJ, McBratney S, Winey M.
Present address: Department of Biology, St Olaf College, Northfield, Minnesota
55057-1098, USA.
A rate-limiting step during translation initiation in eukaryotic cells involves
binding of the initiation factor eIF4E to the 7-methylguanosine-containing cap
of mRNAs. Overexpression of eIF4E leads to malignant transformation [1-3], and
eIF4E is elevated in many human cancers [4-7]. In mammalian cells, three
eIF4E-binding proteins each interact with eIF4E and inhibit its function [8-10].
In yeast, EAP1 encodes a protein that binds eIF4E and inhibits cap-dependent
translation in vitro [11]. A point mutation in the canonical eIF4E-binding motif
of Eap1p blocks its interaction with eIF4E [11]. Here, we characterized the
genetic interactions between EAP1 and NDC1, a gene whose function is required
for duplication of the spindle pole body (SPB) [12], the centrosome-equivalent
organelle in yeast that functions as the centrosome. We found that the deletion
of EAP1 is lethal when combined with the ndc1-1 mutation. Mutations in NDC1 or
altered NDC1 gene dosage lead to genetic instability [13,14]. Yeast strains
lacking EAP1 also exhibit genetic instability. We tested whether these
phenotypes are due to loss of EAP1 function in regulating translation. We found
that both the synthetic lethal phenotype and the genetic instability phenotypes
are rescued by a mutant allele of EAP1 that is unable to bind eIF4E. Our
findings suggest that Eap1p carries out an eIF4E-independent function to
maintain genetic stability, most likely involving SPBs.
PMID: 11114520 [PubMed - indexed for MEDLINE]
315: Proc Natl Acad Sci U S A 2000 Dec 19;97(26):14400-5
Activation of the myocyte enhancer factor-2 transcription factor by
calcium/calmodulin-dependent protein kinase-stimulated binding of 14-3-3 to
histone deacetylase 5.
McKinsey TA, Zhang CL, Olson EN.
Department of Molecular Biology, The University of Texas Southwestern Medical
Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA.
Skeletal muscle differentiation is controlled by interactions between myocyte
enhancer factor-2 (MEF2) and myogenic basic helix-loop-helix transcription
factors. Association of MEF2 with histone deacetylases (HDAC) -4 and -5 results
in repression of MEF2 target genes and inhibition of myogenesis.
Calcium/calmodulin-dependent protein kinase (CaMKNonsense-mediated mRNA decay (NMD), also called mRNA surveillance, is an
important pathway used by all organisms that have been tested to degrade mRNAs
that prematurely terminate translation and, as a consequence, eliminate the
production of aberrant proteins that could be potentially harmful. In mammalian
cells, NMD appears to involve splicing-dependent alterations to mRNA as well as
ribosome-associated components of the translational apparatus. To date, human
(h) Upf1 protein (p) (hUpf1p), a group 1 RNA helicase named after its
Saccharomyces cerevisiae orthologue that functions in both translation
termination and NMD, has been the only factor shown to be required for NMD in
mammalian cells. Here, we describe human orthologues to S. cerevisiae Upf2p and
S. cerevisiae Upf3p (Caenorhabditis elegans SMG-4) based on limited amino acid
similarities. The existence of these orthologues provides evidence for a higher
degree of evolutionary conservation of NMD than previously appreciated.
Interestingly, human orthologues to S. cerevisiae Upf3p (C. elegans SMG-4)
derive from two genes, one of which is X-linked and both of which generate
multiple isoforms due to alternative pre-mRNA splicing. We demonstrate using
immunoprecipitations of epitope-tagged proteins transiently produced in HeLa
cells that hUpf2p interacts with hUpf1p, hUpf3p-X, and hUpf3p, and we define the
domains required for the interactions. Furthermore, we find by using indirect
immunofluorescence that hUpf1p is detected only in the cytoplasm, hUpf2p is
detected primarily in the cytoplasm, and hUpf3p-X localizes primarily to nuclei.
The finding that hUpf3p-X is a shuttling protein provides additional indication
that NMD has both nuclear and cytoplasmic components.
PMID: 11113196 [PubMed - indexed for MEDLINE]
317: Biochemistry 2000 Dec 19;39(50):15462-74
Synthesis and biophysical analysis of transmembrane domains of a Saccharomyces
cerevisiae G protein-coupled receptor.
Xie H, Ding FX, Schreiber D, Eng G, Liu SF, Arshava B, Arevalo E, Becker JM,
Naider F.
Department of Chemistry, The College of Staten Island Staten Island, New York
10314, USA.
The Ste2p receptor for alpha-factor, a tridecapeptide mating pheromone of the
yeast Saccharomyces cerevisiae, belongs to the G protein-coupled family of
receptors. In this paper we report on the synthesis of peptides corresponding to
five of the seven transmembrane domains (M1-M5) and two homologues of the sixth
transmembrane domain corresponding to the wild-type sequence and a mutant
sequence found in a constitutively active receptor. The secondary structures of
all new transmembrane peptides and previously synthesized peptides corresponding
to domains 6 and 7 were assessed using a detailed CD analysis in
trifluoroethanol, trifluoroethanol-water mixtures, sodium dodecyl sulfate
micelles, and dimyristoyl phosphatidyl choline bilayers. Tryptophan fluorescence
quenching experiments were used to assess the penetration of the membrane
peptides into lipid bilayers. All peptides were predominantly (40-80%) helical
in trifluoroethanol and most trifluoroethanol-water mixtures. In contrast, two
of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVIFTGDNFKKKG) and M6-31
(KQFDSFHILLINleSAQSLLVPSIIFILAYSLK) formed stable beta-sheet structures in both
sodium dodecyl sulfate micelles and DMPC bilayers. Polyacrylamide gel
electrophoresis showed that these two peptides formed high molecular aggregates
in the presence of SDS whereas all other peptides moved as monomeric species.
The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS) corresponding to the sequence
in the constitutive mutant was predominantly helical under a variety of
conditions, whereas the homologous wild-type sequence
(KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to form
beta-structures. These results demonstrate a connection between a conformational
shift in secondary structure, as detected by biophysical techniques, and
receptor function. The aggregation of particular transmembrane domains may also
reflect a tendency for intermolecular interactions that occur in the membrane
environment facilitating formation of receptor dimers or multimers.
PMID: 11112532 [PubMed - indexed for MEDLINE]
318: Biochim Biophys Acta 2000 Dec 15;1529(1-3):63-88
Sterol methyl transferase: enzymology and inhibition.
Nes WD.
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX,
79409-1061, USA. u0nes@ttacs.ttu.edu
Sterol C-methylations catalyzed by the (S)-adenosyl-L-methionine:
Delta(24)-sterol methyl transferase (SMT) have provided the focus for study of
electrophilic alkylations, a reaction type of functional importance in C-C bond
formation of natural products. SMTs occur generally in nature, but do not occur
in animal systems, suggesting that the difference in sterol synthetic pathways
can be exploited therapeutically and in insect-plant interactions. The SMT genes
from several plants and fungi have been cloned, sequenced and expressed in
bacteria or yeast and bioengineered into tobacco or tomato plants. These enzymes
share significant amino acid sequence similarity in the putative sterol and
AdoMet binding sites. Investigations of the molecular recognition of sterol
fitness and studies with stereospecifically labeled substrates as well as
various sterol analogs assayed with native or mutant SMTs from fungi and plants
have been carried out recently in our own and other laboratories. These analyses
have led to an active-site model, referred to as the 'steric-electric plug'
model, which is consistent with a non-covalent mechanism involving the
intermediacy of a 24beta-methyl (or ethyl) sterol bound to the ternary complex.
Despite the seeming differences between fungal and plant SMT activities the
recent data indicate that a distinct SMT or family of SMTs exist in these
organisms which bind and transform sterols according to a similar mechanistic
plan. Vascular plants have been found to express different complements of
C(1)/C(2)-activities in the form of at least three SMT isoforms. This enzyme
multiplicity can be a target of regulatory control to affect phytosterol
homeostasis in transgenic plants. The state of our current understanding of SMT
enzymology and inhibition is presented.
Publication Types:
Review
Review, Tutorial
PMID: 11111078 [PubMed - indexed for MEDLINE]
319: J Comput Biol 2000;7(3-4):601-20
Using Bayesian networks to analyze expression data.
Friedman N, Linial M, Nachman I, Pe'er D.
School of Computer Science and Engineering, Hebrew University, Jerusalem,
Israel. nir@cs.huji.ac.il
DNA hybridization arrays simultaneously measure the expression level for
thousands of genes. These measurements provide a "snapshot" of transcription
levels within the cell. A major challenge in computational biology is to
uncover, from such measurements, gene/protein interactions and key biological
features of cellular systems. In this paper, we propose a new framework for
discovering interactions between genes based on multiple expression
measurements. This framework builds on the use of Bayesian networks for
representing statistical dependencies. A Bayesian network is a graph-based model
of joint multivariate probability distributions that captures properties of
conditional independence between variables. Such models are attractive for their
ability to describe complex stochastic processes and because they provide a
clear methodology for learning from (noisy) observations. We start by showing
how Bayesian networks can describe interactions between genes. We then describe
a method for recovering gene interactions from microarray data using tools for
learning Bayesian networks. Finally, we demonstrate this method on the S.
cerevisiae cell-cycle measurements of Spellman et al. (1998).
PMID: 11108481 [PubMed - indexed for MEDLINE]
320: Mol Cell 2000 Nov;6(5):1169-82
The molecular basis of FHA domain:phosphopeptide binding specificity and
implications for phospho-dependent signaling mechanisms.
Durocher D, Taylor IA, Sarbassova D, Haire LF, Westcott SL, Jackson SP, Smerdon
SJ, Yaffe MB.
Wellcome Trust/Cancer Research Campaign Institute of Cancer and Developmental
Biology and Department of Zoology University of Cambridge CB2 1QR, Cambridge,
United Kingdom.
Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules
that appear to function through interactions with phosphorylated target
molecules. We have used oriented peptide library screening to determine the
optimal phosphopeptide binding motifs recognized by several FHA domains,
including those within a number of DNA damage checkpoint kinases, and determined
the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide,
at 1.6 A resolution. The structure reveals a striking similarity to the MH2
domains of Smad tumor suppressor proteins and reveals a mode of peptide binding
that differs from SH2, 14-3-3, or PTB domain complexes. These results have
important implications for DNA damage signaling and CHK2-dependent tumor
suppression, and they indicate that FHA domains play important and unsuspected
roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes.
PMID: 11106755 [PubMed - indexed for MEDLINE]
321: Genetics 2000 Dec;156(4):1503-17
Genetic and physical interactions between factors involved in both cell cycle
progression and pre-mRNA splicing in Saccharomyces cerevisiae.
Ben-Yehuda S, Dix I, Russell CS, McGarvey M, Beggs JD, Kupiec M.
Department of Molecular Microbiology and Biotechnology, Tel Aviv University,
Ramat Aviv 69978, Israel.
The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different
cellular processes: pre-mRNA splicing and cell cycle progression. The
Prp17/Cdc40 protein participates in the second step of the splicing reaction
and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature
arrest in the G2 phase of the cell cycle. Here we describe the identification of
nine genes that, when mutated, show synthetic lethality with the
prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p,
Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and
SYF3, represent genes also involved in cell cycle progression and in pre-mRNA
splicing. Syf1p and Syf3p are highly conserved proteins containing several
copies of a repeated motif, which we term RTPR. This newly defined motif is
shared by proteins involved in RNA processing and represents a subfamily of the
known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens
and biochemical analysis, we show that the SYF gene products interact with each
other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss
the role played by these proteins in splicing and cell cycle progression.
PMID: 11102353 [PubMed - indexed for MEDLINE]
322: Nat Struct Biol 2000 Dec;7(12):1156-64
Crystal structures of ribosome anti-association factor IF6.
Groft CM, Beckmann R, Sali A, Burley SK.
Laboratories of Molecular Biophysics, The Rockefeller University, 1230 York
Avenue, New York, New York 10021, USA.
Ribosome anti-association factor eIF6 (originally named according to translation
initiation terminology as eukaryotic initiation factor 6) binds to the large
ribosomal subunit, thereby preventing inappropriate interactions with the small
subunit during initiation of protein synthesis. We have determined the X-ray
structures of two IF6 homologs, Methanococcus jannaschii archaeal aIF6 and
Sacchromyces cerevisiae eIF6, revealing a phylogenetically conserved 25 kDa
protein consisting of five quasi identical alpha/beta subdomains arrayed about a
five-fold axis of pseudosymmetry. Yeast eIF6 prevents ribosomal subunit
association. Comparative protein structure modeling with other known archaeal
and eukaryotic homologs demonstrated the presence of two conserved surface
regions, one or both of which may bind the large ribosomal subunit.
PMID: 11101899 [PubMed - indexed for MEDLINE]
323: Nat Biotechnol 2000 Dec;18(12):1257-61
Comment in:
Nat Biotechnol. 2000 Dec;18(12):1242-3.
A network of protein-protein interactions in yeast.
Schwikowski B, Uetz P, Fields S.
The Institute for Systems Biology, 4225 Roosevelt Way NE, Suite 200, Seattle, WA
98105, USA.
A global analysis of 2,709 published interactions between proteins of the yeast
Saccharomyces cerevisiae has been performed, enabling the establishment of a
single large network of 2,358 interactions among 1,548 proteins. Proteins of
known function and cellular location tend to cluster together, with 63% of the
interactions occurring between proteins with a common functional assignment and
76% occurring between proteins found in the same subcellular compartment.
Possible functions can be assigned to a protein based on the known functions of
its interacting partners. This approach correctly predicts a functional category
for 72% of the 1,393 characterized proteins with at least one partner of known
function, and has been applied to predict functions for 364 previously
uncharacterized proteins.
PMID: 11101803 [PubMed - indexed for MEDLINE]
324: Proc Natl Acad Sci U S A 2000 Dec 5;97(25):13732-7
A new screen for protein interactions reveals that the Saccharomyces cerevisiae
high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1
promoter.
Laser H, Bongards C, Schuller J, Heck S, Johnsson N, Lehming N.
Max-Delbruck-Laboratorium in der Max-Planck-Gesellschaft, Carl-von-Linne-Weg 10,
50829 Cologne, Germany.
The split-ubiquitin assay detects protein interactions in vivo. To identify
proteins interacting with Gal4p and Tup1p, two transcriptional regulators, we
converted the split-ubiquitin assay into a generally applicable screen for
binding partners of specific proteins in vivo. A library of genomic
Saccharomyces cerevisiae DNA fragments fused to the N-terminal half of ubiquitin
was constructed and transformed into yeast strains carrying either Gal4p or
Tup1p as a bait. Both proteins were C-terminally extended by the C-terminal half
of ubiquitin followed by a modified Ura3p with an arginine in position 1, a
destabilizing residue in the N-end rule pathway. The bait fusion protein alone
is stable and enzymatically active. However, upon interaction with its prey, a
native-like ubiquitin is reconstituted. RUra3p is then cleaved off by the
ubiquitin-specific proteases and rapidly degraded by the N-end rule pathway. In
both screens, Nhp6B was identified as a protein in close proximity to Gal4p as
well as to Tup1p. Direct interaction between either protein and Nhp6B was
confirmed by coprecipitation assays. Genetic analysis revealed that Nhp6B, a
member of the HMG1 family of DNA-binding proteins, can influence transcriptional
activation as well as repression at a specific locus in the chromosome of the
yeast S. cerevisiae.
PMID: 11095729 [PubMed - indexed for MEDLINE]
325: Nucleic Acids Res 2000 Dec 1;28(23):4665-73
Signaling through regulated transcription factor interaction: mapping of a
regulatory interaction domain in the Myb-related Bas1p.
Pinson B, Kongsrud TL, Ording E, Johansen L, Daignan-Fornier B, Gabrielsen OS.
Department of Biochemistry, University of Oslo, PO Box 1041, Blindern, N-0316
Oslo 3, Norway.
Gene activation in eukaryotes is inherently combinatorial depending on
cooperation between different transcription factors. An example where this
cooperation seems to be directly exploited for regulation is the Bas1p/Bas2p
couple in yeast. Bas1p is a Myb-related transcription factor that acts together
with the homeodomain-related Bas2p (Pho2p) to regulate purine and histidine
biosynthesis genes in response to extracellular purine limitation. We show that
fusion of the two factors abolished adenine repression, suggesting that what is
regulated by adenine is the Bas1p-Bas2p interaction. Analysis of Bas1p deletions
revealed a critical domain (Bas1p interaction and regulatory domain, BIRD)
acting in two-hybrid assays as an adenine-dependent Bas1p-Bas2p interaction
domain. BIRD had a dual function, as an internal repressor of a centrally
located Bas1p transactivation domain on the ADE1 promoter and as a
Bas2p-dependent activator on the HIS4 promoter. This promoter-dependent behavior
reflected a differential binding to the two promoters in vivo. On ADE1 Bas1p
bound the promoter efficiently by itself, but required adenine limitation and
Bas2p interaction through BIRD for derepression. On HIS4 efficient promoter
binding and derepression required both factors and adenine limitation. We
propose a promoter-dependent model for adenine regulation in yeast based on
controlled Bas1p-Bas2p interactions through BIRD and exploited differentially by
the two promoters.
PMID: 11095676 [PubMed - indexed for MEDLINE]
326: Proc Natl Acad Sci U S A 2000 Nov 21;97(24):13203-8
Comment in:
Proc Natl Acad Sci U S A. 2000 Nov 21;97(24):12935-6.
A computationally directed screen identifying interacting coiled coils from
Saccharomyces cerevisiae.
Newman JR, Wolf E, Kim PS.
Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research,
Department of Biology, Massachusetts Institute of Technology, Nine Cambridge
Center, Cambridge, MA 02142, USA.
Computational methods can frequently identify protein-interaction motifs in
otherwise uncharacterized open reading frames. However, the identification of
candidate ligands for these motifs (e.g., so that partnering can be determined
experimentally in a directed manner) is often beyond the scope of current
computational capabilities. One exception is provided by the coiled-coil
interaction motif, which consists of two or more alpha helices that wrap around
each other: the ligands for coiled-coil sequences are generally other
coiled-coil sequences, thereby greatly simplifying the motif/ligand recognition
problem. Here, we describe a two-step approach to identifying protein-protein
interactions mediated by two-stranded coiled coils that occur in Saccharomyces
cerevisiae. Coiled coils from the yeast genome are first predicted
computationally, by using the multicoil program, and associations between coiled
coils are then determined experimentally by using the yeast two-hybrid assay. We
report 213 unique interactions between 162 putative coiled-coil sequences. We
evaluate the resulting interactions, focusing on associations identified between
components of the spindle pole body (the yeast centrosome).
PMID: 11087867 [PubMed - indexed for MEDLINE]
327: Proc Natl Acad Sci U S A 2000 Nov 21;97(24):13080-5
Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase.
Caruthers JM, Johnson ER, McKay DB.
Department of Structural Biology, Stanford University School of Medicine,
Stanford, CA 94305, USA.
The eukaryotic translation initiation factor 4A (eIF4A) is a member of the
DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples an
ATPase activity to RNA binding and unwinding. Previous work has provided the
structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those
results, we have solved the structure of the carboxyl-terminal domain of eIF4A
with data to 1.75 A resolution; it has a parallel alpha-beta topology that
superimposes, with minor variations, on the structures and conserved motifs of
the equivalent domain in other, distantly related helicases. Using data to 2.8 A
resolution and molecular replacement with the refined model of the
carboxyl-terminal domain, we have completed the structure of full-length eIF4A;
it is a "dumbbell" structure consisting of two compact domains connected by an
extended linker. By using the structures of other helicases as a template,
compact structures can be modeled for eIF4A that suggest (i) helicase motif IV
binds RNA; (ii) Arg-298, which is conserved in the DEA(D/H)-box RNA helicase
family but is absent from many other helicases, also binds RNA; and (iii) motifs
V and VI "link" the carboxyl-terminal domain to the amino-terminal domain
through interactions with ATP and the DEA(D/H) motif, providing a mechanism for
coupling ATP binding and hydrolysis with conformational changes that modulate
RNA binding.
PMID: 11087862 [PubMed - indexed for MEDLINE]
328: Biochemistry 2000 Nov 21;39(46):14103-12
Biochemical and structural analysis of the interaction between the UBA(2) domain
of the DNA repair protein HHR23A and HIV-1 Vpr.
Withers-Ward ES, Mueller TD, Chen IS, Feigon J.
Department of Microbiology, University of California at Los Angeles, Los
Angeles, California 90095, USA.
The DNA repair protein HHR23A is a highly conserved protein that functions in
nucleotide excision repair. HHR23A contains two ubiquitin associated domains
(UBA) that are conserved in a number of proteins with diverse functions involved
in ubiquitination, UV excision repair, and signaling pathways via protein
kinases. The cellular binding partners of UBA domains remain unclear; however,
we previously found that the HHR23A UBA(2) domain interacts specifically with
the HIV-1 Vpr protein. Analysis of the low resolution solution structure of
HHR23A UBA(2) revealed a hydrophobic loop region of the UBA(2) domain that we
predicted was the interface for protein/protein interactions. Here we present
results of in vitro binding studies that demonstrate the requirement of this
hydrophobic loop region for interaction with human immunodeficiency virus
(HIV-1) Vpr. A single point mutation of the Pro at residue 333 to a Glu totally
abolishes the binding of HIV-1 Vpr to UBA(2). High resolution NMR structures of
the binding deficient UBA(2) mutant P333E as well as of the wild-type UBA(2)
domain were determined to compare the effect of this mutation on the structure.
Small but significant differences are observed only locally at the site of the
mutation. The biochemical and structural analysis confirms the function of the
HHR23A UBA(2) GFP-loop as the protein/protein interacting domain.
PMID: 11087358 [PubMed - indexed for MEDLINE]
329: Curr Biol 2000 Nov 2;10(21):1375-8
The spindle checkpoint of Saccharomyces cerevisiae responds to separable
microtubule-dependent events.
Daum JR, Gomez-Ospina N, Winey M, Burke DJ.
Department of Biochemistry and Molecular Genetics, University of Virginia
Medical Center, Charlottesville, Virginia 22908-0733, USA.
The spindle checkpoint regulates microtubule-based chromosome segregation and
helps to maintain genomic stability [1,2]. Mutational inactivation of spindle
checkpoint genes has been implicated in the progression of several types of
human cancer. Recent evidence from budding yeast suggests that the spindle
checkpoint is complex. Order-of-function experiments have defined two separable
pathways within the checkpoint. One pathway, defined by MAD2, controls the
metaphase-to-anaphase transition and the other, defined by BUB2, controls the
exit from mitosis [3-6]. The relationships between the separate branches of the
checkpoint, and especially the events that trigger the pathways, have not been
defined. We localized a Bub2p-GFP fusion protein to the cytoplasmic side of the
spindle pole body and used a kar9 mutant to show that cells with misoriented
spindles are arrested in anaphase of mitosis. We used a kar9 bub2 double mutant
to show that the arrest is BUB2 dependent. We conclude that the separate
pathways of the spindle checkpoint respond to different classes of microtubules.
The MAD2 branch of the pathway responds to kinetochore microtubule interactions
and the BUB2 branch of the pathway operates within the cytoplasm, responding to
spindle misorientation.
PMID: 11084338 [PubMed - indexed for MEDLINE]
330: Anal Chem 2000 Nov 1;72(21):5151-7
A fluorescent indicator for detecting protein-protein interactions in vivo based
on protein splicing.
Ozawa T, Nogami S, Sato M, Ohya Y, Umezawa Y.
Department of Chemistry, School of Science, University of Tokyo, Japan.
We describe a new method with general applicability for monitoring any
protein-protein interaction in vivo. The principle is based on a protein
splicing system, which involves a self-catalyzed excision of protein splicing
elements, or inteins, from flanking polypeptide sequences, or exteins, leading
to formation of a new protein in which the exteins are linked directly by a
peptide bond. As the exteins, split N- and C-terminal halves of enhanced green
fluorescent protein (EGFP) were used. When a single peptide consisting of an
intein derived from Saccharomyces cerevisiae intervening the split EGFP was
expressed in Escherichia coli, the two external regions of EGFP were ligated,
thereby forming the EGFP corresponding fluorophore. Genetic alteration of the
intein, which involved large deletion of the central region encoding 104 amino
acids, was performed. In the expression of the residual N- and C-terminal intein
fragments each fused to the split EGFP exteins, the splicing in trans did not
proceed. However, upon coexpression of calmodulin and its target peptide M13,
each connected to the N- and C-terminal inteins, fluorescence of EGFP was
observed. These results demonstrate that interaction of calmodulin and M13
triggers the refolding of intein, which induces the protein splicing, thereby
folding the ligated extein correctly for yielding the EGFP fluorophore. This
method opens a new way not only to screen protein-protein interactions but also
to visualize the interaction in vivo in transgenic animals.
PMID: 11080857 [PubMed - indexed for MEDLINE]
331: EMBO J 2000 Nov 15;19(22):6141-9
The structural basis for the recognition of acetylated histone H4 by the
bromodomain of histone acetyltransferase gcn5p.
Owen DJ, Ornaghi P, Yang JC, Lowe N, Evans PR, Ballario P, Neuhaus D, Filetici
P, Travers AA.
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK and
Centro di studio per gli Acidi Nucleici, CNR, c/o Dipartimento di Genetica e
Biologia Molecolare, Universita 'La Sapienza', P.le A.Moro 5, 00185 Roma, Italy.
The bromodomain is an approximately 110 amino acid module found in histone
acetyltransferases and the ATPase component of certain nucleosome remodelling
complexes. We report the crystal structure at 1.9 A resolution of the
Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide
corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine
16. We show that this bromodomain preferentially binds to peptides containing an
N:-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact
with the bromodomain. The primary interaction is the N:-acetyl lysine binding in
a cleft with the specificity provided by the interaction of the amide nitrogen
of a conserved asparagine with the oxygen of the acetyl carbonyl group. A
network of water-mediated H-bonds with protein main chain carbonyl groups at the
base of the cleft contributes to the binding. Additional side chain binding
occurs on a shallow depression that is hydrophobic at one end and can
accommodate charge interactions at the other. These findings suggest that the
Gcn5p bromodomain may discriminate between different acetylated lysine residues
depending on the context in which they are displayed.
PMID: 11080160 [PubMed - indexed for MEDLINE]
332: Methods Enzymol 2000;328:297-321
Using the yeast three-hybrid system to detect and analyze RNA-protein
interactions.
Kraemer B, Zhang B, SenGupta D, Fields S, Wickens M.
Department of Biochemistry, University of Wisconsin, Madison 53706, USA.
PMID: 11075352 [PubMed - indexed for MEDLINE]
333: Methods Enzymol 2000;328:111-27
The yeast tribid system: cDNA expression cloning of protein interactions
dependent on posttranslational modifications.
Kochan JP, Volpers C, Osborne MA.
Department of Metabolic Diseases, Hoffmann-La Roche, Inc., Nutley, New Jersey
07110, USA.
PMID: 11075342 [PubMed - indexed for MEDLINE]
334: Methods Enzymol 2000;328:89-103
Yeast three-hybrid system for detecting ligand-receptor interactions.
Griffith EC, Licitra EJ, Liu JO.
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139,
USA.
PMID: 11075340 [PubMed - indexed for MEDLINE]
335: Methods Enzymol 2000;328:47-59
Analysis and identification of protein-protein interactions using protein
recruitment systems.
Aronheim A, Karin M.
Department of Molecular Genetics, Technion Israel Institute of Technology,
Haifa, Israel.
PMID: 11075337 [PubMed - indexed for MEDLINE]
336: Methods Enzymol 2000;328:3-14
High-throughput screening for protein-protein interactions using two-hybrid
assay.
Cagney G, Uetz P, Fields S.
Banting and Best Department of Medical Research, University of Toronto, Ontario,
Canada.
PMID: 11075334 [PubMed - indexed for MEDLINE]
337: Mol Cell Biol 2000 Dec;20(23):8944-57
Novel Upf2p orthologues suggest a functional link between translation initiation
and nonsense surveillance complexes.
Mendell JT, Medghalchi SM, Lake RG, Noensie EN, Dietz HC.
Institute of Genetic Medicine, Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205, USA.
Transcripts harboring premature signals for translation termination are
recognized and rapidly degraded by eukaryotic cells through a pathway known as
nonsense-mediated mRNA decay (NMD). In addition to protecting cells by
preventing the translation of potentially deleterious truncated peptides,
studies have suggested that NMD plays a broader role in the regulation of the
steady-state levels of physiologic transcripts. In Saccharomyces cerevisiae,
three trans-acting factors (Upf1p to Upf3p) are required for NMD. Orthologues of
Upf1p have been identified in numerous species, showing that the NMD machinery,
at least in part, is conserved through evolution. In this study, we demonstrate
additional functional conservation of the NMD pathway through the identification
of Upf2p homologues in Schizosaccharomyces pombe and humans (rent2). Disruption
of S. pombe UPF2 established that this gene is required for NMD in fission
yeast. rent2 was demonstrated to interact directly with rent1, a known
trans-effector of NMD in mammalian cells. Additionally, fragments of rent2 were
shown to possess nuclear targeting activity, although the native protein
localizes to the cytoplasmic compartment. Finally, novel functional domains of
Upf2p and rent2 with homology to eukaryotic initiation factor 4G (eIF4G) and
other translational regulatory proteins were identified. Directed mutations
within these so-called eIF4G homology (4GH) domains were sufficient to abolish
the function of S. pombe Upf2p. Furthermore, using the two-hybrid system, we
obtained evidence for direct interaction between rent2 and human eIF4AI and
Sui1, both components of the translation initiation complex. Based on these
findings, a novel model in which Upf2p and rent2 effects decreased translation
and accelerated decay of nonsense transcripts through competitive interactions
with eIF4G-binding partners is proposed.
PMID: 11073994 [PubMed - indexed for MEDLINE]
338: Mol Cell Biol 2000 Dec;20(23):8879-88
A specificity and targeting subunit of a human SWI/SNF family-related
chromatin-remodeling complex.
Nie Z, Xue Y, Yang D, Zhou S, Deroo BJ, Archer TK, Wang W.
Laboratory of Genetics, National Institute on Aging, National Institutes of
Health, Baltimore, Maryland 21224, USA.
The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation
by assisting transcription machinery to gain access to targets in chromatin.
This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from
humans and SWI/SNF and Rsc from Saccharomyces cerevisiae. However, the
relationship between the human and yeast complexes is unclear because all human
subunits published to date are similar to those of both yeast SWI/SNF and Rsc.
Also, the two human complexes have many identical subunits, making it difficult
to distinguish their structures or functions. Here we describe the cloning and
characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250
contains structural motifs conserved in yeast SWI1 but not in any Rsc
components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog
of the Drosophila melanogaster Osa protein, which has been shown to interact
with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved
domains that could be important for its function. First, it has an AT-rich DNA
interaction-type DNA-binding domain, which can specifically bind a DNA sequence
known to be recognized by a SWI/SNF family-related complex at the beta-globin
locus. Second, BAF250 stimulates glucocorticoid receptor-dependent
transcriptional activation, and the stimulation is sharply reduced when the
C-terminal region of BAF250 is deleted. This region of BAF250 is capable of
interacting directly with the glucocorticoid receptor in vitro. Our data suggest
that BAF250 confers specificity to the human BAF complex and may recruit the
complex to its targets through either protein-DNA or protein-protein
interactions.
PMID: 11073988 [PubMed - indexed for MEDLINE]
339: Mol Cell Biol 2000 Dec;20(23):8709-19
In vivo requirement of activator-specific binding targets of mediator.
Park JM, Kim HS, Han SJ, Hwang MS, Lee YC, Kim YJ.
Genome Regulation Center, Creative Research Initiative, Samsung Biomedical
Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746,
Korea.
There has been no unequivocal demonstration that the activator binding targets
identified in vitro play a key role in transcriptional activation in vivo. To
examine whether activator-Mediator interactions are required for gene
transcription under physiological conditions, we performed functional analyses
with Mediator components that interact specifically with natural yeast
activators. Different activators interact with Mediator via distinct binding
targets. Deletion of a distinct activator binding region of Mediator completely
compromised gene activation in vivo by some, but not all, transcriptional
activators. These demonstrate that the activator-specific targets in Mediator
are essential for transcriptional activation in living cells, but their
requirement was affected by the nature of the activator-DNA interaction and the
existence of a postrecruitment activation process.
PMID: 11073972 [PubMed - indexed for MEDLINE]
340: J Bacteriol 2000 Dec;182(23):6638-44
InvB is a type III secretion chaperone specific for SspA.
Bronstein PA, Miao EA, Miller SI.
Department of Microbiology, University of Washington, Seattle, Washington 98195,
USA.
A wide variety of gram-negative bacteria utilize a specialized apparatus called
the type III secretion system (TTSS) to translocate virulence factors directly
into the cytoplasm of eukaryotic cells. These translocated effectors contribute
to the pathogen's ability to infect and replicate within plant and animal hosts.
The amino terminus of effector proteins contains sequences that are necessary
and sufficient for both secretion and translocation by TTSS. Portions of these
sequences contain binding sites for type III chaperones, which facilitate
efficient secretion and translocation of specific effectors through TTSS. In
this study, we have utilized the yeast two-hybrid assay to identify
protein-protein interactions between effector and chaperone proteins encoded
within Salmonella pathogenicity island 1 (SPI-1). Several interactions were
identified including a novel interaction between the effector protein, SspA
(SipA), and a putative chaperone, InvB. InvB was demonstrated to bind to the
amino terminus of SspA in the bacterial cytoplasm. Furthermore, InvB acts as a
type III chaperone for the efficient secretion and translocation of SspA by
SPI-1. InvB also permitted translocation of SspA through the SPI-2 TTSS,
indicating that it is an important regulator in the recognition of SspA as a
target of TTSS. Finally, it was determined that InvB does not alter the
transcription of sspA but that its absence results in reduced SspA protein
levels in Salmonella enterica serovar Typhimurium.
PMID: 11073906 [PubMed - indexed for MEDLINE]
341: Nucleic Acids Res 2000 Nov 15;28(22):4523-30
Predicting regulons and their cis-regulatory motifs by comparative genomics.
Manson McGuire A, Church GM.
Department of Genetics, Warren Alpert Building, Room 513, Harvard Medical
School, 200 Longwood Avenue, Boston, MA 02115, USA.
We have combined and compared three techniques for predicting functional
interactions based on comparative genomics (methods based on conserved operons,
protein fusions and correlated evolution) and optimized these methods to predict
coregulated sets of genes in 24 complete genomes, including Saccharomyces
cerevisiae, Caenorhabditis elegans and 22 prokaryotes. The method based on
conserved operons was the most useful for this purpose. Upstream regions of the
genes comprising these predicted regulons were then used to search for
regulatory motifs in 22 prokaryotic genomes using the motif-discovery program
AlignACE. Many significant upstream motifs, including five known Escherichia
coli regulatory motifs, were identified in this manner. The presence of a
significant regulatory motif was used to refine the members of the predicted
regulons to generate a final set of predicted regulons that share significant
regulatory elements.
PMID: 11071941 [PubMed - indexed for MEDLINE]
342: Nucleic Acids Res 2000 Nov 15;28(22):4460-6
A network of yeast basic helix-loop-helix interactions.
Robinson KA, Koepke JI, Kharodawala M, Lopes JM.
Department of Biological Sciences, Wayne State University, 5047 Gullen Mall,
Detroit, MI 48202, USA.
The Ino4 protein belongs to the basic helix-loop-helix (bHLH) family of
proteins. It is known to form a dimer with Ino2p, which regulates phospholipid
biosynthetic genes. Mammalian bHLH proteins have been shown to form multiple
dimer combinations. However, this flexibility in dimerization had not been
documented for yeast bHLH proteins. Using the yeast two-hybrid assay and a
biochemical assay we show that Ino4p dimerizes with the Pho4p, Rtg1p, Rtg3p and
Sgc1p bHLH proteins. Screening a yeast cDNA library identified three additional
proteins that interact with Ino4p: Bck2p, YLR422W and YNR064C. The interaction
with Bck2p prompted us to examine if any of the Bck2p-associated functions
affect expression of phospholipid biosynthetic genes. We found that hyperosmotic
growth conditions altered the growth phase regulation of a phospholipid
biosynthetic gene, CHO1. There are two recent reports of initial whole genome
yeast two-hybrid interactions. Interestingly, one of these reports identified
five proteins that interact with Ino4p: Ino2p, Hcs1p, Apl2p, YMR317W and
YNL279W. Ino2p is the only protein in common with the data presented here. Our
finding that Ino4p interacts with five bHLH proteins suggests that Ino4p is
likely to be a central player in the coordination of multiple biological
processes.
PMID: 11071933 [PubMed - indexed for MEDLINE]
343: Mol Biol Cell 2000 Nov;11(11):3859-71
Sec62p, a component of the endoplasmic reticulum protein translocation
machinery, contains multiple binding sites for the Sec-complex.
Wittke S, Dunnwald M, Johnsson N.
Max-Delbruck-Laboratorium, D-50829 Koln, Germany.
SEC62 encodes an essential component of the Sec-complex that is responsible for
posttranslational protein translocation across the membrane of the endoplasmic
reticulum in Saccharomyces cerevisiae. The specific role of Sec62p in
translocation was not known and difficult to identify because it is part of an
oligomeric protein complex in the endoplasmic reticulum membrane. An in vivo
competition assay allowed us to characterize and dissect physical and functional
interactions between Sec62p and components of the Sec-complex. We could show
that Sec62p binds via its cytosolic N- and C-terminal domains to the
Sec-complex. The N-terminal domain, which harbors the major interaction site,
binds directly to the last 14 residues of Sec63p. The C-terminal binding site of
Sec62p is less important for complex stability, but adjoins the region in Sec62p
that might be involved in signal sequence recognition.
PMID: 11071912 [PubMed - indexed for MEDLINE]
344: Mol Biol Cell 2000 Nov;11(11):3689-702
Bud6 directs sequential microtubule interactions with the bud tip and bud neck
during spindle morphogenesis in Saccharomyces cerevisiae.
Segal M, Bloom K, Reed SI.
Department of Molecular Biology, The Scripps Research Institute, La Jolla,
California 92037, USA.
In budding yeast, spindle polarity relies on a precise temporal program of
cytoplasmic microtubule-cortex interactions throughout spindle assembly. Loss of
Clb5-dependent kinase activity under conditions of attenuated Cdc28 function
disrupts this program, resulting in diploid-specific lethality. Here we show
that polarity loss is tolerated by haploids due to a more prominent contribution
of microtubule-neck interactions to spindle orientation inherent to haploids.
These differences are mediated by the relative partition of Bud6 between the bud
tip and bud neck, distinguishing haploids from diploids. Bud6 localizes
initially to the bud tip and accumulates at the neck concomitant with spindle
assembly. bud6Delta mutant phenotypes are consistent with Bud6's role as a
cortical cue for cytoplasmic microtubule capture. Moreover, mutations that
affect Bud6 localization and partitioning disrupt the sequential program of
microtubule-cortex interactions accordingly. These data support a model whereby
Bud6 sequentially cues microtubule capture events at the bud tip followed by
capture events at the bud neck, necessary for correct spindle morphogenesis and
polarity.
PMID: 11071900 [PubMed - indexed for MEDLINE]
345: Proc Natl Acad Sci U S A 2000 Nov 7;97(23):12583-8
Interaction of yeast kinetochore proteins with centromere-protein/transcription
factor Cbf1.
Hemmerich P, Stoyan T, Wieland G, Koch M, Lechner J, Diekmann S.
Institut fuer Molekulare Biotechnologie, Abteilung Molekularbiologie,
Beutenbergstrasse 11, 07745 Jena, Germany. phemmer@imb-jena.de
The centromere-kinetochore complex of Saccharomyces cerevisiae is a specialized
chromosomal substructure that mediates attachment of duplicated chromosomes to
the mitotic spindle by a regulated network of protein-DNA and protein-protein
interactions. We have used in vitro assays to analyze putative molecular
interactions between components of the yeast centromerekinetochore complex.
Glutathione S-transferase pull-down experiments showed the direct interaction of
in vitro translated p110, p64, and p58 of the essential CBF3 kinetochore protein
complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip)
that specifically binds to the CDEI region on the centromere DNA. Furthermore,
recombinant p64 and p23 each stimulated the in vitro DNA binding activity of
Cbf1p. The N-terminal 70 amino acids of p23 were sufficient to mediate this
effect. P64 could also promote the multimerization activity of Cbf1p in the
presence of centromere DNA in vitro. These results show the direct physical
interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components
can promote the binding of Cbf1p to its binding site in the yeast kinetochore. A
functional comparison of the centromere binding proteins with transcription
factors binding at MET16 promoters reveals the strong analogy between
centromeres and the MET16 promoter.
PMID: 11070082 [PubMed - indexed for MEDLINE]
346: Genes Dev 2000 Nov 1;14(21):2737-44
Ssn6-Tup1 interacts with class I histone deacetylases required for repression.
Watson AD, Edmondson DG, Bone JR, Mukai Y, Yu Y, Du W, Stillman DJ, Roth SY.
Department of Biochemistry and Molecular Biology, University of Texas M.D.
Anderson Cancer Center, Houston, Texas 77030, USA.
Ssn6-Tup1 regulates multiple genes in yeast, providing a paradigm for
corepressor functions. Tup1 interacts directly with histones H3 and H4, and
mutation of these histones synergistically compromises Ssn6-Tup1-mediated
repression. In vitro, Tup1 interacts preferentially with underacetylated
isoforms of H3 and H4, suggesting that histone acetylation may modulate Tup1
functions in vivo. Here we report that histone hyperacetylation caused by
combined mutations in genes encoding the histone deacetylases (HDACs) Rpd3,
Hos1, and Hos2 abolishes Ssn6-Tup1 repression. Unlike HDAC mutations that do not
affect repression, this combination of mutations causes concomitant
hyperacetylation of both H3 and H4. Strikingly, two of these class I HDACs
interact physically with Ssn6-Tup1. These findings suggest that Ssn6-Tup1
actively recruits deacetylase activities to deacetylate adjacent nucleosomes and
promote Tup1-histone interactions.
PMID: 11069890 [PubMed - indexed for MEDLINE]
347: J Cell Sci 2000 Dec;113 Pt 23:4143-9
The FHA domain mediates phosphoprotein interactions.
Li J, Lee GI, Van Doren SR, Walker JC.
Division of Biological Sciences and Department of Biochemistry, University of
Missouri-Columbia, Columbia, MO 65211, USA.
The forkhead-associated (FHA) domain is a phosphopeptide-binding domain first
identified in a group of forkhead transcription factors but is present in a wide
variety of proteins from both prokaryotes and eukaryotes. In yeast and human,
many proteins containing an FHA domain are found in the nucleus and involved in
DNA repair, cell cycle arrest, or pre-mRNA processing. In plants, the FHA domain
is part of a protein that is localized to the plasma membrane and participates
in the regulation of receptor-like protein kinase signaling pathways. Recent
studies show that a functional FHA domain consists of 120-140 amino acid
residues, which is significantly larger than the sequence motif first described.
Although FHA domains do not exhibit extensive sequence similarity, they share
similar secondary and tertiary structures, featuring a sandwich of two
anti-parallel (beta)-sheets. One intriguing finding is that FHA domains may bind
phosphothreonine, phosphoserine and sometimes phosphotyrosine, distinguishing
them from other well-studied phosphoprotein-binding domains. The diversity of
proteins containing FHA domains and potential differences in binding
specificities suggest the FHA domain is involved in coordinating diverse
cellular processes.
Publication Types:
Review
Review Literature
PMID: 11069759 [PubMed - indexed for MEDLINE]
348: Arch Biochem Biophys 2000 Oct 15;382(2):262-74
Interaction of insulin-like growth factor binding protein-4, Miz-1, leptin,
lipocalin-type prostaglandin D synthase, and granulin precursor with the
N-terminal half of type III hexokinase.
Sui D, Wilson JE.
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Insulin-like growth factor binding protein-4, Miz-1, leptin, prostaglandin D
synthase, and granulin precursor were identified as proteins interacting with
the N-terminal half of mammalian Type III hexokinase (HKIII) in the yeast
two-hybrid method. These interactions were confirmed by in vitro binding
studies. All five of these proteins, and their mRNAs, were present in PC12
cells, as shown by immunoblotting and RT-PCR, respectively. All were
coimmunoprecipitated from PC12 extracts with an antibody against HKIII, but not
with anti-Type I hexokinase. Moreover, all of these proteins were
coimmunoprecipitated using antileptin as precipitating antibody, indicating the
existence of a macromolecular complex including these five proteins and HKIII.
Transfection of M+R 42 cells with HKIII-green fluorescent protein (GFP) reporter
constructs gave a diffuse intracellular fluorescence. Cotransfection with leptin
or Miz-1 resulted in distinctly different localization of the HKIII-GFP fusion
protein, at intracellular sites coincident with localization of leptin-GFP or
Miz-1-GFP reporter constructs.
PMID: 11068878 [PubMed - indexed for MEDLINE]
349: Genetics 2000 Nov;156(3):973-81
CSE4 genetically interacts with the Saccharomyces cerevisiae centromere DNA
elements CDE I and CDE II but not CDE III. Implications for the path of the
centromere dna around a cse4p variant nucleosome.
Keith KC, Fitzgerald-Hayes M.
Department of Biochemistry and Molecular Biology, Program in Molecular and
Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003,
USA.
Each Saccharomyces cerevisiae chromosome contains a single centromere composed
of three conserved DNA elements, CDE I, II, and III. The histone H3 variant,
Cse4p, is an essential component of the S. cerevisiae centromere and is thought
to replace H3 in specialized nucleosomes at the yeast centromere. To investigate
the genetic interactions between Cse4p and centromere DNA, we measured the
chromosome loss rates exhibited by cse4 cen3 double-mutant cells that express
mutant Cse4 proteins and carry chromosomes containing mutant centromere DNA
(cen3). When compared to loss rates for cells carrying the same cen3 DNA mutants
but expressing wild-type Cse4p, we found that mutations throughout the Cse4p
histone-fold domain caused surprisingly large increases in the loss of
chromosomes carrying CDE I or CDE II mutant centromeres, but had no effect on
chromosomes with CDE III mutant centromeres. Our genetic evidence is consistent
with direct interactions between Cse4p and the CDE I-CDE II region of the
centromere DNA. On the basis of these and other results from genetic,
biochemical, and structural studies, we propose a model that best describes the
path of the centromere DNA around a specialized Cse4p-nucleosome.
PMID: 11063678 [PubMed - indexed for MEDLINE]
350: Genetics 2000 Nov;156(3):943-51
Synthetic interactions of the post-Golgi sec mutations of Saccharomyces
cerevisiae.
Finger FP, Novick P.
Department of Cell Biology, Yale University School of Medicine, New Haven,
Connecticut 06520-8002, USA.
In the budding yeast Saccharomyces cerevisiae, synthetic lethality has been
extensively used both to characterize interactions between genes previously
identified as likely to be involved in similar processes as well as to uncover
new interactions. We have performed a large study of the synthetic lethal
interactions of the post-Golgi sec mutations. Included in this study are the
interactions of the post-Golgi sec mutations with each other, with mutations
affecting earlier stages of the secretory pathway, with selected mutations
affecting the actin cytoskeleton, and with selected cell division cycle (cdc)
mutations affecting processes thought to be important for or involving
secretion, such as polarity establishment and cytokinesis. Synthetic negative
interactions of the post-Golgi sec mutations appear (as predicted) to be largely
stage specific, although there are some notable exceptions. The significance of
these results is discussed in the context of both secretory pathway function and
the utility of synthetic lethality studies and their interpretation.
PMID: 11063675 [PubMed - indexed for MEDLINE]
351: Virology 2000 Nov 10;277(1):127-35
The human T-cell leukemia virus type I (HTLV-I) X region encoded protein p13(II)
interacts with cellular proteins.
Hou X, Foley S, Cueto M, Robinson MA.
Laboratory of Immunogenetics, Twinbrook II Facility, National Institute of
Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn
Drive, Rockville, Maryland, 20852, USA.
Interactions between the Human T-cell leukemia virus type I (HTLV-I) gene
product p13(II) and cellular proteins were investigated using the yeast
two-hybrid system. Variant forms of p13(II) were derived from two HTLV-I
molecular clones, K30p and K34p, that differ in both virus production and in
vivo and in vitro infectivity. Two nucleotide differences between the p13 from
K30p (p13K30) and K34p (p13K34) result in a Trp-Arg substitution at amino acid
17 and the truncation of the 25 carboxyl-terminal residues of p13K34. A cDNA
library from an HTLV-I-infected rabbit T-cell line was screened with p13K30 and
p13K34 as bait. Products of two cDNA clones, C44 and C254, interacted with
p13K34 but not with p13K30. Interactions were further confirmed using the
GST-fusion protein coprecipitation assay. Sequence analysis of C44 and C254 cDNA
clones revealed similarities to members of the nucleoside monophosphate kinase
superfamily and actin-binding protein 280, respectively. Further analysis of the
function of these two proteins and the consequence of their interaction with p13
may help elucidate a role for p13 in virus production, infectivity, or the
pathogenesis of HTLV-I. Copyright 2000 Academic Press.
PMID: 11062043 [PubMed - indexed for MEDLINE]
352: Biochem Biophys Res Commun 2000 Nov 2;277(3):589-93
Combined transformation and genetic technique verification of protein-protein
interactions in the yeast two-hybrid system.
Tyagi S, Lal SK.
Virology Group, International Centre for Genetic Engineering and Biotechnology,
New Delhi, 1100069, India.
The yeast two-hybrid system is frequently used to identify protein-protein
interactions. The assay is based on the functional reconstitution of a
transcriptional activator. Since an indirect phenotype of the positive clones is
the basis for selection of positive interacting clones, the two-hybrid screens
are vulnerable to false positives. Here we report a screening protocol based on
the sequential use of the cotransformation approach followed by the genetic
method for verifying true two-hybrid interactions. Using this procedure, we have
screened a cDNA library and have been able to isolate true positives from the
yeast two-hybrid screen. Copyright 2000 Academic Press.
PMID: 11061998 [PubMed - indexed for MEDLINE]
353: J Biol Chem 2001 Jan 26;276(4):2608-15
A hybrid between Na+,K+-ATPase and H+,K+-ATPase is sensitive to palytoxin,
ouabain, and SCH 28080.
Farley RA, Schreiber S, Wang SG, Scheiner-Bobis G.
Department of Physiology and Biophysics, Keck School of Medicine, University of
Southern California, Los Angeles 90033, USA. rfarley@hsc.usc.edu
Na(+),K(+)-ATPase is inhibited by cardiac glycosides such as ouabain, and
palytoxin, which do not inhibit gastric H(+),K(+)-ATPase. Gastric
H(+),K(+)-ATPase is inhibited by SCH28080, which has no effect on
Na(+),K(+)-ATPase. The goal of the current study was to identify amino acid
sequences of the gastric proton-potassium pump that are involved in recognition
of the pump-specific inhibitor SCH 28080. A chimeric polypeptide consisting of
the rat sodium pump alpha3 subunit with the peptide Gln(905)-Val(930) of the
gastric proton pump alpha subunit substituted in place of the original
Asn(886)-Ala(911) sequence was expressed together with the gastric beta subunit
in the yeast Saccharomyces cerevisiae. Yeast cells that express this subunit
combination are sensitive to palytoxin, which interacts specifically with the
sodium pump, and lose intracellular K(+) ions. The palytoxin-induced K(+) efflux
is inhibited by the sodium pump-specific inhibitor ouabain and also by the
gastric proton pump-specific inhibitor SCH 28080. The IC(50) for SCH 28080
inhibition of palytoxin-induced K(+) efflux is 14.3 +/- 2.4 microm, which is
similar to the K(i) for SCH 28080 inhibition of ATP hydrolysis by the gastric
H(+),K(+)-ATPase. In contrast, palytoxin-induced K(+) efflux from cells
expressing either the native alpha3 and beta1 subunits of the sodium pump or the
alpha3 subunit of the sodium pump together with the beta subunit of the gastric
proton pump is inhibited by ouabain but not by SCH 28080. The acquisition of SCH
28080 sensitivity by the chimera indicates that the Gln(905)-Val(930) peptide of
the gastric proton pump is likely to be involved in the interactions of the
gastric proton-potassium pump with SCH 28080.
PMID: 11054424 [PubMed - indexed for MEDLINE]
354: Biophys J 2000 Nov;79(5):2624-31
PMP1 18-38, a yeast plasma membrane protein fragment, binds phosphatidylserine
from bilayer mixtures with phosphatidylcholine: a (2)H-NMR study.
Roux M, Beswick V, Coic YM, Huynh-Dinh T, Sanson A, Neumann JM.
Departement de Biologie Cellulaire et Moleculaire, Section de Biophysique des
Proteines et des Membranes, CEA and URA CNRS 2096, Centre d'Etudes de Saclay,
91191 Gif sur Yvette Cedex, France. roux@dsvidf.cea.fr
PMP1 is a 38-residue plasma membrane protein of the yeast Saccharomyces
cerevisiae that regulates the activity of the H(+)-ATPase. The cytoplasmic
domain conformation results in a specific interfacial distribution of five basic
side chains, thought to strongly interact with anionic phospholipids. We have
used the PMP1 18-38 fragment to carry out a deuterium nuclear magnetic resonance
((2)H-NMR) study for investigating the interactions between the PMP1 cytoplasmic
domain and phosphatidylserines. For this purpose, mixed bilayers of 1-palmitoyl,
2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl,
2-oleoyl-sn-glycero-3-phosphoserine (POPS) were used as model membranes
(POPC/POPS 5:1, m/m). Spectra of headgroup- and chain-deuterated POPC and POPS
phospholipids, POPC-d4, POPC-d31, POPS-d3, and POPS-d31, were recorded at
different temperatures and for various concentrations of the PMP1 fragment. Data
obtained from POPS deuterons revealed the formation of specific peptide-POPS
complexes giving rise to a slow exchange between free and bound PS lipids,
scarcely observed in solid-state NMR studies of lipid-peptide/protein
interactions. The stoichiometry of the complex (8 POPS per peptide) was
determined and its significance is discussed. The data obtained with
headgroup-deuterated POPC were rationalized with a model that integrates the
electrostatic perturbation induced by the cationic peptide on the negatively
charged membrane interface, and a "spacer" effect due to the intercalation of
POPS/PMP1f complexes between choline headgroups.
PMID: 11053135 [PubMed - indexed for MEDLINE]
355: Endocrine 2000 Aug;13(1):55-62
c-Jun targets amino terminus of androgen receptor in regulating
androgen-responsive transcription.
Bubulya A, Zhou XF, Shen XQ, Fisher CJ, Shemshedini L.
Department of Biology, University of Toledo, OH 43606, USA.
The human androgen receptor (hAR) is a member of the nuclear receptor
superfamily and functions as a ligand-inducible transcription factor. We have
previously proposed that c-Jun mediates the transcriptional activity of this
receptor. The modular nature of hAR was used in this study to generate several
fusions with the heterologous DNA-binding domain of the yeast transcription
factor GAL4 in an attempt to identify the c-Jun-responsive domains within the
receptor. Our results suggest that the target of c-Jun action is the amino
terminus (AB region) of the receptor and that hAR amino acids 502-521 are
critical for the c-Jun response. Additionally, amino acids 503-555 were shown to
harbor an autonomous transactivation that is stimulated by c-Jun. Furthermore,
we demonstrated that transcription intermediary factor-2 (TIF-2), a coactivator
that acts on the activation function-2, stimulates the full-length hAR. These
results suggest that c-Jun and TIF-2 can work together as coactivators on the
hAR by targeting distinct portions of the receptor.
PMID: 11051047 [PubMed - indexed for MEDLINE]
356: Mol Cell Biol 2000 Nov;20(22):8548-59
Saccharomyces cerevisiae cdc42p GTPase is involved in preventing the recurrence
of bud emergence during the cell cycle.
Richman TJ, Johnson DI.
Department of Microbiology and Molecular Genetics and Markey Center for
Molecular Genetics, University of Vermont Burlington, Vermont 05405, USA.
The Saccharomyces cerevisiae Cdc42p GTPase interacts with multiple regulators
and downstream effectors through an approximately 25-amino-acid effector domain.
Four effector domain mutations, Y32K, F37A, D38E, and Y40C, were introduced into
Cdc42p and characterized for their effects on these interactions. Each mutant
protein showed differential interactions with a number of downstream effectors
and regulators and various levels of functionality. Specifically, Cdc42(D38E)p
showed reduced interactions with the Cla4p p21-activated protein kinase and the
Bem3p GTPase-activating protein and cdc42(D38E) was the only mutant allele able
to complement the Deltacdc42 null mutant. However, the mutant protein was only
partially functional, as indicated by a temperature-dependent multibudded
phenotype seen in conjunction with defects in both septin ring localization and
activation of the Swe1p-dependent morphogenetic checkpoint. Further analysis of
this mutant suggested that the multiple buds emerged consecutively with a
premature termination of bud enlargement preceding the appearance of the next
bud. Cortical actin, the septin ring, Cla4p-green fluorescent protein (GFP), and
GFP-Cdc24p all predominantly localized to one bud at a time per multibudded
cell. These data suggest that Cdc42(D38E)p triggers a morphogenetic defect
post-bud emergence, leading to cessation of bud growth and reorganization of the
budding machinery to another random budding site, indicating that Cdc42p is
involved in prevention of the initiation of supernumerary buds during the cell
cycle.
PMID: 11046150 [PubMed - indexed for MEDLINE]
357: Mol Cell Biol 2000 Nov;20(22):8343-51
Functional interaction between Ssu72 and the Rpb2 subunit of RNA polymerase II
in Saccharomyces cerevisiae.
Pappas DL Jr, Hampsey M.
Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood
Johnson Medical School, Piscataway, New Jersey 08854, USA.
SSU72 is an essential gene encoding a phylogenetically conserved protein of
unknown function that interacts with the general transcription factor TFIIB. A
recessive ssu72-1 allele was identified as a synthetic enhancer of a TFIIB
(sua7-1) defect, resulting in a heat-sensitive (Ts(-)) phenotype and a dramatic
downstream shift in transcription start site selection. Here we describe a new
allele, ssu72-2, that confers a Ts(-) phenotype in a SUA7 wild-type background.
In an effort to further define Ssu72, we isolated suppressors of the ssu72-2
mutation. One suppressor is allelic to RPB2, the gene encoding the
second-largest subunit of RNA polymerase II (RNAP II). Sequence analysis of the
rpb2-100 suppressor defined a cysteine replacement of the phylogenetically
invariant arginine residue at position 512 (R512C), located within homology
block D of Rpb2. The ssu72-2 and rpb2-100 mutations adversely affected
noninduced gene expression, with no apparent effects on activated transcription
in vivo. Although isolated as a suppressor of the ssu72-2 Ts(-) defect, rpb2-100
enhanced the transcriptional defects associated with ssu72-2. The Ssu72 protein
interacts directly with purified RNAP II in a coimmunoprecipitation assay,
suggesting that the genetic interactions between ssu72-2 and rpb2-100 are a
consequence of physical interactions. These results define Ssu72 as a highly
conserved factor that physically and functionally interacts with the RNAP II
core machinery during transcription initiation.
PMID: 11046131 [PubMed - indexed for MEDLINE]
358: J Biol Chem 2001 Jan 12;276(2):1051-6
Biochemical analysis of the eIF2beta gamma complex reveals a structural function
for eIF2alpha in catalyzed nucleotide exchange.
Nika J, Rippel S, Hannig EM.
Department of Molecular and Cell Biology, The University of Texas at Dallas,
Richardson, Texas 75083, USA.
Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and
delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-dependent
manner. Initiation requires hydrolysis of eIF2-bound GTP, which releases an
eIF2.GDP complex that is recycled to the GTP form by the nucleotide exchange
factor eIF2B. The alpha-subunit of eIF2 plays a critical role in regulating
nucleotide exchange via phosphorylation at serine 51, which converts eIF2 into a
competitive inhibitor of the eIF2B-catalyzed exchange reaction. We purified a
form of eIF2 (eIF2betagamma) completely devoid of the alpha-subunit to further
study the role of eIF2alpha in eIF2 function. These studies utilized a yeast
strain genetically altered to bypass a deletion of the normally essential
eIF2alpha structural gene (SUI2). Removal of the alpha-subunit did not appear to
significantly alter binding of guanine nucleotide or Met-tRNA(i)(Met) ligands by
eIF2 in vitro. Qualitative assays to detect 43 S initiation complex formation
and eIF5-dependent GTP hydrolysis revealed no differences between eIF2betagamma
and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of
eIF2B-catalyzed nucleotide exchange revealed that the absence of the
alpha-subunit increased K(m) for eIF2betagamma.GDP by an order of magnitude,
with a smaller increase in V(max). These data indicate that eIF2alpha is
required for structural interactions between eIF2 and eIF2B that promote
wild-type rates of nucleotide exchange. We suggest that this function
contributes to the ability of the alpha-subunit to control the rate of
nucleotide exchange through reversible phosphorylation.
PMID: 11042214 [PubMed - indexed for MEDLINE]
359: J Biol Chem 2001 Jan 12;276(2):1204-10
Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase.
Panisko EA, McAlister-Henn L.
Department of Biochemistry, University of Texas Health Science Center, San
Antonio, Texas 78229-3900, USA.
Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase is an octamer
composed of four each of two nonidentical but related subunits designated IDH1
and IDH2. IDH2 was previously shown to contain the catalytic site, whereas IDH1
contributes regulatory properties including cooperativity with respect to
isocitrate and allosteric activation by AMP. In this study, interactions between
IDH1 and IDH2 were detected using the yeast two-hybrid system, but interactions
between identical subunit polypeptides were not detected with this or other
methods. A model for heterodimeric interactions between the subunits is
therefore proposed for this enzyme. A corollary of this model, based on the
three-dimensional structure of the homologous enzyme from Escherichia coli, is
that some interactions between subunits occur at isocitrate binding sites. Based
on this model, two residues (Lys-183 and Asp-217) in the regulatory IDH1 subunit
were predicted to be important in the catalytic site of IDH2. We found that
individually replacing these residues with alanine results in mutant enzymes
that exhibit a drastic reduction in catalysis both in vitro and in vivo. Also
based on this model, the two analogous residues (Lys-189 and Asp-222) of the
catalytic IDH2 subunit were predicted to contribute to the regulatory site of
IDH1. A K189A substitution in IDH2 was found to produce a decrease in activation
of the enzyme by AMP and a loss of cooperativity with respect to isocitrate. A
D222A substitution in IDH2 produces similar regulatory defects and a substantial
reduction in V(max) in the absence of AMP. Collectively, these results suggest
that the basic structural/functional unit of yeast isocitrate dehydrogenase is a
heterodimer of IDH1 and IDH2 subunits and that each subunit contributes to the
isocitrate binding site of the other.
PMID: 11042198 [PubMed - indexed for MEDLINE]
360: J Biol Chem 2001 Jan 19;276(3):2023-30
The potency and specificity of the interaction between the IA3 inhibitor and its
target aspartic proteinase from Saccharomyces cerevisiae.
Phylip LH, Lees WE, Brownsey BG, Bur D, Dunn BM, Winther JR, Gustchina A, Li M,
Copeland T, Wlodawer A, Kay J.
School of Biosciences, Cardiff University, P. O. Box 911, Cardiff CF10 3US,
Wales, United Kingdom.
The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor
has little intrinsic secondary structure. IA3 showed subnanomolar potency toward
its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any
of a large number of aspartic proteinases with similar sequences/structures from
a wide variety of other species. Systematic truncation and mutagenesis of the
IA3 polypeptide revealed that the inhibitory activity is located in the
N-terminal half of the sequence. Crystal structures of different forms of IA3
complexed with proteinase A showed that residues in the N-terminal half of the
IA3 sequence became ordered and formed an almost perfect alpha-helix in the
active site of the enzyme. This potent, specific interaction was directed
primarily by hydrophobic interactions made by three key features in the
inhibitory sequence. Whereas IA3 was cut as a substrate by the nontarget
aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3
polypeptide escapes cleavage by being stabilized in a helical conformation upon
interaction with the active site of proteinase A. This results, paradoxically,
in potent selective inhibition of the target enzyme.
PMID: 11042188 [PubMed - indexed for MEDLINE]
361: J Biol Chem 2001 Jan 19;276(3):2122-31
Plant initiation factor 3 subunit composition resembles mammalian initiation
factor 3 and has a novel subunit.
Burks EA, Bezerra PP, Le H, Gallie DR, Browning KS.
Department of Chemistry and Biochemistry and the Institute for Cellular and
Molecular Biology, University of Texas, Austin, Texas 78712, USA.
Eukaryotic initiation factor 3 (Short id=314>eIF3) is a multisubunit complex that is required
for binding of mRNA to 40 S ribosomal subunits, stabilization of ternary complex
binding to 40 S subunits, and dissociation of 40 and 60 S subunits. These
functions and the complex nature of eIF3 suggest multiple interactions with many
components of the translational machinery. Recently, the subunits of mammalian
and Saccharomyces cerevisiae eIF3 were identified, and substantial differences
in the subunit composition of mammalian and S. cerevisiae were observed.
Mammalian eIF3 consists of 11 nonidentical subunits, whereas S. cerevisiae eIF3
consists of up to eight nonidentical subunits. Only five of the subunits of
mammalian and S. cerevisiae are shared in common, and these five subunits
comprise a "core" complex in S. cerevisiae. eIF3 from wheat consists of at least
10 subunits, but their relationship to either the mammalian or S. cerevisiae
eIF3 subunits is unknown. Peptide sequences derived from purified wheat eIF3
subunits were used to correlate each subunit with mammalian and/or S. cerevisiae
subunits. The peptide sequences were also used to identify Arabidopsis thaliana
cDNAs for each of the eIF3 subunits. We report seven new cDNAs for A. thaliana
eIF3 subunits. A. thaliana eIF3 was purified and characterized to confirm that
the subunit composition and activity of wheat and A. thaliana eIF3 were similar.
We report that plant eIF3 closely resembles the subunit composition of mammalian
eIF3, having 10 out of 11 subunits in common. Further, we find a novel subunit
in the plant eIF3 complex not present in either mammalian or S. cerevisiae eIF3.
These results suggest that plant and mammalian eIF3 evolved similarly, whereas
S. cerevisiae has diverged.
PMID: 11042177 [PubMed - indexed for MEDLINE]
362: J Biotechnol 2001 Nov 17;84(1):87-91
Improved resistance to transition metals of a cobalt-substituted alcohol
dehydrogenase 1 from Saccharomyces cerevisiae.
Cavaletto M, Pessione E, Vanni A, Giunta C.
Dipartimento di Biologia Animale e dell'Uomo, Universita di Torino, Via
Accademia Albertina 13, 10123, Torino, Italy. maria.calvetto@unito.it
Cobalt-substituted alcohol dehydrogenase 1 was purified from a yeast culture of
Saccharomyces cerevisiae. Its reactivity towards different transition metals was
tested and compared with the native zinc enzyme. The cobalt enzyme displayed a
catalytic efficiency 100-fold higher than that of the zinc enzyme. Copper,
nickel and cadmium exerted a mixed-type inhibition, with a scale of inhibition
efficiency: Cu(2+)>Ni(2+)>Cd(2+). In general, a higher resistance of the
modified protein to the inhibitory action of transition metals was observed,
with two orders of magnitude for copper I(50). The presence of nickel in the
complexes enzyme-coenzyme-inhibitor-substrate resulted in a decrease of the
ampholytic nature of the catalytic site. On the contrary, cadmium and copper
exerted an enhancement of this parameter. Electrostatic or other types of
interactions may be involved in conferring a good resistance in the basic pH
range, making cobalt enzyme very suitable for biotechnological processes.
PMID: 11035192 [PubMed - indexed for MEDLINE]
363: J Biol Chem 2001 Jan 5;276(1):395-405
Mutations in the TATA-binding protein, affecting transcriptional activation,
show synthetic lethality with the TAF145 gene lacking the TAF N-terminal domain
in Saccharomyces cerevisiae.
Kobayashi A, Miyake T, Ohyama Y, Kawaichi M, Kokubo T.
Division of Gene Function in Animals, Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
The general transcription factor TFIID, which is composed of the TATA
box-binding protein (TBP) and a set of TBP-associated factors (TAFs), is crucial
for both basal and regulated transcription by RNA polymerase II. The N-terminal
small segment of yeast TAF145 (yTAF145) binds to TBP and thereby inhibits TBP
function. To understand the physiological role of this inhibitory domain, which
is designated as TAND (TAF N-terminal domain), we screened mutations,
synthetically lethal with the TAF145 gene lacking TAND (taf145 Delta TAND), in
Saccharomyces cerevisiae by exploiting a red/white colony-sectoring assay. Our
screen yielded several recessive nsl (Delta TAND synthetic lethal) mutations,
two of which, nsl1-1 and nsl1-2, define the same complementation group. The NSL1
gene was found to be identical to the SPT15 gene encoding TBP. Interestingly,
both temperature-sensitive nsl1/spt15 alleles, which harbor the single amino
acid substitutions, S118L and P65S, respectively, were defective in
transcriptional activation in vivo. Several other previously characterized
activation-deficient spt15 alleles also displayed synthetic lethal interactions
with taf145 Delta TAND, indicating that TAND and TBP carry an overlapping but as
yet unidentified function that is specifically required for transcriptional
regulation.
PMID: 11035037 [PubMed - indexed for MEDLINE]
364: Mol Biol Cell 2000 Oct;11(10):3629-43
Yeast exocytic v-SNAREs confer endocytosis.
Gurunathan S, Chapman-Shimshoni D, Trajkovic S, Gerst JE.
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100,
Israel.
In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and
Snc2) confer the docking and fusion of secretory vesicles at the cell surface.
As no v-SNARE has been shown to confer endocytosis, we examined whether yeast
lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1
(SNC1(ala43)), are deficient in the endocytic uptake of components from the cell
surface. We found that both SNC and temperature-shifted SNC1(ala43) yeast are
deficient in their ability to deliver the soluble dye FM4-64 to the vacuole.
Under conditions in which vesicles accumulate, FM4-64 stained primarily the
cytoplasm as well as fragmented vacuoles. In addition, alpha-factor-stimulated
endocytosis of the alpha-factor receptor, Ste2, was fully blocked, as evidenced
using a Ste2-green fluorescent protein fusion protein as well as metabolic
labeling studies. This suggests a direct role for Snc v-SNAREs in the retrieval
of membrane proteins from the cell surface. Moreover, this idea is supported by
genetic and physical data that demonstrate functional interactions with t-SNAREs
that confer endosomal transport (e.g., Tlg1,2). Notably, Snc1(ala43) was found
to be nonfunctional in cells lacking Tlg1 or Tlg2. Thus, we propose that
synaptobrevin/VAMP family members are engaged in anterograde and retrograde
protein sorting steps between the Golgi and the plasma membrane.
PMID: 11029060 [PubMed - indexed for MEDLINE]
365: Mol Biol Cell 2000 Oct;11(10):3381-96
Identification of a new vertebrate nucleoporin, Nup188, with the use of a novel
organelle trap assay.
Miller BR, Powers M, Park M, Fischer W, Forbes DJ.
Department of Biology, University of California at San Diego, La Jolla,
California 92093, USA.
The study of the nuclear pore in vertebrates would benefit from a strategy to
directly identify new nucleoporins and interactions between those nucleoporins.
We have developed a novel two-step "organelle trap" assay involving affinity
selection and in vitro pore assembly. In the first step, soluble proteins
derived from Xenopus egg extracts are applied to a column containing a ligand of
interest. The bound proteins are then tagged by biotinylation and eluted. In the
second step, potential nucleoporins are selected for by virtue of their ability
to assemble into annulate lamellae, a cytoplasmic mimic of nuclear pores. The
incorporated proteins are then recognized by their biotin tag. Here we use the
lectin wheat germ agglutinin (WGA) as ligand; WGA inhibits nuclear transport and
has been shown to directly bind three known nucleoporins from Xenopus extract,
Nup62, Nup98, and Nup214, all of which contain N-acetylglucosamine residues.
Under reduced-stringency conditions, three additional proteins bind to
WGA-Sepharose and are revealed by the organelle trap assay. We identified all
three as partner nucleoporins. Two were discovered to be Xenopus Nup93 and
Nup205. The third is a novel vertebrate nucleoporin, Nup188. This new vertebrate
protein, Xenopus Nup188, exists in a complex with xNup93 and xNup205. The
Nup93-Nup188-Nup205 complex does not bind directly to WGA but binds indirectly
via the N-acetylglucosamine-modified nucleoporins. A gene encoding human Nup188
was also identified. The discovery of vertebrate Nup188, related to a yeast
nucleoporin, and its novel protein-protein interactions illustrates the power of
the two-step organelle trap assay and identifies new building blocks for
constructing the nuclear pore.
PMID: 11029043 [PubMed - indexed for MEDLINE]
366: Nucleic Acids Res 2000 Oct 15;28(20):3897-903
Localisation of the DmCdc45 DNA replication factor in the mitotic cycle and
during chorion gene amplification.
Loebel D, Huikeshoven H, Cotterill S.
Department of Biochemistry and Immunology, St Georges Hospital Medical School,
Cranmer Terrace, London SW17 0RE, UK.
The cdc45 protein was originally identified in Saccharomyces cerevisiae and
shown to be essential for initiation of eukaryotic DNA replication. Subsequent
isolation and characterisation of the corresponding genes from fission yeast,
Xenopus and mammals also support a replication role for the protein in these
species. They further suggest that during the course of its function cdc45
interacts with a number of other replication proteins, including minichromosome
maintenance proteins, the origin recognition complex and DNA polymerase alpha.
We have cloned the gene coding for cdc45 protein from Drosophila melanogaster.
We have analysed the expression pattern of the cdc45 protein throughout the cell
cycle and the life cycle using a combination of indirect immunofluorescence and
subcellular fractionation. Our data show that cellular localisation and
developmental regulation of the protein is consistent with a role in DNA
replication. DmCdc45 is predominantly expressed in proliferating cells. In
addition, its subcellular location is nuclear during interphase and the protein
shows association with chromatin. The chromatin-associated form of the protein
shows a post-translational modification, which may be involved in control of the
action of the protein. DmCdc45 shows interactions with mcm proteins, however,
the interactions detected show some specificity, perhaps suggesting a
preferential association with particular mcm proteins. In addition we show that
a stoichiometric mcm interaction may not be obligatory for the function of cdc45
in follicle cell replication, because, unlike the mcm proteins, DmCdc45
localises to the chorion amplification foci in the follicle cells of the ovary.
PMID: 11024168 [PubMed - indexed for MEDLINE]
367: J Cell Biol 2000 Oct 2;151(1):167-78
Spontaneous release of cytosolic proteins from posttranslational substrates
before their transport into the endoplasmic reticulum.
Plath K, Rapoport TA.
Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical
School, Boston, Massachusetts 02115, USA.
In posttranslational translocation in yeast, completed protein substrates are
transported across the endoplasmic reticulum membrane through a translocation
channel formed by the Sec complex. We have used photo-cross-linking to
investigate interactions of cytosolic proteins with a substrate synthesized in a
reticulocyte lysate system, before its posttranslational translocation through
the channel in the yeast membrane. Upon termination of translation, the signal
recognition particle (SRP) and the nascent polypeptide-associated complex (NAC)
are released from the polypeptide chain, and the full-length substrate interacts
with several different cytosolic proteins. At least two distinct complexes exist
that contain among other proteins either 70-kD heat shock protein (Hsp70) or
tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1
(TRiC/CCT), which keep the substrate competent for translocation. None of the
cytosolic factors appear to interact specifically with the signal sequence.
Dissociation of the cytosolic proteins from the substrate is accelerated to the
same extent by the Sec complex and an unspecific GroEL trap, indicating that
release occurs spontaneously without the Sec complex playing an active role.
Once bound to the Sec complex, the substrate is stripped of all cytosolic
proteins, allowing it to subsequently be transported through the membrane
channel without the interference of cytosolic binding partners.
PMID: 11018062 [PubMed - indexed for MEDLINE]
368: J Cell Biol 2000 Oct 2;151(1):15-28
Tea2p is a kinesin-like protein required to generate polarized growth in fission
yeast.
Browning H, Hayles J, Mata J, Aveline L, Nurse P, McIntosh JR.
Department of Molecular, Cellular, and Developmental Biology, University of
Colorado, Boulder, Colorado 80309-0347, USA. browninh@icrf.icnet.uk
Cytoplasmic microtubules are critical for establishing and maintaining cell
shape and polarity. Our investigations of kinesin-like proteins (klps) and
morphological mutants in the fission yeast Schizosaccharomyces pombe have
identified a kinesin-like gene, tea2(+), that is required for cells to generate
proper polarized growth. Cells deleted for this gene are often bent during
exponential growth and initiate growth from improper sites as they exit
stationary phase. They have a reduced cytoplasmic microtubule network and
display severe morphological defects in genetic backgrounds that produce long
cells. The tip-specific marker, Tea1p, is mislocalized in both tea2-1 and
tea2Delta cells, indicating that Tea2p function is necessary for proper
localization of Tea1p. Tea2p is localized to the tips of the cell and in a
punctate pattern within the cell, often coincident with the ends of cytoplasmic
microtubules. These results suggest that this kinesin promotes microtubule
growth, possibly through interactions with the microtubule end, and that it is
important for establishing and maintaining polarized growth along the long axis
of the cell.
PMID: 11018050 [PubMed - indexed for MEDLINE]
369: J Biol Chem 2001 Jan 5;276(1):488-94
Identification and characterization of two novel components of the
Prp19p-associated complex, Ntc30p and Ntc20p.
Chen CH, Tsai WY, Chen HR, Wang CH, Cheng SC.
Institute of Microbiology and Immunology, National Yang-Ming University,
Shih-Pai 112, Taiwan, Republic of China.
The yeast Saccharomyces cerevisiae Prp19p protein is an essential splicing
factor and a spliceosomal component. It is not tightly associated with small
nuclear RNAs (snRNAs) but is associated with a protein complex consisting of at
least eight proteins. We have identified two novel components of the
Prp19p-associated complex, Ntc30p and Ntc20p. Like other identified components
of the complex, both Ntc30p and Ntc20p are associated with the spliceosome in
the same manner as Prp19p immediately after or concurrently with dissociation of
U4, indicating that the entire complex may bind to the spliceosome as an intact
form. Neither Ntc30p nor Ntc20p directly interacts with Prp19p, but both
interact with another component of the complex, Ntc85p. Immunoprecipitation
analysis revealed an ordered interactions of these components in formation of
the Prp19p-associated complex. Although null mutants of NTC30 or NTC20 showed no
obvious growth phenotype, deletion of both genes impaired yeast growth resulting
in accumulation of precursor mRNA. Extracts prepared from such a strain were
defective in pre-mRNA splicing in vitro, but the splicing activity could be
restored upon addition of the purified Prp19p-associated complex. These results
indicate that Ntc30p and Ntc20p are auxiliary splicing factors the functions of
which may be modulating the function of the Prp19p-associated complex.
PMID: 11018040 [PubMed - indexed for MEDLINE]
370: Nat Struct Biol 2000 Oct;7(10):894-902
Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly.
Munson M, Chen X, Cocina AE, Schultz SM, Hughson FM.
Department of Molecular Biology, Princeton University, Princeton, New Jersey
08544, USA.
In the eukaryotic secretory and endocytic pathways, transport vesicles shuttle
cargo among intracellular organelles and to and from the plasma membrane. Cargo
delivery entails fusion of the transport vesicle with its target, a process
thought to be mediated by membrane bridging SNARE protein complexes. Temporal
and spatial control of intracellular trafficking depends in part on regulating
the assembly of these complexes. In vitro, SNARE assembly is inhibited by the
closed conformation adopted by the syntaxin family of SNAREs. To visualize this
closed conformation directly, the X-ray crystal structure of a yeast syntaxin,
Sso1p, has been determined and refined to 2.1 A resolution. Mutants designed to
destabilize the closed conformation exhibit accelerated rates of SNARE assembly.
Our results provide insight into the mechanism of SNARE assembly and its
intramolecular and intermolecular regulation.
PMID: 11017200 [PubMed - indexed for MEDLINE]
371: Nat Genet 2000 Oct;26(2):141-2
Prediction of protein interactions: metabolic enzymes are frequently involved in
gene fusion.
Tsoka S, Ouzounis CA.
Computational Genomics Group, Research Programme, The European Bioinformatics
Institute, EMBL Cambridge Outstation, Cambridge, UK.
PMID: 11017064 [PubMed - indexed for MEDLINE]
372: Nat Biotechnol 2000 Oct;18(10):1075-9
Erratum in:
Nat Biotechnol 2000 Dec;18(12):1318
Use of G-protein fusions to monitor integral membrane protein-protein
interactions in yeast.
Ehrhard KN, Jacoby JJ, Fu XY, Jahn R, Dohlman HG.
Department of Pharmacology, Yale University School of Medicine, New Haven, CT
06536, USA.
The control of protein-protein interactions is a fundamental aspect of cell
regulation. Here we describe a new approach to detect the interaction of two
proteins in vivo. By this method, one binding partner is an integral membrane
protein whereas the other is soluble but fused to a G-protein gamma-subunit. If
the binding partners interact, G-protein signaling is disrupted. We demonstrate
interaction between known binding partners, syntaxin 1a with neuronal Sec1
(nSec1), and the fibroblast-derived growth factor receptor 3 (FGFR3) with SNT-1.
In addition, we describe a genetic screen to identify nSec1 mutants that are
expressed normally, but are no longer able to bind to syntaxin 1a. This provides
a convenient method to study interactions of integral membrane proteins, a class
of molecules that has been difficult to study by existing biochemical or genetic
methods.
PMID: 11017046 [PubMed - indexed for MEDLINE]
373: Mol Gen Genet 2000 Sep;264(1-2):89-97
Genetic analysis of the Saccharomyces cerevisiae Sgs1 helicase defines an
essential function for the Sgs1-Top3 complex in the absence of SRS2 or TOP1.
Duno M, Thomsen B, Westergaard O, Krejci L, Bendixen C.
Section for Molecular Genetics, Danish Institute of Agricultural Sciences,
Tjele.
The Saccharomyces cerevisiae gene SGS1 encodes a DNA helicase that shows
homology to the Escherichia coli protein RecQ and the products of the BLM and
WRN genes in humans, which are defective in Bloom's and Werner's syndrome,
respectively. Recently, it has been proposed that this helicase is involved in
maintaining the integrity of the rDNA and that loss of Sgs1 function leads to
accelerated aging. Sgs1 has been isolated on the basis of its genetic
interaction with both topoisomerase I and topoisomerase III, as well as in a
two-hybrid screen for proteins that interact with the C-terminal portion of
topoisomerase II. We have defined the minimal structural elements of Sgs1
required for its interactions with the three topoisomerases, and demonstrate
that the complex phenotypes associated with sgs1 mutants are a consequence of a
dysfunctional Sgs1-Top3 complex. We also report that the synthetic relationship
between mutations in SGS1 and SRS2, which encodes another helicase implicated in
recombinational repair, likewise result from a dysfunctional Sgs1-Top3
interaction. Our findings indicate that Sgs1 may act on different DNA structures
depending on the activity of topoisomerase I, Srs2 and topoisomerase III.
PMID: 11016837 [PubMed - indexed for MEDLINE]
374: Genetics 2000 Oct;156(2):535-47
Synthetic lethal interactions suggest a role for the Saccharomyces cerevisiae
Rtf1 protein in transcription elongation.
Costa PJ, Arndt KM.
Department of Biological Sciences, University of Pittsburgh, Pittsburgh,
Pennsylvania 15260, USA.
Strong evidence indicates that transcription elongation by RNA polymerase II
(pol II) is a highly regulated process. Here we present genetic results that
indicate a role for the Saccharomyces cerevisiae Rtf1 protein in transcription
elongation. A screen for synthetic lethal mutations was carried out with an rtf1
deletion mutation to identify factors that interact with Rtf1 or regulate the
same process as Rtf1. The screen uncovered mutations in SRB5, CTK1, FCP1, and
POB3. These genes encode an Srb/mediator component, a CTD kinase, a CTD
phosphatase, and a protein involved in the regulation of transcription by
chromatin structure, respectively. All of these gene products have been directly
or indirectly implicated in transcription elongation, indicating that Rtf1 may
also regulate this process. In support of this view, we show that RTF1
functionally interacts with genes that encode known elongation factors,
including SPT4, SPT5, SPT16, and PPR2. We also show that a deletion of RTF1
causes sensitivity to 6-azauracil and mycophenolic acid, phenotypes correlated
with a transcription elongation defect. Collectively, our results suggest that
Rtf1 may function as a novel transcription elongation factor in yeast.
PMID: 11014804 [PubMed - indexed for MEDLINE]
375: Genetics 2000 Oct;156(2):523-34
Suppressors of mdm20 in yeast identify new alleles of ACT1 and TPM1 predicted to
enhance actin-tropomyosin interactions.
Singer JM, Hermann GJ, Shaw JM.
Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.
The actin cytoskeleton is required for many aspects of cell division in yeast,
including mitochondrial partitioning into growing buds (mitochondrial
inheritance). Yeast cells lacking MDM20 function display defects in both
mitochondrial inheritance and actin organization, specifically, a lack of
visible actin cables and enhanced sensitivity to Latrunculin A. mdm20 mutants
also exhibit a temperature-sensitive growth phenotype, which we exploited to
isolate second-site suppressor mutations. Nine dominant suppressors selected in
an mdm20/mdm20 background rescue temperature-sensitive growth defects and
mitochondrial inheritance defects and partially restore actin cables in haploid
and diploid mdm20 strains. The suppressor mutations define new alleles of ACT1
and TPM1, which encode actin and the major form of tropomyosin in yeast,
respectively. The ACT1 mutations cluster in a region of the actin protein
predicted to contact tropomyosin, suggesting that they stabilize actin cables by
enhancing actin-tropomyosin interactions. The characteristics of the mutant ACT1
and TPM1 alleles and their potential effects on protein structure and binding
are discussed.
PMID: 11014803 [PubMed - indexed for MEDLINE]
376: Biochem Pharmacol 2000 Oct 15;60(8):1009-13
Protein recruitment systems for the analysis of protein-protein interactions.
Aronheim A.
Department of Molecular Genetics, the B. Rappaport Faculty of Medicine,
Technion-Israel Institute of Technology, Haifa 31096, Israel.
aronheim@tx.technion.ac.il
Following the completion of genome projects in a number of organisms, it is
becoming evident that a relatively large proportion of the genes identified
encode for proteins that have no sequence homology with known proteins. One
possible approach towards understanding protein function is to identify the
proteins with which a particular protein associates. Although very powerful, the
most commonly used genetic method, the two-hybrid system, is limited in its
ability to detect all possible protein-protein interactions. The development of
novel approaches, such as the protein recruitment systems, provides attractive
alternatives towards identification of protein-protein interactions where other
methods have failed to function.
Publication Types:
Review
Review, Tutorial
PMID: 11007935 [PubMed - indexed for MEDLINE]
377: J Biol Chem 2000 Nov 24;275(47):36498-501
Functional interaction of proliferating cell nuclear antigen with MSH2-MSH6 and
MSH2-MSH3 complexes.
Clark AB, Valle F, Drotschmann K, Gary RK, Kunkel TA.
Laboratory of Molecular Genetics and Laboratory of Structural Biology, National
Institute of Environmental Health Sciences, Research Triangle Park, North
Carolina 27709, USA.
Eukaryotic DNA mismatch repair requires the concerted action of several
proteins, including proliferating cell nuclear antigen (PCNA) and heterodimers
of MSH2 complexed with either MSH3 or MSH6. Here we report that MSH3 and MSH6,
but not MSH2, contain N-terminal sequence motifs characteristic of proteins that
bind to PCNA. MSH3 and MSH6 peptides containing these motifs bound PCNA, as did
the intact Msh2-Msh6 complex. This binding was strongly reduced when alanine was
substituted for conserved residues in the motif. Yeast strains containing
alanine substitutions in the PCNA binding motif of Msh6 or Msh3 had elevated
mutation rates, indicating that these interactions are important for genome
stability. When human MSH3 or MSH6 peptides containing the PCNA binding motif
were added to a human cell extract, mismatch repair activity was inhibited at a
step preceding DNA resynthesis. Thus, MSH3 and MSH6 interactions with PCNA may
facilitate early steps in DNA mismatch repair and may also be important for
other roles of these eukaryotic MutS homologs.
PMID: 11005803 [PubMed - indexed for MEDLINE]
378: Biochim Biophys Acta 2000 Aug 15;1459(2-3):316-24
Haem-polypeptide interactions during cytochrome c maturation.
Thony-Meyer L.
Institute of Microbiology, ETH Zurich, Schmelzbergstrasse 7, CH-8092, Zurich,
Switzerland. lthoeny@micro.biol.ethz.ch
Cytochrome c maturation involves the translocation of a polypeptide, the
apocytochrome, and its cofactor, haem, through a membrane, before the two
molecules are ligated covalently. This review article focuses on the current
knowledge on the journey of haem during this process, which is known best in the
Gram-negative bacterium Escherichia coli. As haem always occurs bound to
protein, its passage across the cytoplasmic membrane and incorporation into the
apocytochrome appears to be mediated by a set of proteinaceous maturation
factors, the Ccm (cytochrome c maturation) proteins. At least three of them,
CcmC, CcmE and CcmF, are thought to interact directly with haem. CcmE binds haem
covalently, thus representing an intermediate of the haem trafficking pathway.
CcmC is required for binding of haem to CcmE, and CcmF for releasing it from
CcmE and transferring it onto the apocytochrome. The mechanism by which haem
crosses the cytoplasmic membrane is currently unknown.
Publication Types:
Review
Review, Tutorial
PMID: 11004446 [PubMed - indexed for MEDLINE]
379: Mol Cell Biol 2000 Oct;20(20):7438-49
A motif shared by TFIIF and TFIIB mediates their interaction with the RNA
polymerase II carboxy-terminal domain phosphatase Fcp1p in Saccharomyces
cerevisiae.
Kobor MS, Simon LD, Omichinski J, Zhong G, Archambault J, Greenblatt J.
Banting and Best Department of Medical Research, University of Toronto, Toronto,
Ontario M5G 1L6, Canada.
Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and
dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of
its largest subunit. We have used deletion and point mutations in Fcp1p, a
TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT
domain, like that of its catalytic domain, is important for cell viability, mRNA
synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy
terminal to its BRCT domain and at its amino terminus were not essential for
viability, deletion of either of these regions affected the phosphorylation
state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound
directly to the first cyclin-like repeat in the core domain of the general
transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These
regulatory interactions with Fcp1p involved closely related amino acid sequence
motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74
(Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain
fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate
transcription when it is artificially tethered to a promoter. These results
suggest strongly that this KEFGK motif in RAP74 mediates its interaction with
Fcp1p in vivo.
PMID: 11003641 [PubMed - indexed for MEDLINE]
380: RNA 2000 Sep;6(9):1289-305
The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is
critical for function.
Lindsey-Boltz LA, Chawla G, Srinivasan N, Vijayraghavan U, Garcia-Blanco MA.
Program in Molecular Cancer Biology, Duke University Medical Center, Durham,
North Carolina 27710, USA.
In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of
the second step of pre-mRNA splicing. The function and interacting factors for
this protein have not been elucidated. We have performed a mutational analysis
of yPrp17p to identify protein domains critical for function. A series of
deletions were made throughout the region spanning the N-terminal 158 amino
acids of the protein, which do not contain any identified structural motifs. The
C-terminal portion (amino acids 160-455) contains a WD domain containing seven
WD repeats. We determined that a minimal functional Prp17p consists of the WD
domain and 40 amino acids N-terminal to it. We generated a three-dimensional
model of the WD repeats in Prp17p based on the crystal structure of the
beta-transducin WD domain. This model was used to identify potentially important
amino acids for in vivo functional characterization. Through analysis of
mutations in four different loops of Prp17p that lie between beta strands in the
WD repeats, we have identified four amino acids, 235TETG238, that are critical
for function. These amino acids are predicted to be surface exposed and may be
involved in interactions that are important for splicing. Temperature-sensitive
prp17 alleles with mutations of these four amino acids are defective for the
second step of splicing and are synthetically lethal with a U5 snRNA loop I
mutation, which is also required for the second step of splicing. These data
reinforce the functional significance of this region within the WD domain of
Prp17p in the second step of splicing.
PMID: 10999606 [PubMed - indexed for MEDLINE]
381: Structure Fold Des 2000 Aug 15;8(8):841-50
Crystal structure and mutational analysis of the Saccharomyces cerevisiae cell
cycle regulatory protein Cks1: implications for domain swapping, anion binding
and protein interactions.
Bourne Y, Watson MH, Arvai AS, Bernstein SL, Reed SI, Tainer JA.
Centre National de la Recherche Scientifique, Marseille, France.
yves@afmb.cnrs-mrs.fr
BACKGROUND: The Saccharomyces cerevisiae protein Cks1 (cyclin-dependent kinase
subunit 1) is essential for cell-cycle progression. The biological function of
Cks1 can be modulated by a switch between two distinct molecular assemblies: the
single domain fold, which results from the closing of a beta-hinge motif, and
the intersubunit beta-strand interchanged dimer, which arises from the opening
of the beta-hinge motif. The crystal structure of a cyclin-dependent kinase
(Cdk) in complex with the human Cks homolog CksHs1 single-domain fold revealed
the importance of conserved hydrophobic residues and charged residues within the
beta-hinge motif. RESULTS: The 3.0 A resolution Cks1 structure reveals the
strict structural conservation of the Cks alpha/beta-core fold and the
beta-hinge motif. The beta hinge identified in the Cks1 structure includes a
novel pivot and exposes a cluster of conserved tyrosine residues that are
involved in Cdk binding but are sequestered in the beta-interchanged Cks homolog
suc1 dimer structure. This Cks1 structure confirms the conservation of the Cks
anion-binding site, which interacts with sidechain residues from the C-terminal
alpha helix of another subunit in the crystal. CONCLUSIONS: The Cks1 structure
exemplifies the conservation of the beta-interchanged dimer and the
anion-binding site in evolutionarily distant yeast and human Cks homologs.
Mutational analyses including in vivo rescue of CKS1 disruption support the dual
functional roles of the beta-hinge residue Glu94, which participates in Cdk
binding, and of the anion-binding pocket that is located 22 A away and on an
opposite face to Glu94. The Cks1 structure suggests a biological role for the
beta-interchanged dimer and the anion-binding site in targeting Cdks to specific
phosphoproteins during cell-cycle progression.
PMID: 10997903 [PubMed - indexed for MEDLINE]
382: Yeast 2000 Sep 30;16(13):1229-41
A network of proteins around Rvs167p and Rvs161p, two proteins related to the
yeast actin cytoskeleton.
Bon E, Recordon-Navarro P, Durrens P, Iwase M, Toh-E A, Aigle M.
Laboratoire de Biologie Cellulaire de la Levure, IBGC, 1 rue Camille
Saint-Saens, 33077 Bordeaux, France.
The Rvs161p and Rvs167p proteins of Saccharomyces cerevisiae, homologues of
higher eukaryotes' amphiphysins, associate with actin and appear to be involved
in several functions related to the actin cytoskeleton. In order to identify
partners of the Rvsp proteins, yeast libraries constructed in two-hybrid vectors
were screened using either Rvs167p or Rvs161p as a bait. The selected
candidates, representing 34 ORFs, were then tested against both Rvsp proteins,
as well as domains of Rvs167p or Rvs161p. Among the most significant ones, 24
ORFs were specific preys of Rvs167p only and two gave interactions with Rvs161p
only. Interestingly, five ORFs were preys of both Rvs161p and Rvs167p (RVS167,
LAS17, YNL094w, YMR192w and YPL249c). Analysis of putative functions of the
candidates confirm involvement of the Rvsp in endocytosis/vesicle traffic, but
also opens possible new fields, such as nuclear functions. Copyright 2000 John
Wiley & Sons, Ltd.
PMID: 10992286 [PubMed - indexed for MEDLINE]
383: J Neurochem 2000 Oct;75(4):1335-51
Regulators of G protein signaling: a bestiary of modular protein binding
domains.
Burchett SA.
Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University
School of Medicine, New Haven, Connecticut, USA.
Members of the newly discovered regulator of G protein signaling (RGS) families
of proteins have a common RGS domain. This RGS domain is necessary for
conferring upon RGS proteins the capacity to regulate negatively a variety of
Galpha protein subunits. However, RGS proteins are more than simply negative
regulators of signaling. RGS proteins can function as effector antagonists, and
recent evidence suggests that RGS proteins can have positive effects on
signaling as well. Many RGS proteins possess additional C- and N-terminal
modular protein-binding domains and motifs. The presence of these additional
modules within the RGS proteins provides for multiple novel regulatory
interactions performed by these molecules. These regions are involved in
conferring regulatory selectivity to specific Galpha-coupled signaling pathways,
enhancing the efficacy of the RGS domain, and the translocation or targeting of
RGS proteins to intracellular membranes. In other instances, these domains are
involved in cross-talk between different Galpha-coupled signaling pathways and,
in some cases, likely serve to integrate small GTPases with these G protein
signaling pathways. This review discusses these C- and N-terminal domains and
their roles in the biology of the brain-enriched RGS proteins. Methods that can
be used to investigate the function of these domains are also discussed.
Publication Types:
Review
Review, Tutorial
PMID: 10987813 [PubMed - indexed for MEDLINE]
384: FEBS Lett 2000 Sep 8;481(1):13-8
Caspase-3 and inhibitor of apoptosis protein(s) interactions in Saccharomyces
cerevisiae and mammalian cells.
Wright ME, Han DK, Hockenbery DM.
Molecular and Cellular Biology Program, Division of Clinical Research, Fred
Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.
Using a heterologous yeast expression assay, we show that inhibitor of apoptosis
proteins (IAPs) suppress caspase-3-mediated cytotoxicity in the order of
XIAP>c-IAP2>c-IAP1>survivin. The same ordering of IAP activities was
demonstrated in mammalian cells expressing an auto-activating caspase-3. The
relative anti-apoptotic activities of each IAP depended on the particular death
stimulus. For IAP-expressing cells treated with camptothecin, survival
correlated with their intrinsic anti-caspase-3 activity. However,
c-IAP1-transfected cells were disproportionately resistant to tumor necrosis
factor-alpha, suggesting that its anti-apoptotic activities extend beyond
caspase-3 or -7 inhibition. Yeast-based caspase assays provide rapid, reliable
information on specificity and activity of the IAPs and aid in identifying
critical targets in mammalian apoptotic pathways.
PMID: 10984607 [PubMed - indexed for MEDLINE]
385: Mol Biol Cell 2000 Sep;11(9):2949-59
Bim1p/Yeb1p mediates the Kar9p-dependent cortical attachment of cytoplasmic
microtubules.
Miller RK, Cheng SC, Rose MD.
Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University,
Princeton, New Jersey 08544, USA.
In Saccharomyces cerevisiae, positioning of the mitotic spindle depends on the
interaction of cytoplasmic microtubules with the cell cortex. In this process,
cortical Kar9p in the bud acts as a link between the actin and microtubule
cytoskeletons. To identify Kar9p-interacting proteins, a two-hybrid screen was
conducted with the use of full-length Kar9p as bait, and three genes were
identified: BIM1, STU2, and KAR9 itself. STU2 encodes a component of the spindle
pole body. Bim1p is the yeast homologue of the human microtubule-binding protein
EB1, which is a binding partner to the adenomatous polyposis coli protein
involved in colon cancer. Eighty-nine amino acids within the third quarter of
Bim1p was sufficient to confer interaction with Kar9p. The two-hybrid
interactions were confirmed with the use of coimmunoprecipitation experiments.
Genetic analysis placed Bim1p in the Kar9p pathway for nuclear migration. Bim1p
was not required for Kar9p's cortical or spindle pole body localization.
However, deletion of BIM1 eliminated Kar9p localization along cytoplasmic
microtubules. Furthermore, in the bim1 mutants, the cytoplasmic microtubules no
longer intersected the cortical dot of Green Fluorescent Protein-Kar9p. These
experiments demonstrate that the interaction of cytoplasmic microtubules with
the Kar9p cortical attachment site requires the microtubule-binding protein
Bim1p.
PMID: 10982392 [PubMed - indexed for MEDLINE]
386: J Virol 2000 Oct;74(19):9167-74
Yeast three-hybrid screening of rous sarcoma virus mutants with randomly
mutagenized minimal packaging signals reveals regions important for gag
interactions.
Lee EG, Linial ML.
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle,
Washington 98109, USA.
We previously showed that the yeast three-hybrid system provides a genetic assay
of both RNA and protein components for avian retroviral RNA encapsidation. In
the current study, we used this assay to precisely define cis-acting
determinants involved in avian leukosis sarcoma virus packaging RNA binding to
Gag protein. In vivo screening of Rous sarcoma virus mutants was performed with
randomly mutated minimal packaging sequences (MPsi) made using PCR amplification
after cotransformation with GagDeltaPR protein into yeast cells. Colonies with
low beta-galactosidase activity were analyzed to locate mutations in MPsi
sequences affecting binding to Gag proteins. This genetic assay delineated
secondary structural elements that are important for efficient RNA binding,
including a single-stranded small bulge containing the initiation codon for
uORF3, as well as adjacent stem structures. This implies a possible tertiary
structure favoring the high-affinity binding sites for Gag. In most cases,
results from the three-hybrid assay were well correlated with those from the
viral RNA packaging assays. The results from random mutagenesis using the rapid
three-hybrid binding assay are consistent with those from site-directed
mutagenesis using in vivo packaging assays.
PMID: 10982363 [PubMed - indexed for MEDLINE]
387: J Biol Chem 2000 Dec 8;275(49):38206-12
Mapping of a minimal apolipoprotein(a) interaction motif conserved in
fibrin(ogen) beta - and gamma -chains.
Klose R, Fresser F, Kochl S, Parson W, Kapetanopoulos A, Fruchart-Najib J, Baier
G, Utermann G.
Institute for Medical Biology and Human Genetics, Universitat Innsbruck, 6020
Innsbruck, Austria.
Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombotic
disease in humans. The physiological function(s) of Lp(a) as well as the precise
mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown.
Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of
the blood clotting cascade has been demonstrated in vitro, but the domains in
fibrin(ogen) critical for interaction are undefined. We used apo(a) kringle IV
subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid
interaction trap system. Among positive clones that emerged from the screen,
clones were identified as fibrinogen beta- and gamma-chains. Peptide-based
pull-down experiments confirmed that the emerging peptide motif, conserved in
the carboxyl-terminal globular domains of the fibrinogen beta and gamma modules
specifically interacts with apo(a)/Lp(a) in human plasma as well as in cell
culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically
expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and
of lysine binding sites in apo(a) for the interaction was evaluated by binding
experiments with apo(a) mutants and a mutated fibrin(ogen) peptid. This
confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major
fibrin(ogen) binding site but also demonstrated lysine-independent interactions.
PMID: 10980194 [PubMed - indexed for MEDLINE]
388: Genetics 2000 Sep;156(1):21-9
Important role for phylogenetically invariant PP2Acalpha active site and
C-terminal residues revealed by mutational analysis in Saccharomyces cerevisiae.
Evans DR, Hemmings BA.
Friedrich Miescher Institute, Basel 4058 Switzerland. drhevans@usa.net
PP2A is a central regulator of eukaryotic signal transduction. The human
catalytic subunit PP2Acalpha functionally replaces the endogenous yeast enzyme,
Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells
were employed to explore the role of invariant PP2Ac residues. The PP2Acalpha
Y127N substitution abolished essential PP2Ac function in vivo and impaired
catalysis severely in vitro, consistent with the prediction from structural
studies that Tyr-127 mediates substrate binding and its side chain interacts
with the key active site residues His-118 and Asp-88. The V159E substitution
similarly impaired PP2Acalpha catalysis profoundly and may cause global
disruption of the active site. Two conditional mutations in the yeast Pph22p
protein, F232S and P240H, were found to cause temperature-sensitive impairment
of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects
conferred by these mutations result from a loss of PP2Ac enzyme activity.
Substitution of the PP2Acalpha C-terminal Tyr-307 residue by phenylalanine
impaired protein function, whereas the Y307D and T304D substitutions abolished
essential function in vivo. Nevertheless, Y307D did not reduce PP2Acalpha
catalytic activity significantly in vitro, consistent with an important role for
the C terminus in mediating essential protein-protein interactions. Our results
identify key residues important for PP2Ac function and characterize new reagents
for the study of PP2A in vivo.
PMID: 10978272 [PubMed - indexed for MEDLINE]
389: J Biol Chem 2000 Nov 24;275(47):37251-6
Functional connections between mediator components and general transcription
factors of Saccharomyces cerevisiae.
Sakurai H, Fukasawa T.
School of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80
Kodatsuno, Kanazawa, Ishikawa 920-0942, Japan. sakurai@kenroku.kanazawa-u.ac.jp
The yeast Gal11 protein is an important component of the Mediator complex in RNA
polymerase II-directed transcription. Gal11 and the general transcription factor
(TF) IIE are involved in regulation of the protein kinase activity of TFIIH that
phosphorylates the carboxyl-terminal domain of RNA polymerase II. We have
previously shown that Gal11 binds the small and large subunits of TFIIE at two
Gal11 domains, A and B, respectively, which are important for normal function of
Gal11 in vivo. Here we demonstrate that Gal11 binds directly to TFIIH through
domain A in vitro. A null mutation in GAL11 caused lethality of cells when
combined with temperature-sensitive mutations in the genes encoding TFIIE or the
carboxyl-terminal domain kinase, indicating the presence of genetic interactions
between Gal11 and these proteins. Mutational depletion of Gal11 or TFIIE caused
inefficient opening of the transcription initiation region, but had no
significant effect on TATA-binding protein occupancy of the TATA sequence in
vivo. These results suggest that the functions of Gal11 and TFIIE are necessary
after recruitment of TATA-binding protein to the TATA box presumably at the step
of stable preinitiation complex formation and/or promoter melting. We illustrate
genetic interactions between Gal11 and other Mediator components such as Med2
and Pgd1/Hrs1/Med3.
PMID: 10973956 [PubMed - indexed for MEDLINE]
390: Genes Dev 2000 Sep 1;14(17):2206-15
Promotion of Rad51-dependent D-loop formation by yeast recombination factor
Rdh54/Tid1.
Petukhova G, Sung P, Klein H.
Department of Molecular Medicine and Institute of Biotechnology, University of
Texas Health Science Center at San Antonio, San Antonio, Texas 78245-3207, USA.
The first DNA joint formed in homologous recombination processes is a D-loop.
Saccharomyces cerevisiae RDH54/TID1-encoded product, a Swi2/Snf2-like factor
involved in recombination, is shown here to promote D-loop formation with Rad51
recombinase. Physical interaction between Rdh54 and Rad51 is functionally
important because Rdh54 does not enhance the recombinase activity of the
Escherichia coli RecA protein. Robust dsDNA-activated ATPase activity in Rdh54
generates unconstrained negative and positive supercoils in DNA. Efficient
D-loop formation occurs with even topologically relaxed DNA, suggesting that via
specific protein-protein interactions, the negative supercoils produced by Rdh54
are used by Rad51 for making DNA joints.
PMID: 10970884 [PubMed - indexed for MEDLINE]
391: FEBS Lett 2000 Aug 25;480(1):37-41
Four years of post-genomic life with 6,000 yeast genes.
Goffeau A.
Unite de Biochimie Physiologique, Universite Catholique de Louvain,
Louvain-la-Neuve, Belgium. goffeau@fysa.ucl.ac.be
Four years after disclosure of the full yeast genome sequence, a series of
resources including tens of thousands of mutant strains, plasmids bearing
isolated genes and disruption cassettes are becoming publicly available.
Deletions of each of the 6,000 putative yeast genes are being screened
systematically for dozens of phenotypic traits. In addition, new global
approaches such as DNA hybridization arrays, quantitative proteomics and
two-hybrid interactions are being steadily improved. They progressively build up
an immense computation network of billions of data points which will, within the
next decade, characterize all molecular interactions occurring in a simple
eukaryotic cell. In this process of acquisition of new basic knowledge, an
international community of over 1,000 laboratories cooperates with a remarkable
willingness to share projects and results.
Publication Types:
Review
Review, Tutorial
PMID: 10967326 [PubMed - indexed for MEDLINE]
392: J Mol Biol 2000 Sep 1;301(5):1097-112
The strength of acidic activation domains correlates with their affinity for
both transcriptional and non-transcriptional proteins.
Melcher K.
Departments of Internal Medicine and Biochemistry, University of Texas
Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75235-8573,
USA. K.Melcher@em.uni-frankfurt.de
Activation domains (ADs) appear to work by making specific protein-protein
contacts with the transcriptional machinery. However, ADs show no apparent
sequence conservation, they can be functionally replaced by a number of random
peptides and unrelated proteins, and their function does not depend on
sustaining a complex tertiary structure. To gain a broader perspective on the
nature of interactions between acidic ADs and several of their proposed targets,
the in vivo strengths of viral, human, yeast, and artificial activation domains
were determined under physiological conditions, and mutant ADs with increased in
vivo potencies were selected. The affinities between ADs and proposed targets
were determined in vitro and all interactions were found to be of low-level
affinity with dissociation constants above 10(-7)M. However, in vivo potencies
of all ADs correlated nearly perfectly with their affinities for transcriptional
proteins. Surprisingly, the weak interactions of the different ADs with at least
two non-transcriptional proteins show the same rank order of binding and AD
mutants selected for increased in vivo strength also have increased affinities
to non-transcriptional proteins. Based on these results, isolated acidic ADs can
bind with relatively low-level specificity and affinity to many different
proteins and the strength of these semi-specific interactions determine the
strength of an AD. I suggest that ADs expose flexible hydrophobic elements in an
aqueous environment to contact hydrophobic patches over short distances,
shifting specificity of activators largely to the DNA colocalization of arrays
of ADs and targets. Copyright 2000 Academic Press.
PMID: 10966808 [PubMed - indexed for MEDLINE]
393: Annu Rev Biochem 2000;69:829-80
Regulation of chromosome replication.
Kelly TJ, Brown GW.
Department of Molecular Biology and Genetics, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205, USA. tkelly@jhmi.edu
The initiation of DNA replication in eukaryotic cells is tightly controlled to
ensure that the genome is faithfully duplicated once each cell cycle. Genetic
and biochemical studies in several model systems indicate that initiation is
mediated by a common set of proteins, present in all eukaryotic species, and
that the activities of these proteins are regulated during the cell cycle by
specific protein kinases. Here we review the properties of the initiation
proteins, their interactions with each other, and with origins of DNA
replication. We also describe recent advances in understanding how the
regulatory protein kinases control the progress of the initiation reaction.
Finally, we describe the checkpoint mechanisms that function to preserve the
integrity of the genome when the normal course of genome duplication is
perturbed by factors that damage the DNA or inhibit DNA synthesis.
Publication Types:
Review
Review, Academic
PMID: 10966477 [PubMed - indexed for MEDLINE]
394: Annu Rev Biochem 2000;69:571-95
Mechanisms and control of mRNA decapping in Saccharomyces cerevisiae.
Tucker M, Parker R.
Department of Molecular and Cellular Biology and Howard Hughes Medical
Institute, University of Arizona, Tucson, Arizona 85721, USA.
The process of mRNA turnover is a critical component of the regulation of gene
expression. In the past few years a discrete set of pathways for the degradation
of polyadenylated mRNAs in eukaryotic cells have been described. A major pathway
of mRNA degradation in yeast occurs by deadenylation of the mRNA, which leads to
a decapping reaction, thereby exposing the mRNA to rapid 5' to 3' exonucleolytic
degradation. A critical step in this pathway is decapping, since it effectively
terminates the existence of the mRNA and is the site of numerous control inputs.
In this review, we discuss the properties of the decapping enzyme and how its
activity is regulated to give rise to differential mRNA turnover. A key point is
that decapping appears to be controlled by access of the enzyme to the cap
structure in a competition with the translation initiation complex. Strikingly,
several proteins required for mRNA decapping show interactions with the
translation machinery and suggest possible mechanisms for the triggering of mRNA
decapping.
Publication Types:
Review
Review, Academic
PMID: 10966469 [PubMed - indexed for MEDLINE]
395: J Neurosci 2000 Sep 1;20(17):6333-9
Cbln3, a novel member of the precerebellin family that binds specifically to
Cbln1.
Pang Z, Zuo J, Morgan JI.
Department of Developmental Neurobiology, St. Jude Children's Research Hospital,
Memphis, Tennessee 38105, USA.
Precerebellin (Cbln1) is the precursor of the brain-specific hexadecapeptide
cerebellin. Although cerebellin has properties of a conventional neuropeptide,
its function is controversial because Cbln1 has structural features
characteristic of circulating atypical collagens. Cbln1 is related to the three
subunits of the complement C1q complex. Therefore, we hypothesized that Cbln1
participated in analogous heteromeric complexes with precerebellin-related
proteins. Using LexA-Cbln1 as bait in a yeast two-hybrid screen, we isolated a
cDNA encoding a novel Cbln1-related protein, designated Cbln3. The gene encoding
cbln3 had the same intron-exon structure as cbln1 but mapped to a different
mouse chromosome (14). The deduced amino acid sequence of Cbln3 was 55%
identical to Cbln1 and also contained a C1q signature domain and signal sequence
for secretion. In addition to binding avidly to Cbln3, Cbln1 also formed
homomeric complexes. In contrast, Cbln3 homomeric association was weak. These
interactions exhibited specificity because C1qB bound to neither Cbln1 nor
Cbln3. Like cbln1, cbln3 was expressed in the cerebellum and dorsal cochlear
nucleus in which it was detected in granule neurons. Because Cbln1 and Cbln3 are
coexpressed in the brain and interact avidly, they may function as a secreted
heteromeric complex in vivo.
PMID: 10964938 [PubMed - indexed for MEDLINE]
396: Biochem Soc Trans 2000;28(4):410-4
Recruitment of chromatin remodelling factors during gene activation via the
glucocorticoid receptor N-terminal domain.
Wallberg AE, Flinn EM, Gustafsson JA, Wright AP.
Department of Biosciences, Karolinska Institutet, Novum, SE-141 57 Huddinge,
Sweden.
We have shown that yeast mutants with defects in the Ada adaptor proteins are
defective in hormone-dependent gene activation by ectopically expressed human
glucocorticoid receptor (GR). Others have shown that the Ada2 protein is
required for physical interactions between some activation domains and TBP
(TATA-binding protein), whereas the Gcn5 (Ada4) protein has a histone
acetyltransferase (HAT) activity. Although all HAT enzymes are able to acetylate
histone substrates, some also acetylate non-histone proteins. Taken together,
these observations suggest that the Ada proteins have the ability to effect
different steps in the process of gene activation. It has recently been shown
that the Ada proteins are present in two distinct protein complexes, the Ada
complex and a larger SAGA complex. Our recent work has focused on determining
(1) which of the Ada-containing complexes mediates gene activation by GR, (2)
whether the HAT activity encoded by GCN5 is required for GR-dependent gene
activation, (3) whether the Ada proteins contribute to GR-mediated activation at
the level of chromatin remodelling and (4) how the role of these HAT complexes
is integrated with other chromatin remodelling activities during GR-mediated
gene activation. Our results suggest a model in which GR recruits the SAGA
complex and that this contributes to chromatin remodelling via a mechanism
involving the acetylation of histones. Furthermore, recruitment of the SWI/SNF
remodelling complex also has a role in GR-mediated activation that is
independent of the role of SAGA. These complexes are similar to analogous
mammalian complexes and therefore these results are likely to be relevant to the
human system.
Publication Types:
Review
Review, Tutorial
PMID: 10961930 [PubMed - indexed for MEDLINE]
397: J Bacteriol 2000 Sep;182(18):5262-6
Lantibiotic biosynthesis: interactions between prelacticin 481 and its putative
modification enzyme, LctM.
Uguen P, Le Pennec JP, Dufour A.
Laboratoire de Biologie et Chimie Moleculaires, Universite de Bretagne Sud,
Vannes, France.
Class AII and AIII lantibiotics and mersacidin are antibacterial peptides
containing unusual residues obtained by posttranslational modifications of
prepeptides, presumably catalyzed by LanM. LctM, the LanM for lacticin 481, is
essential for the production of this class AII lantibiotic. Using the yeast
two-hybrid system, we showed direct contact between the prelacticin 481 and
LctM, supporting the proposed LctM function. Sixteen domains are conserved
between the 10 known LanM proteins, whereas three additional domains were found
only in class AII LanM proteins and in MrsM, the LanM for mersacidin. All the
truncated LctM proteins that we tested presented impaired LctA-binding activity.
PMID: 10960114 [PubMed - indexed for MEDLINE]
398: J Biol Chem 2000 Nov 10;275(45):34837-40
Interactions of Cdk7 and Kin28 with Hint/PKCI-1 and Hnt1 histidine triad
proteins.
Korsisaari N, Makela TP.
Haartman Institute & Biocentrum Helsinki, P. O. Box 21, University of Helsinki,
00014 Helsinki, Finland.
Cyclin-dependent kinase 7 (Cdk7) forms a trimeric complex with cyclin H and Mat1
to form the mammalian Cdk-activating kinase, CAK, as well as a part of the basal
transcription factor TFIIH, where Cdk7 phosphorylates the C-terminal domain
(CTD) of the large subunit of RNA polymerase II. Here, we report a novel
interaction between Cdk7 and a histidine triad (HIT) family protein,
Hint/PKCI-1. This interaction was initially observed in a yeast two-hybrid study
and subsequently verified by co-immunoprecipitation and subcellular localization
studies, where overexpression of Cdk7 leads to partial relocalization of Hint to
the nucleus. The physical association is independent of cyclin H binding or Cdk7
kinase activity and is conserved between the related Sacharomyces cerevisiae CTD
kinase Kin28 and the HIT protein Hnt1. Furthermore, combination of a disruption
of HNT1 and a KIN28 temperature-sensitive allele in S. cerevisiae led to highly
elongated cell morphology and reduced colony formation, indicating a genetic
interaction between KIN28 and HNT1. The physical and genetic interactions of
Hint and Hnt1 with Cdk7 and Kin28 suggest a role for this class of histidine
triad proteins in the regulation of Cdk7 and Kin28 functions.
PMID: 10958787 [PubMed - indexed for MEDLINE]
399: Mol Cell Biol 2000 Sep;20(18):7037-48
The N terminus of the centromere H3-like protein Cse4p performs an essential
function distinct from that of the histone fold domain.
Chen Y, Baker RE, Keith KC, Harris K, Stoler S, Fitzgerald-Hayes M.
Department of Biochemistry and Molecular Biology, University of Massachusetts at
Amherst, 01003, USA.
Cse4p is an evolutionarily conserved histone H3-like protein that is thought to
replace H3 in a specialized nucleosome at the yeast (Saccharomyces cerevisiae)
centromere. All known yeast, worm, fly, and human centromere H3-like proteins
have highly conserved C-terminal histone fold domains (HFD) but very different N
termini. We have carried out a comprehensive and systematic mutagenesis of the
Cse4p N terminus to analyze its function. Surprisingly, only a 33-amino-acid
domain within the 130-amino-acid-long N terminus is required for Cse4p
N-terminal function. The spacing of the essential N-terminal domain (END)
relative to the HFD can be changed significantly without an apparent effect on
Cse4p function. The END appears to be important for interactions between Cse4p
and known kinetochore components, including the Ctf19p/Mcm21p/Okp1p complex.
Genetic and biochemical evidence shows that Cse4p proteins interact with each
other in vivo and that nonfunctional cse4 END and HFD mutant proteins can form
functional mixed complexes. These results support different roles for the Cse4p
N terminus and the HFD in centromere function and are consistent with the
proposed Cse4p nucleosome model. The structure-function characteristics of the
Cse4p N terminus are relevant to understanding how other H3-like proteins, such
as the human homolog CENP-A, function in kinetochore assembly and chromosome
segregation.
PMID: 10958698 [PubMed - indexed for MEDLINE]
400: Mol Pharmacol 2000 Sep;58(3):560-8
Probing the interaction of the cytotoxic bisdioxopiperazine ICRF-193 with the
closed enzyme clamp of human topoisomerase IIalpha.
Patel S, Jazrawi E, Creighton AM, Austin CA, Fisher LM.
Molecular Genetics Group, Department of Biochemistry and Immunology, St.
George's Hospital Medical School, University of London, London, United Kingdom.
Topoisomerase II is an ATP-operated protein clamp that captures a DNA helix and
transports it through another DNA duplex, allowing chromosome segregation at
mitosis. A number of cytotoxic bisdioxopiperazines such as ICRF-193 target
topoisomerase II by binding and trapping the closed enzyme clamp. To investigate
this unusual mode of action, we have used yeast to select plasmid-borne human
topoisomerase IIalpha alleles resistant to ICRF-193. Mutations in topoisomerase
IIalpha of Leu-169 to Phe (L169F) (in the N-terminal ATPase domain) and Ala-648
to Pro (A648P) (in the core domain) were identified as conferring >50-fold and
5-fold resistance to ICRF-193 in vivo, respectively. The L169F mutation, located
next to the Walker A box ATP-binding sequence, resulted in a mutant enzyme
displaying ICRF-193-resistant topoisomerase and ATPase activities and whose
closed clamp was refractory to ICRF-193-mediated trapping as an annulus on
closed circular DNA. These data imply that the mutation interferes directly with
ICRF-193 binding to the N-terminal ATPase gate. In contrast, the A648P enzyme
displayed topoisomerase activities exhibiting wild-type sensitivity to ICRF-193.
We suggest that the inefficient trapping of the A648P closed clamp results
either from the observed increased ATP requirement, or more likely, from lowered
salt stability, perhaps involving destabilization of ICRF-193 interactions with
the B'-B' interface in the core domain. These results provide evidence for at
least two different phenotypic classes of ICRF-193 resistance mutations and
suggest that bisdioxopiperazine action involves the interplay of both the ATPase
and core domains of topoisomerase IIalpha.
PMID: 10953049 [PubMed - indexed for MEDLINE]
401: Biotechniques 2000 Aug;29(2):278-9, 282-4, 286-8
Streamlined yeast colorimetric reporter activity assays using scanners and plate
readers.
Serebriiskii IG, Toby GG, Golemis EA.
Fox Chase Cancer Center, Philadelphia, PA, USA. ig_serebriiskii@fccc.edu
Two-hybrid systems have become favored tools for detection and analysis of
protein interactions because of their low cost and ease of use compared to
biochemical or biophysical interaction technologies. It is possible to augment
the utility of two-hybrid systems and derivative systems such as dual-bait
two-hybrid systems by adapting strategies that speed the analysis of the
relative strength of a series of protein-protein associations. This report
describes two simple techniques that employ either a flatbed scanner or a plate
reader to quantitate the activity of colorimetric reporters such as LacZ or GusA
commonly used in two-hybrid approaches.
Publication Types:
Technical Report
PMID: 10948429 [PubMed - indexed for MEDLINE]
402: DNA Cell Biol 2000 Jul;19(7):447-57
Loss control of Mcm5 interaction with chromatin in cdc6-1 mutated in CDC-NTP
motif.
Feng L, Hu Y, Wang B, Wu L, Jong A.
Division of Hematology/Oncology, Childrens Hospital Los Angeles, and University
of Southern California, School of Medicine, 90027, USA.
Saccharomyces cerevisiae Cdc6 plays an essential role in establishing and
maintaining the prereplicative complex (pre-RC) by interacting with the origin
recognition complex (ORC) and associating with chromatin origins. These
interactions are required to load minichromosome maintenance proteins (MCMs) and
other initiator proteins onto replication origins. Although the
temperature-sensitive cdc6 mutant, cdc6-1, has been widely used for these
studies, the molecular mechanism of the cdc6-1 mutation has been unclear. In
this study, we have identified a base substitution at Gly260-->Asp, near the
CDC-NTP motif. Using a chromatin immunoprecipitation assay (CHIP), we found that
cdc6-1 fails to load Mcm5 onto the replication origins. Chromatin fractions were
used to study Mcm5 binding in both the wildtype and mutant background. These
studies indicated that Cdc6 is also involved in unloading Mcm5 from chromatin.
Specifically, the cdc6-1 mutation protein, cdc6(G260D), which failed to load
Mcm5 onto replication origins, also failed to unload the Mcm5 protein.
Furthermore, the overexpression of wildtype CDC6 accelerated the unloading of
Mcm5 from chromatin fractions. In the absence of functional Cdc6, the Mcm5
protein showed nonorigin binding to chromatin with the cell cycle arrested at
the G1S phase transition. Our results suggested that the cdc6(G260D) mutant
protein fails to assemble an operational replicative complex and that wildtype
Cdc6 plays a role in preventing re-replication by controlling the unloading the
MCMs from chromatin origins.
PMID: 10945234 [PubMed - indexed for MEDLINE]
403: EMBO J 2000 Aug 15;19(16):4372-82
The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally
with the eIF4F translation initiation complex.
Vilela C, Velasco C, Ptushkina M, McCarthy JE.
Posttranscriptional Control Group, Department of Biomolecular Sciences,
University of Manchester Institute of Science and Technology PO Box 88,
Manchester M60 1QD, UK.
Dcp1 plays a key role in the mRNA decay process in Saccharomyces cerevisiae,
cleaving off the 5' cap to leave an end susceptible to exonucleolytic
degradation. The eukaryotic initiation factor complex eIF4F, which in yeast
contains the core components eIF4E and eIF4G, uses the cap as a binding site,
serving as an initial point of assembly for the translation apparatus, and also
binds the poly(A) binding protein Pab1. We show that Dcp1 binds to eIF4G and
Pab1 as free proteins, as well as to the complex eIF4E-eIF4G-Pab1. Dcp1
interacts with the N-terminal region of eIF4G but does not compete significantly
with eIF4E or Pab1 for binding to eIF4G. Most importantly, eIF4G acts as a
function-enhancing recruitment factor for Dcp1. However, eIF4E blocks this
effect as a component of the high affinity cap-binding complex eIF4E-eIF4G.
Indeed, cooperative enhancement of the eIF4E-cap interaction stabilizes yeast
mRNAs in vivo. These data on interactions at the interface between translation
and mRNA decay suggest how events at the 5' cap and 3' poly(A) tail might be
coupled.
PMID: 10944120 [PubMed - indexed for MEDLINE]
404: J Biol Chem 2000 Nov 24;275(47):36541-9
Structural basis for the species-specific activity of TFIIS.
Shimasaki NB, Kane CM.
Department of Molecular and Cell Biology, University of California, Berkeley,
California 94720-3202, USA.
Many proteins involved in eukaryotic transcription are similar in function and
in sequence between organisms. Despite the sequence similarities, there are many
factors that do not function across species. For example, transcript elongation
factor TFIIS is highly conserved among eukaryotes, and yet the TFIIS protein
from Saccharomyces cerevisiae cannot function with mammalian RNA polymerase II
and vice versa. To determine the reason for this species specificity, chimeras
were constructed linking three structurally independent regions of the TFIIS
proteins from yeast and human cells. Two independently folding domains, II and
III, have been examined previously using NMR (). Yeast domain II alone is able
to bind yeast RNA polymerase II with the same affinity as the full-length TFIIS
protein, and this domain was expected to confer the species selectivity. Domain
III has previously been shown to be readily exchanged between mammalian and
yeast factors. However, the results presented here indicate that domain II is
insufficient to confer species selectivity, and a primary determinant lies in a
30-amino acid highly conserved linker region connecting domain II with domain
III. These 30 amino acids may physically orient domains II and III to support
functional interactions between TFIIS and RNA polymerase II.
PMID: 10940308 [PubMed - indexed for MEDLINE]
405: Mol Cell Biol 2000 Sep;20(17):6435-48
Cell cycle-dependent binding of yeast heat shock factor to nucleosomes.
Venturi CB, Erkine AM, Gross DS.
Department of Biochemistry and Molecular Biology, Louisiana State University
Health Sciences Center, Shreveport, Louisiana 71130, USA.
In the nucleus, transcription factors must contend with the presence of
chromatin in order to gain access to their cognate regulatory sequences. As most
nuclear DNA is assembled into nucleosomes, activators must either invade a
stable, preassembled nucleosome or preempt the formation of nucleosomes on newly
replicated DNA, which is transiently free of histones. We have investigated the
mechanism by which heat shock factor (HSF) binds to target nucleosomal heat
shock elements (HSEs), using as our model a dinucleosomal heat shock promoter
(hsp82-DeltaHSE1). We find that activated HSF cannot bind a stable,
sequence-positioned nucleosome in G(1)-arrested cells. It can do so readily,
however, following release from G(1) arrest or after the imposition of either an
early S- or late G(2)-phase arrest. Surprisingly, despite the S-phase
requirement, HSF nucleosomal binding activity is restored in the absence of
hsp82 replication. These results contrast with the prevailing paradigm for
activator-nucleosome interactions and implicate a nonreplicative,
S-phase-specific event as a prerequisite for HSF binding to nucleosomal sites in
vivo.
PMID: 10938121 [PubMed - indexed for MEDLINE]
406: Mol Cell Biol 2000 Sep;20(17):6426-34
Protein kinase A and mitogen-activated protein kinase pathways antagonistically
regulate fission yeast fbp1 transcription by employing different modes of action
at two upstream activation sites.
Neely LA, Hoffman CS.
Department of Biology, Boston College, Massachusetts 02467, USA.
A significant challenge to our understanding of eukaryotic transcriptional
regulation is to determine how multiple signal transduction pathways converge on
a single promoter to regulate transcription in divergent fashions. To study
this, we have investigated the transcriptional regulation of the
Schizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP
(cAMP)-dependent protein kinase A (PKA) pathway and is activated by a
stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study,
we identified and characterized two cis-acting elements in the fbp1 promoter
required for activation of fbp1 transcription. Upstream activation site 1
(UAS1), located approximately 900 bp from the transcriptional start site,
resembles a cAMP response element (CRE) that is the binding site for the
atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to
UAS1 is positively regulated by the MAPK pathway and negatively regulated by
PKA. UAS2, located approximately 250 bp from the transcriptional start site,
resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by
transcriptional activators and repressors regulated by both the PKA and MAPK
pathways, although atf1 itself is not present in these complexes.
Transcriptional regulation of fbp1 promoter constructs containing only UAS1 or
UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We
conclude that the PKA and MAPK signal transduction pathways regulate fbp1
transcription at UAS1 and UAS2, but that the antagonistic interactions between
these pathways involve different mechanisms at each site.
PMID: 10938120 [PubMed - indexed for MEDLINE]
407: Mol Cell Biol 2000 Sep;20(17):6244-58
Gic2p may link activated Cdc42p to components involved in actin polarization,
including Bni1p and Bud6p (Aip3p).
Jaquenoud M, Peter M.
Swiss Institute for Experimental Cancer Research, 1066 Epalinges/VD,
Switzerland.
Gic2p is a Cdc42p effector which functions during cytoskeletal organization at
bud emergence and in response to pheromones, but it is not understood how Gic2p
interacts with the actin cytoskeleton. Here we show that Gic2p displayed
multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting
that Gic2p may regulate their function in vivo. In support of this idea, Gic2p
cofractionated with Bud6p and Spa2p and interacted with Bud6p by
coimmunoprecipitation and two-hybrid analysis. Importantly, localization of
Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the
Gic proteins but did not require an intact actin cytoskeleton. We identified a
conserved domain in Gic2p which was necessary for its polarization function but
dispensable for binding to Cdc42p-GTP and its localization to the site of
polarization. Expression of a mutant Gic2p harboring a single-amino-acid
substitution in this domain (Gic2p(W23A)) interfered with polarized growth in a
dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the
incipient bud site. We propose that at bud emergence, Gic2p functions as an
adaptor which may link activated Cdc42p to components involved in actin
organization and polarized growth, including Bni1p, Spa2p, and Bud6p.
PMID: 10938101 [PubMed - indexed for MEDLINE]
408: Biochemistry 2000 Aug 15;39(32):9909-16
Effects of 5' leader and 3' trailer structures on pre-tRNA processing by nuclear
RNase P.
Ziehler WA, Day JJ, Fierke CA, Engelke DR.
Department of Biological Chemistry and Department of Chemistry, University of
Michigan, Ann Arbor, Michigan 48109-0606, USA.
Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader preceding the
aminoacyl acceptor stem and a 3' trailer extending beyond this stem. An early
step in pre-tRNA maturation is removal of the 5' leader by the endoribonuclease,
RNase P. Extensive pairing between leader and trailer sequences has previously
been demonstrated to block RNase P cleavage, suggesting that the 5' leader and
3' trailer sequences might need to be separated for the substrate to be
recognized by the eukaryotic holoenzyme. To address whether the nuclear RNase P
holoenzyme recognizes the 5' leader and 3' trailer sequences independently,
interactions of RNase P with pre-tRNA(Tyr) containing either the 5' leader, the
3' trailer, or both were examined. Kinetic analysis revealed little effect of
the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage
using the various pre-tRNA derivatives. However, the presence of a 3' trailer
that pairs with the 5' leader increases the K(m) of pre-tRNA slightly, in
agreement with previous results. Similarly, competition studies demonstrate that
removal of a complementary 3' trailer lowers the apparent K(I), consistent with
the structure between these two sequences interfering with their interaction
with the enzyme. Deletion of both the 5' and 3' extensions to give mature
termini resulted in the least effective competitor. Further studies showed that
the nuclear holoenzyme, but not the B. subtilis holoenzyme, had a high affinity
for single-stranded RNA in the absence of attached tRNA structure. The data
suggest that yeast nuclear RNase P contains a minimum of two binding sites
involved in substrate recognition, one that interacts with tRNA and one that
interacts with the 3' trailer. Furthermore, base pairing between the 5' leader
and 3' trailer hinders recognition.
PMID: 10933810 [PubMed - indexed for MEDLINE]
409: Eur J Biochem 2000 Aug;267(16):5156-67
Flavin-protein interactions in flavocytochrome b2 as studied by NMR after
reconstitution of the enzyme with 13C- and 15N-labelled flavin.
Fleischmann G, Lederer F, Muller F, Bacher A, Ruterjans H.
Institut fur Biophysikalische Chemie, J.W. Goethe-Universitat, Biozentrum N230,
Frankfurt, Germany.
A new procedure was devised for reversibly removing the flavin from
flavocytochrome b2. It allowed reconstitution with selectively enriched 13C- and
15N-labelled FMN for an NMR analysis of the chemical shifts of the enriched
positions as well as that of 31P. From these measurements, it was possible to
deduce information about the hydrogen-bonding pattern of FMN in the protein, the
hybridization states of the nitrogen atoms and (in part) the pi-electron
distribution. The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1)
and N(5) form hydrogen bonds to the apoenzyme in both redox states.
Nevertheless, according to 15N-chemical shifts, the bond from the protein to
N(3) is very weak in both redox states, whereas that to N(5) is strong for the
oxidized state, and is weakened upon flavin reduction. On the other hand, the
13C-NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger
hydrogen bonds with the enzyme than most other flavoproteins in both redox
states. From coupling constant measurements it is shown that the N(3) proton is
not solvent accessible. Although no N-H coupling constant could be measured for
N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated
in flavocytochrome b2 as in all other known flavoproteins. With respect to
N(10), it is more sp3-hybridized in the oxidized state than in free FMN, whereas
the other nitrogen atoms show a nearly planar structure. In the reduced state,
N(5) and N(10) in bound FMN are both more sp3-hybridized than in free FMN, but
N(5) exhibits a higher degree of sp3-hybridization than N(10), which is only
slightly shifted out of the isoalloxazine plane. In addition, two-electron
reduction of the enzyme leads to anion formation on N(1), as indicated by its
15N-chemical shift of N(1) and characteristic upfield shifts of the resonances
of C(2), C(4) and C(4a) compared to the oxidized state, as observed for most
flavoproteins. 31P-NMR measurements show that the phosphate geometry has changed
in enzyme bound FMN compared to the free flavin in water, indicating a strong
interaction of the phosphate group with the apoenzyme.
PMID: 10931200 [PubMed - indexed for MEDLINE]
410: Biochim Biophys Acta 2000 Jul 31;1467(1):207-18
The yeast mitochondrial transport proteins: new sequences and consensus
residues, lack of direct relation between consensus residues and transmembrane
helices, expression patterns of the transport protein genes, and protein-protein
interactions with other proteins.
Belenkiy R, Haefele A, Eisen MB, Wohlrab H.
Boston Biomedical Research Institute and Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Watertown, MA 02472, USA.
Mitochondrial transport proteins (MTP) typically are homodimeric with a 30-kDa
subunit with six transmembrane helices. The subunit possesses a sequence motif
highly similar to Pro X Asp/Glu X X Lys/Arg X Arg within each of its three
similar 10-kDa segments. Four (YNL083W, YFR045W, YPR021C, YDR470C) of the 35
yeast (S. cerevisiae) MTP genes were resequenced since the masses of their
proteins deviate significantly from the typical 30 kDa. We now find these four
proteins to have 545, 285, 902, and 502 residues, respectively. Together with
only four other MTPs, the sequences of YPR021C and YDR470C show substitutions of
some of the five residues that are absolutely conserved among the 12 MTPs with
identified transport function and 17 other MTPs. We do now find these five
consensus residues also in the new sequences of YNL083W and YFR045W. Additional
analyses of the 35 yeast MTPs show that the location of transmembrane helix
sequences do not correlate with the general consensus residues of the MTP
family; protein segments connecting the six transmembrane helices and facing the
intermembrane space are not uniformly short (about 20 residues) or long (about
40 residues) when facing the matrix; most MTPs have at least one transmembrane
helix for which the sum of the negative hydropathy values of all residues yields
a very small negative value, suggesting a membrane location bordering polar
faces of other transmembrane helices or a non-transmembrane location. The extra
residues of the three large MTPs are hydrophilic and at the N-terminal. The
200-residue N-terminal segment of YNL083W has four putative Ca2+-binding sites.
The 500-residue N-terminal segment of YPR021C shows sequence similarity to
enzymes of nucleic acid metabolism. cDNA microarray data show that YNL083W is
expressed solely during sporulation, while the expressions of YFR045W, YPR021C,
and YDR470C are induced by various stress situations. These results also show
that the 35 MTP genes are expressed under a rather diverse set of metabolic
conditions that may help identify the function of the proteins. Interestingly,
yeast two-hybrid screens, that will also be useful in identifying the function
of MTPs, indicate that MIR1, AAC3, YOR100C, and YPR011C do interact with
non-MTPs.
PMID: 10930523 [PubMed - indexed for MEDLINE]
411: J Biol Chem 2000 Oct 20;275(42):33158-66
Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues
5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is
found in an exosome-sized complex in plant and yeast cells.
Chekanova JA, Shaw RJ, Wills MA, Belostotsky DA.
Department of Biological Sciences and the Center for Molecular Genetics, State
University of New York at Albany, Albany, New York 12222, USA.
Eukaryotic 3'-->5' exonucleolytic activities are essential for a wide variety of
reactions of RNA maturation and metabolism, including processing of rRNA, small
nuclear RNA, and small nucleolar RNA, and mRNA decay. Two related but distinct
forms of a complex containing 10 3'-->5' exonucleases, the exosome, are found in
yeast nucleus and cytoplasm, respectively, and related complexes exist in human
cells. Here we report on the characterization of the AtRrp41p, an Arabidopsis
thaliana homolog of the Saccharomyces cerevisiae exosome subunit Rrp41p (Ski6p).
Purified recombinant AtRrp41p displays a processive phosphorolytic exonuclease
activity and requires a single-stranded poly(A) tail on a substrate RNA as a
"loading pad." The expression of the Arabidopsis RRP41 cDNA in yeast rescues the
5.8 S rRNA processing and 3'-->5' mRNA degradation defects of the yeast ski6-100
mutant. However, neither of these defects can explain the conditional lethal
phenotype of the ski6-100 strain. Importantly, AtRrp41p shares additional
function(s) with the yeast Rrp41p which are essential for cell viability because
it also rescues the rrp41 (ski6) null mutant. AtRrp41p is found predominantly in
a high molecular mass complex in Arabidopsis and in yeast cells, and it
interacts in vitro with the yeast Rrp44p and Rrp4p exosome subunits, suggesting
that it can participate in evolutionarily conserved interactions that could be
essential for the integrity of the exosome complex.
PMID: 10930416 [PubMed - indexed for MEDLINE]
412: Genetics 2000 Aug;155(4):1667-82
Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in
the splicing factor Prp8 that influence spliceosome activation.
Kuhn AN, Brow DA.
Department of Biomolecular Chemistry, University of Wisconsin Medical School,
Madison, Wisconsin 53706-1532, USA.
The highly conserved splicing factor Prp8 has been implicated in multiple stages
of the splicing reaction. However, assignment of a specific function to any part
of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks
recognizable functional or structural motifs. We have used a large-scale screen
for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity
caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with
high resolution five distinct regions of PRP8 involved in the control of
spliceosome activation. Genetic interactions between two of these regions reveal
a potential long-range intramolecular fold. Identification of a yeast two-hybrid
interaction, together with previously reported results, implicates two other
regions in direct and indirect contacts to the U1 snRNP. In contrast to the
suppressor mutations in PRP8, loss-of-function mutations in the genes for two
other splicing factors implicated in U4/U6 unwinding, Prp44
(Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On
the basis of these results we propose a model in which allosteric changes in
Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1
snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44
and Prp24.
PMID: 10924465 [PubMed - indexed for MEDLINE]
413: Genetics 2000 Aug;155(4):1593-606
POB3 is required for both transcription and replication in the yeast
Saccharomyces cerevisiae.
Schlesinger MB, Formosa T.
Department of Biochemistry, University of Utah School of Medicine, Salt Lake
City, Utah 84132, USA.
Spt16 and Pob3 form stable heterodimers in Saccharomyces cerevisiae, and
homologous proteins have also been purified as complexes from diverse
eukaryotes. This conserved factor has been implicated in both transcription and
replication and may affect both by altering the characteristics of chromatin.
Here we describe the isolation and properties of a set of pob3 mutants and
confirm that they have defects in both replication and transcription. Mutation
of POB3 caused the Spt(-) phenotype, spt16 and pob3 alleles displayed severe
synthetic defects, and elevated levels of Pob3 suppressed some spt16 phenotypes.
These results are consistent with previous reports that Spt16 and Pob3 act in a
complex that modulates transcription. Additional genetic interactions were
observed between pob3 mutations and the genes encoding several DNA replication
factors, including POL1, CTF4, DNA2, and CHL12. pob3 alleles caused sensitivity
to the ribonucleotide reductase inhibitor hydroxyurea, indicating a defect in a
process requiring rapid dNTP synthesis. Mutation of the S phase checkpoint gene
MEC1 caused pob3 mutants to lose viability rapidly under restrictive conditions,
revealing defects in a process monitored by Mec1. Direct examination of DNA
contents by flow cytometry showed that S phase onset and progression were
delayed when POB3 was mutated. We conclude that Pob3 is required for normal
replication as well as for transcription.
PMID: 10924459 [PubMed - indexed for MEDLINE]
414: Genetics 2000 Aug;155(4):1543-59
Functional interaction between the PKC1 pathway and CDC31 network of SPB
duplication genes.
Khalfan W, Ivanovska I, Rose MD.
Department of Molecular Biology, Princeton University, Princeton, New Jersey
08544-1014, USA.
The earliest known step in yeast spindle pole body (SPB) duplication requires
Cdc31p and Kar1p, two physically interacting SPB components, and Dsk2p and
Rad23p, a pair of ubiquitin-like proteins. Components of the PKC1 pathway were
found to interact with these SPB duplication genes in two independent genetic
screens. Initially, SLG1 and PKC1 were obtained as high-copy suppressors of
dsk2Delta rad23Delta and a mutation in MPK1 was synthetically lethal with
kar1-Delta17. Subsequently, we demonstrated extensive genetic interactions
between the PKC1 pathway and the SPB duplication mutants that affect Cdc31p
function. The genetic interactions are unlikely to be related to the cell-wall
integrity function of the PKC1 pathway because the SPB mutants did not exhibit
cell-wall defects. Overexpression of multiple PKC1 pathway components suppressed
the G2/M arrest of the SPB duplication mutants and mutations in MPK1 exacerbated
the cell cycle arrest of kar1-Delta17, suggesting a role for the PKC1 pathway in
SPB duplication. We also found that mutations in SPC110, which encodes a major
SPB component, showed genetic interactions with both CDC31 and the PKC1 pathway.
In support of the model that the PKC1 pathway regulates SPB duplication, one of
the phosphorylated forms of Spc110p was absent in pkc1 and mpk1Delta mutants.
PMID: 10924456 [PubMed - indexed for MEDLINE]
415: J Biol Chem 2000 Nov 3;275(44):34068-72
Interactions between Spc2p and other components of the endoplasmic reticulum
translocation sites of the yeast Saccharomyces cerevisiae.
Antonin W, Meyer HA, Hartmann E.
Abteilung Biochemie II, Zentrum Biochemie und Molekulare Zellbiologie,
Universitat Gottingen, Heinrich-Duker Weg 12, Gottingen 37073, Germany.
In yeast, the endoplasmic reticulum membrane proteins Sec11p and Spc3p are
essential for the cleavage of signal peptides of nascent polypeptide chains
during their passage through translocation sites. Genetic and biochemical
experiments demonstrate that Sec11p and Spc3p are tightly associated with two
other proteins, Spc1p and Spc2p, whose functions are largely unknown. Using
anti-Spc2p antibodies, we show here that this heterotetrameric complex
associates with Sbh1p and Sbh2p, the beta-subunits of the Sec61p complex and the
Ssh1p complex, respectively. Depletion of Spc2p decreased the enzymatic activity
of the SPC in vitro, led to a loss of Spc1p, and led to a down-regulation of the
amount of Sec11p and Spc3p in the endoplasmic reticulum. Moreover, the deletion
of Spc2p also decreased the expression level of Sbh2p. These data implicate that
Spc2p not only enhances the enzymatic activity of the SPC but also facilitates
the interactions between different components of the translocation site.
PMID: 10921929 [PubMed - indexed for MEDLINE]
416: EMBO J 2000 Aug 1;19(15):4164-74
Elongation arrest is a physiologically important function of signal recognition
particle.
Mason N, Ciufo LF, Brown JD.
Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology,
University of Edinburgh, Swann Building, The King's Buildings, Mayfield Road,
Edinburgh EH9 3JR, UK.
Signal recognition particle (SRP) targets proteins for co-translational
insertion through or into the endoplasmic reticulum membrane. Mammalian SRP
slows nascent chain elongation by the ribosome during targeting in vitro. This
'elongation arrest' activity requires the SRP9/14 subunit of the particle and
interactions of the C-terminus of SRP14. We have purified SRP from Saccharomyces
cerevisiae and demonstrated that it too has elongation arrest activity. A yeast
SRP containing Srp14p truncated at its C-terminus (delta C29) did not maintain
elongation arrest, was substantially deficient in promoting translocation and
interfered with targeting by wild-type SRP. In vivo, this mutation conferred a
constitutive defect in the coupling of protein translation and translocation and
temperature-sensitive growth, but only a slight defect in protein translocation.
In combination, these data indicate that the primary defect in SRP delta C29 is
in elongation arrest, and that this is a physiologically important and conserved
function of eukaryotic SRP.
PMID: 10921896 [PubMed - indexed for MEDLINE]
417: Mutat Res 2000 Jun 30;451(1-2):151-67
Mismatch repair proteins and mitotic genome stability.
Harfe BD, Jinks-Robertson S.
Department of Biology, Emory University, 1510 Clifton Road, Atlanta, GA 30322,
USA.
Mismatch repair (MMR) proteins play a critical role in maintaining the mitotic
stability of eukaryotic genomes. MMR proteins repair errors made during DNA
replication and in their absence, mutations accumulate at elevated rates. In
addition, MMR proteins inhibit recombination between non-identical DNA
sequences, and hence prevent genome rearrangements resulting from interactions
between repetitive elements. This review provides an overview of the
anti-mutator and anti-recombination functions of MMR proteins in the yeast
Saccharomyces cerevisiae.
Publication Types:
Review
Review, Tutorial
PMID: 10915870 [PubMed - indexed for MEDLINE]
418: Mutat Res 2000 Jun 30;451(1-2):71-89
Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae.
Lewis LK, Resnick MA.
Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute
of Environmental Health Sciences, PO Box 12233, 111 Alexander Drive, NIH,
Research Triangle Park, NC 27709, USA.
The ends of chromosomal DNA double-strand breaks (DSBs) can be accurately
rejoined by at least two discrete pathways, homologous recombination and
nonhomologous end-joining (NHEJ). The NHEJ pathway is essential for repair of
specific classes of DSB termini in cells of the budding yeast Saccharomyces
cerevisiae. Endonuclease-induced DSBs retaining complementary single-stranded
DNA overhangs are repaired efficiently by end-joining. In contrast, damaged DSB
ends (e.g., termini produced by ionizing radiation) are poor substrates for this
pathway. NHEJ repair involves the functions of at least 10 genes, including
YKU70, YKU80, DNL4, LIF1, SIR2, SIR3, SIR4, RAD50, MRE11, and XRS2. Most or all
of these genes are required for efficient recombination-independent
recircularization of linearized plasmids and for rejoining of EcoRI
endonuclease-induced chromosomal DSBs in vivo. Several NHEJ mutants also display
aberrant processing and rejoining of DSBs that are generated by HO endonuclease
or formed spontaneously in dicentric plasmids. In addition, all NHEJ genes
except DNL4 and LIF1 are required for stabilization of telomeric repeat
sequences. Each of the proteins involved in NHEJ appears to bind, directly or
through protein associations, with the ends of linear DNA. Enzymatic and/or
structural roles in the rejoining of DSB termini have been postulated for
several proteins within the group. Most yeast NHEJ genes have homologues in
human cells and many biochemical activities and protein:protein interactions
have been conserved in higher eucaryotes. Similarities and differences between
NHEJ repair in yeast and mammalian cells are discussed.
Publication Types:
Review
Review, Tutorial
PMID: 10915866 [PubMed - indexed for MEDLINE]
419: Methods Enzymol 2000;322:297-322
Exploiting the utility of yeast in the context of programmed cell death.
Torgler CN, Brown R, Meldrum E.
Glaxo Wellcome Medicines Research Centre, Cell Biology Unit, Stevenage, United
Kingdom.
Many researchers have explored the extent to which yeast can be used to dissect
the mechanisms of programmed cell death in higher cells. Yeast has been used as
a system to analyze protein-protein interactions and structure-function
relationships, and as a cloning tool to identify novel higher eukaryote
regulators of apoptosis. In addition, classic genetic strategies in yeast have
been used to analyze the mechanisms of action of core pathway members. The
purpose of this chapter is to describe the strategies pursued and act as a
source for the technical details necessary to exploit the yeast Saccharomyces
cerevisiae and Schizosaccharomyces pombe in the context of programmed cell
death.
PMID: 10914027 [PubMed - indexed for MEDLINE]
420: Mol Cell Biol 2000 Aug;20(16):5960-73
Genetic interactions between TFIIS and the Swi-Snf chromatin-remodeling complex.
Davie JK, Kane CM.
Department of Molecular and Cell Biology, University of California, Berkeley,
California 94720-3202, USA.
The eukaryotic transcript elongation factor TFIIS enables RNA polymerase II to
read through blocks to elongation in vitro and interacts genetically with a
variety of components of the transcription machinery in vivo. In Saccharomyces
cerevisiae, the gene encoding TFIIS (PPR2) is not essential, and disruption
strains exhibit only mild phenotypes and an increased sensitivity to
6-azauracil. The nonessential nature of TFIIS encouraged the use of a synthetic
lethal screen to elucidate the in vivo roles of TFIIS as well as provide more
information on other factors involved in the regulation of transcript
elongation. Several genes were identified that are necessary for either cell
survival or robust growth when the gene encoding TFIIS has been disrupted. These
include UBP3, KEX2, STT4, and SWI2/SNF2. SWI1 and SNF5 disruptions were also
synthetically lethal with ppr2Delta, suggesting that the reduced ability to
remodel chromatin confers the synthetic phenotype. The synthetic phenotypes show
marked osmosensitivity and cytoskeletal defects, including a terminal
hyperelongated bud phenotype with the Swi-Snf complex. These results suggest
that genes important in osmoregulation, cell membrane synthesis and integrity,
and cell division may require the Swi-Snf complex and TFIIS for efficient
transcription. The detection of these genetic interactions provides another
functional link between the Swi-Snf complex and the elongation machinery.
PMID: 10913179 [PubMed - indexed for MEDLINE]
421: Glycobiology 2000 Jul;10(7):737-44
Evidence for interaction of yeast protein kinase C with several subunits of
oligosaccharyl transferase.
Park H, Lennarz WJ.
Department of Biochemistry and Cell Biology, and Institute for Cell and
Developmental Biology, SUNY at Stony Brook, 11794, USA.
Oligosaccharyltransferase (OT) in Saccharomyces cerevisiae is an enzyme complex
consisting of 8 transmembrane proteins located in the endoplasmic reticulum
(ER). Studies on potential protein-protein interactions in OT using a two-hybrid
library screen revealed that protein kinase C (Pkc1p) interacted with the
lumenal domains of several OT subunits. Additional genetic experiments revealed
that overexpression of two OT subunits rescued the growth defect caused by
overexpression of a Pkc1 active site mutant, implying that there are specific
genetic interactions between PKC1 and OT. These in vivo findings were
complemented by in vitro studies that showed that several of the OT subunits
bound to a fusion protein consisting of glutathione S-transferase linked via its
C-terminus to Pkc1p. Assays of OT activity, in which glycosylation of a simple
acceptor peptide was assayed in microsomes from wild-type and a pkc1 null
revealed a 50% reduction in activity in the microsomes from the null strain. In
contrast, strains containing null mutations of two other genes known to be
downstream of Pkc1p in the PKC1-MAP kinase pathway had a level of OT activity
comparable to that of wild-type cells. These in vivo and in vitro experiments
suggest that in yeast cells Pkc1p may be involved in regulation of the
N-glycosylation of proteins.
PMID: 10910977 [PubMed - indexed for MEDLINE]
422: Structure Fold Des 2000 Jul 15;8(7):751-62
X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed
with FMN at 1.8 A resolution.
Safo MK, Mathews I, Musayev FN, di Salvo ML, Thiel DJ, Abraham DJ, Schirch V.
Institute for Structural Biology and Drug Discovery, Virginia Commonwealth
University, Richmond, VA 23219, USA. msafo@hsc.vcu.edu
BACKGROUND: Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes
the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP), a
cofactor used by many enzymes involved in amino acid metabolism. The enzyme
oxidizes either the 4'-hydroxyl group of pyridoxine 5'-phosphate (PNP) or the
4'-primary amine of pyridoxamine 5'-phosphate (PMP) to an aldehyde. PNPOx is a
homodimeric enzyme with one flavin mononucleotide (FMN) molecule non-covalently
bound to each subunit. A high degree of sequence homology among the 15 known
members of the PNPOx family suggests that all members of this group have similar
three-dimensional folds. RESULTS: The crystal structure of PNPOx from E. coli
has been determined to 1.8 A resolution. The monomeric subunit folds into an
eight-stranded beta sheet surrounded by five alpha-helical structures. Two
monomers related by a twofold axis interact extensively along one-half of each
monomer to form the dimer. There are two clefts at the dimer interface that are
symmetry-related and extend from the top to the bottom of the dimer. An FMN
cofactor that makes interactions with both subunits is located in each of these
two clefts. CONCLUSIONS: The structure is quite similar to the recently
deposited 2.7 A structure of Saccharomyces cerevisiae PNPOx and also,
remarkably, shares a common structural fold with the FMN-binding protein from
Desulfovibrio vulgaris and a domain of chymotrypsin. This high-resolution E.
coli PNPOx structure permits predictions to be made about residues involved in
substrate binding and catalysis. These predictions provide testable hypotheses,
which can be answered by making site-directed mutants.
PMID: 10903950 [PubMed - indexed for MEDLINE]
423: Nucleic Acids Res 2000 Jul 15;28(14):2634-42
The analysis of chimeric human/rainbow trout estrogen receptors reveals amino
acid residues outside of P- and D-boxes important for the transactivation
function.
Petit FG, Valotaire Y, Pakdel F.
Equipe d'Endocrinologie Moleculaire de la Reproduction, UPRES-A CNRS 6026,
Universite de Rennes I, 35042 Rennes cedex, France.
The amino acid sequence of rainbow trout estrogen receptor (rtER) is highly
conserved in the C domain but presents few similarities in the A/B and E domains
with human estrogen receptor alpha (hER) [NR3A1]. A previous study has shown
that rtER and hER have differential functional activities in yeast Saccharomyces
cerevisiae. To determine the domain(s) responsible for these differences,
chimeric human/rainbow trout estrogen receptors were constructed. The A/B, C/D
or E/F regions of rtER were replaced by corresponding regions of hER and
expressed in yeast cells. Ligand-binding and transcription activation abilities
of these hybrid receptors were compared with those of wild-type rtER or hER.
Surprisingly, our data revealed that the human C/D domains play an important
role in the magnitude of transactivation of ER. Two other chimeric ERs carrying
either a C or D domain of hER showed that the C domain was responsible for this
effect whereas the D domain did not affect hybrid receptor activities. Moreover,
a chimeric hER carrying the C domain of rtER showed maximal transcriptional
activity similar to that observed with rtER. Gel shift assays showed that,
whereas rtER and hER present a similar binding affinity to an estrogen response
element (ERE) element, the rtER C domain is responsible for a weaker DNA binding
stability compared to those of hER. In addition, the human C domain allows
approximately 2 times faster association of ER to an ERE. Utilization of
reporter genes containing one or three EREs confirms that rtER requires
protein-protein interactions for its stabilization on DNA and that the C domain
is involved in this stabilization. Moreover, AF-1 may be implicated in this
synergistic effect of EREs. Interestingly, although E domains of these two
receptors are much less conserved, replacement of this domain in rtER by its
human counterpart resulted in higher estradiol sensitivity but no increase in
the magnitude of transactivation. Data from the chimeric receptors, rtER(hC) and
hER(rtC), demonstrated that rtER AF-1 and AF-2 activation domains activated
transcription in the presence of estradiol similar to both AF-1 and AF-2 hER.
This implies that these domains, which show poor sequence homology, may interact
with similar basal transcription factors.
PMID: 10908317 [PubMed - indexed for MEDLINE]
424: Yeast 2000 Jun 30;17(2):95-110
Genome-wide protein interaction screens reveal functional networks involving
Sm-like proteins.
Fromont-Racine M, Mayes AE, Brunet-Simon A, Rain JC, Colley A, Dix I, Decourty
L, Joly N, Ricard F, Beggs JD, Legrain P.
Genetique des Interactions Macromoleculaires, CNRS (URA 1300), Institut Pasteur,
Paris Cedex 15, France.
A set of seven structurally related Sm proteins forms the core of the snRNP
particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the
genomic sequence of Saccharomyces cerevisiae has identified a number of open
reading frames that potentially encode structurally similar proteins termed Lsm
(Like Sm) proteins. With the aim of analysing all possible interactions between
the Lsm proteins and any protein encoded in the yeast genome, we performed
exhaustive and iterative genomic two-hybrid screens, starting with the Lsm
proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins
were found that suggest the existence of a Lsm complex or complexes. These Lsm
interactions apparently involve the conserved Sm domain that also mediates
interactions between the Sm proteins. The screens also reveal functionally
significant interactions with splicing factors, in particular with Prp4 and
Prp24, compatible with genetic studies and with the reported association of Lsm
proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions
with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1,
point to roles for Lsm complexes in distinct RNA metabolic processes, that are
confirmed in independent functional studies. These results provide compelling
evidence that two-hybrid screens yield functionally meaningful information about
protein-protein interactions and can suggest functions for uncharacterized
proteins, especially when they are performed on a genome-wide scale. Copyright
2000 John Wiley & Sons, Ltd.
PMID: 10900456 [PubMed - indexed for MEDLINE]
425: Yeast 2000 Jun 30;17(2):88-94
Yeast two-hybrid systems and protein interaction mapping projects for yeast and
worm.
Walhout AJ, Boulton SJ, Vidal M.
Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School,
Boston, MA 02115, USA.
The availability of complete genome sequences necessitates the development of
standardized functional assays to analyse the tens of thousands of predicted
gene products in high-throughput experimental settings. Such approaches are
collectively referred to as 'functional genomics'. One approach to investigate
the properties of a proteome of interest is by systematic analysis of
protein-protein interactions. So far, the yeast two-hybrid system is the most
commonly used method for large-scale, high-throughput identification of
potential protein-protein interactions. Here, we discuss several technical
features of variants of the two-hybrid systems in light of data recently
obtained from different protein interaction mapping projects for the budding
yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans.
Copyright 2000 John Wiley & Sons, Ltd.
Publication Types:
Review
Review, Tutorial
PMID: 10900455 [PubMed - indexed for MEDLINE]
426: Proc Natl Acad Sci U S A 2000 Aug 1;97(16):8967-72
The spliceosomal snRNP core complex of Trypanosoma brucei: cloning and
functional analysis reveals seven Sm protein constituents.
Palfi Z, Lucke S, Lahm HW, Lane WS, Kruft V, Bragado-Nilsson E, Seraphin B,
Bindereif A.
Institut fur Biochemie, Justus-Liebig-Universitat Giessen, Heinrich-Buff-Ring
58, D-35392 Giessen, Germany.
Each of the trypanosome small nuclear ribonucleoproteins (snRNPs) U2, U4/U6, and
U5, as well as the spliced leader (SL) RNP, contains a core of common proteins,
which we have previously identified. This core is unusual because it is not
recognized by anti-Sm Abs and it associates with an Sm-related sequence in the
trypanosome small nuclear RNAs (snRNAs). Using peptide sequences derived from
affinity-purified U2 snRNP proteins, we have cloned cDNAs for five common
proteins of 8.5, 10, 12.5, 14, and 15 kDa of Trypanosoma brucei and identified
them as Sm proteins SmF (8.5 kDa), -E (10 kDa), -D1 (12.5 kDa), -G (14 kDa), and
-D2 (15 kDa), respectively. Furthermore, we found the trypanosome SmB (T.
brucei) and SmD3 (Trypanosoma cruzi) homologues through database searches, thus
completing a set of seven canonical Sm proteins. Sequence comparisons of the
trypanosome proteins revealed several deviations in highly conserved positions
from the Sm consensus motif. We have identified a network of specific
heterodimeric and -trimeric Sm protein interactions in vitro. These results are
summarized in a model of the trypanosome Sm core, which argues for a strong
conservation of the Sm particle structure. The conservation extends also to the
functional level, because at least one trypanosome Sm protein, SmG, was able to
specifically complement a corresponding mutation in yeast.
PMID: 10900267 [PubMed - indexed for MEDLINE]
427: Nature 2000 Jul 6;406(6791):94-8
Forkhead-like transcription factors recruit Ndd1 to the chromatin of
G2/M-specific promoters.
Koranda M, Schleiffer A, Endler L, Ammerer G.
Department of Biochemistry and Molecular Cell Biology, Ludwig Boltzmann
Forschungsstelle, University of Vienna, Austria.
Many cell-cycle-specific events are supported by stage-specific gene expression.
In budding yeast, at least three different nuclear factors seem to cooperate in
the periodic activation of G2/M-specific genes. Here we show, by using chromatin
immunoprecipitation polymerase chain reaction assays, that a positive regulator,
Ndd1, becomes associated with G2/M promoter regions in manner that depends on
the stage in cell cycle. Its recruitment depends on a permanent protein-DNA
complex consisting of the MADS box protein, Mcm1, and a recently identified
partner Fkh2, a forkhead/winged helix related transcription factor. The
lethality of Ndd1 depletion is suppressed by fkh2 null mutations, which
indicates that Fkh2 may also have a negative regulatory role in the
transcription of G2/M-induced RNAs. We conclude that Ndd1-Fkh2 interactions may
be the transcriptionally important process targeted by Cdk activity.
PMID: 10894549 [PubMed - indexed for MEDLINE]
428: Mol Cell Biol 2000 Aug;20(15):5700-11
Histone-histone interactions and centromere function.
Glowczewski L, Yang P, Kalashnikova T, Santisteban MS, Smith MM.
Department of Microbiology and Cancer Center, University of Virginia,
Charlottesville, Virginia 22908, USA.
Cse4p is a structural component of the core centromere of Saccharomyces
cerevisiae and is a member of the conserved CENP-A family of specialized histone
H3 variants. The histone H4 allele hhf1-20 confers defects in core centromere
chromatin structure and mitotic chromosome transmission. We have proposed that
Cse4p and histone H4 interact through their respective histone fold domains to
assemble a nucleosome-like structure at centromeric DNA. To test this model, we
targeted random mutations to the Cse4p histone fold domain and isolated three
temperature-sensitive cse4 alleles in an unbiased genetic screen. Two of the
cse4 alleles contain mutations at the Cse4p-H4 interface. One of these requires
two widely separated mutations demonstrating long-range cooperative interactions
in the structure. The third cse4 allele is mutated at its helix 2-helix 3
interface, a region required for homotypic H3 fold dimerization. Overexpression
of wild-type Cse4p and histone H4 confer reciprocal allele-specific suppression
of cse4 and hhf1 mutations, providing strong evidence for Cse4p-H4 protein
interaction. Overexpression of histone H3 is dosage lethal in cse4 mutants,
suggesting that histone H3 competes with Cse4p for histone H4 binding. However,
the relative resistance of the Cse4p-H4 pathway to H3 interference argues that
centromere chromatin assembly must be highly regulated.
PMID: 10891506 [PubMed - indexed for MEDLINE]
429: Biochemistry 2000 Jul 11;39(27):7886-94
Investigations of the active site of Saccharomyces cerevisiae
dolichyl-phosphate-mannose synthase using fluorescent labeled dolichyl-phosphate
derivatives.
Xing J, Forsee WT, Lamani E, Maltsev SD, Danilov LL, Shibaev VN, Schutzbach JS,
Cheung HC, Jedrzejas MJ.
Department of Biochemistry and Molecular Genetics and Department of
Microbiology, The University of Alabama at Birmingham, 933 19th Street South,
Birmingham, Alabama 35295-2041, USA.
Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the
biosynthesis of N-linked oligosaccharides as well as for O-linked
oligosaccharides on yeast glycoproteins, and for the synthesis of the
glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins.
It is synthesized by Dol-P-Man synthase which is the only glycosyltransferase in
the dolichol pathway that has been expressed as an active protein, solubilized
and purified in large enough quantities for structural investigations. Earlier
studies showed that the enzyme is closely associated with membranes of
endoplasmic reticulum with unique lipid requirements for its maximal activity.
This potential target of antibiotic therapy is now being investigated at the
molecular level to establish information about the structure of the enzyme as
well as determine the nature and properties of the enzyme-phospholipid
interactions. In this paper, we have determined the activities of the
fluorescent labeled dolichyl-phosphate derivatives as well as the intramolecular
distances between amino acid residues near the active site and/or the
fluorophores of the substrate derivatives using fluorescence energy resonance
transfer. These results also show that the conserved consensus sequence is not
required by Dol-P-Man synthase neither for the recognition of Dol-P nor for the
catalytic activity.
PMID: 10891068 [PubMed - indexed for MEDLINE]
430: Methods Enzymol 2000;318:374-84
Translational repression assay procedure: a method to study RNA-protein
interactions in yeast.
Paraskeva E, Hentze MW.
Zentrum fur Molekulare Biologie, Universitat Heidelberg, Germany.
PMID: 10890000 [PubMed - indexed for MEDLINE]
431: J Biochem Biophys Methods 2000 Jul 10;44(1-2):95-107
Fast, isotope-free methods for the assay of thiamine-binding proteins and for
the determination of their affinities to thiamine-related compounds.
Mickowska B, Dulinski R, Kozik A.
Department of Biochemistry, The Jan Zurzycki Institute of Molecular Biology,
Jagiellonian University, Al. Mickiewicza 3, 31-120, Krakow, Poland.
A fast, isotope-free method for the determination of parameters for the
interactions of proteins with thiamine and related compounds was developed. The
free and bound forms of a ligand (thiamine or a fluorogenic analogue) were
separated by ultrafiltration using commercially available centrifugal protein
microconcentrators (Nanosep, Pall Filtron). The free thiamine concentration in
the filtrate was analysed by (i) a pre-column derivatisation of thiamine to
thiochrome with the use of alkaline potassium hexacyanoferrate(III) followed by
reverse-phase HPLC (isocratic, analytical ODS column, 10 mM potassium phosphate,
pH 7.8, 5% tetrahydrofuran) with fluorometric detection (excitation at 365 nm,
emission at 430 nm), or (ii) an ion-pair reverse-phase HPLC (isocratic, ODS
column, 0.08% trifluoroacetic acid-0.08% sodium octanesulfonate-25%
tetrahydrofuran) with post-column derivatisation and fluorometric detection. The
'saturation-binding' version (single ligand added in increasing doses to the
protein samples) of this method allowed the determination of low micromolar
concentrations of thiamine-binding proteins and of the dissociation constants of
their complexes with thiamine or fluorogenic thiamine analogues in the range of
0.3-10 microM. Using the other, 'competitive displacement' version (constant
amount of thiamine plus increasing doses of a competing ligand), dissociation
constants at least one order of magnitude higher could successfully be
determined.
PMID: 10889280 [PubMed - indexed for MEDLINE]
432: Mol Biol Cell 2000 Jul;11(7):2335-47
The Skn7 response regulator of Saccharomyces cerevisiae interacts with Hsf1 in
vivo and is required for the induction of heat shock genes by oxidative stress.
Raitt DC, Johnson AL, Erkine AM, Makino K, Morgan B, Gross DS, Johnston LH.
Division of Yeast Genetics, National Institute for Medical Research, The
Ridgeway, London NW7 1AA, United Kingdom. desmond_raitt@dfci.harvard.edu
The Skn7 response regulator has previously been shown to play a role in the
induction of stress-responsive genes in yeast, e.g., in the induction of the
thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor,
Hsf1, is central to the induction of another set of stress-inducible genes,
namely the heat shock genes. These two regulatory trans-activators, Hsf1 and
Skn7, share certain structural homologies, particularly in their DNA-binding
domains and the presence of adjacent regions of coiled-coil structure, which are
known to mediate protein-protein interactions. Here, we provide evidence that
Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to
the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore,
we demonstrate that a strain deleted for the SKN7 gene and containing a
temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress.
Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of
heat shock genes in response specifically to oxidative stress. We further show
that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus
under normal growth conditions as well as during oxidative stress.
PMID: 10888672 [PubMed - indexed for MEDLINE]
433: J Inorg Biochem 2000 May 30;80(1-2):161-8
Oxo-vanadium as a spin probe for the investigation of the metal coordination
environment of imidazole glycerol phosphate dehydratase.
Petersen J, Hawkes TR, Lowe DJ.
Nitrogen Fixation Laboratory, John Innes Centre, Norwich, UK.
Imidazole glycerol phosphate dehydratase (IGPD) catalyses the dehydration of
imidazole glycerol phosphate to imidazole acetol phosphate, an important late
step in the biosynthesis of histidine. IGPD, isolated as a low molecular weight
and inactive apo-form, assembles with specific divalent metal cations to form a
catalytically active high molecular weight metalloenzyme. Oxo-vanadium ions also
assemble the protein into, apparently, the same high molecular weight form but,
uniquely, yield a protein without catalytic activity. The VO2+ derivative of
IGPD has been investigated by electron paramagnetic resonance (EPR), electron
nuclear double resonance (ENDOR) and electron spin echo envelope modulation
(ESEEM) spectroscopy. The spin Hamiltonian parameters indicate the presence of
multiple 14N nuclei in the inner coordination sphere of VO2+ which is
corroborated by ENDOR and ESEEM spectra showing resonances attributable to
interactions with 14N nuclei. The isotropic superhyperfine coupling component of
about 7 MHz determined by ENDOR is consistent with a nitrogen of coordinated
histidine imidazole(s). The ESEEM Fourier-transform spectra further support the
notion that the VO2+ substituted enzyme contains inner-sphere nitrogen ligands.
The isotropic and anisotropic 14N superhyperfine coupling components are similar
to those reported for other equatorially coordinated enzymatic histidine
imidazole systems. ESEEM resonances from axial 14N ligands are discussed.
Publication Types:
Review
Review, Tutorial
PMID: 10885480 [PubMed - indexed for MEDLINE]
434: Mol Cell 2000 May;5(5):865-76
Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions
and a regulatory element essential for cell growth in yeast.
Mossessova E, Lima CD.
Biochemistry Department, Weill Medical College of Cornell University, New York,
New York 10021, USA.
Modification of cellular proteins by the ubiquitin-like protein SUMO is
essential for nuclear processes and cell cycle progression in yeast. The Ulp1
protease catalyzes two essential functions in the SUMO pathway: (1) processing
of full-length SUMO to its mature form and (2) deconjugation of SUMO from
targeted proteins. Selective reduction of the proteolytic reaction produced a
covalent thiohemiacetal transition state complex between a Ulp1 C-terminal
fragment and its cellular substrate Smt3, the yeast SUMO homolog. The Ulp1-Smt3
crystal structure and functional testing of elements within the conserved
interface elucidate determinants of SUMO recognition, processing, and
deconjugation. Genetic analysis guided by the structure further reveals a
regulatory element N-terminal to the proteolytic domain that is required for
cell growth in yeast.
PMID: 10882122 [PubMed - indexed for MEDLINE]
435: Eur J Biochem 2000 Jul;267(14):4566-76
Reconstitution of ethanolic fermentation in permeabilized spheroplasts of
wild-type and trehalose-6-phosphate synthase mutants of the yeast Saccharomyces
cerevisiae.
Noubhani A, Bunoust O, Rigoulet M, Thevelein JM.
Laboratorium voor Moleculaire Celbiologie, Institute of Botany and Microbiology,
Katholieke Universiteit Leuven, Flanders, Belgium.
In the yeast Saccharomyces cerevisiae, TPS1-encoded trehalose-6-phosphate
synthase (TPS) exerts an essential control on the influx of glucose into
glycolysis, presumably by restricting hexokinase activity. Deletion of TPS1
results in severe hyperaccumulation of sugar phosphates and near absence of
ethanol formation. To investigate whether trehalose 6-phosphate (Tre6P) is the
sole mediator of hexokinase inhibition, we have reconstituted ethanolic
fermentation from glucose in permeabilized spheroplasts of the wild-type,
tps1Delta and tps2Delta (Tre6P phosphatase) strains. For the tps1Delta strain,
ethanol production was significantly lower and was associated with
hyperaccumulation of Glu6P and Fru6P. A tps2Delta strain shows reduced
accumulation of Glu6P and Fru6P both in intact cells and in permeabilized
spheroplasts. These results are not consistent with Tre6P being the sole
mediator of hexokinase inhibition. Reconstitution of ethanolic fermentation in
permeabilized spheroplasts with glycolytic intermediates indicates additional
target site(s) for the Tps1 control. Addition of Tre6P partially shifts the
ethanol production rate and the metabolite pattern in permeabilized tps1Delta
spheroplasts to those of the wild-type strain, but only with glucose as
substrate. This is observed at a very high ratio of glucose to Tre6P. Inhibition
of hexokinase activity by Tre6P is less efficiently counteracted by glucose in
permeabilized spheroplasts compared to cell extracts, and this effect is largely
abolished by deletion of TPS2 but not TPS1. In permeabilized spheroplasts,
hexokinase activity is significantly lower in a tps2Delta strain compared to a
wild-type strain and this difference is strongly reduced by additional deletion
of TPS1. These results indicate that Tps1-mediated protein-protein interactions
are important for control of glucose influx into yeast glycolysis, that Tre6P
inhibition of hexokinase might not be competitive with respect to glucose in
vivo and that also Tps2 appears to play a role in the control of hexokinase
activity.
PMID: 10880982 [PubMed - indexed for MEDLINE]
436: Genetics 2000 Jul;155(3):1069-81
MPH1, a yeast gene encoding a DEAH protein, plays a role in protection of the
genome from spontaneous and chemically induced damage.
Scheller J, Schurer A, Rudolph C, Hettwer S, Kramer W.
Abteilung Molekulare Genetik und Praparative Molekularbiologie, Institut fur
Mikrobiologie und Genetik, Georg-August-Universitat Gottingen, 37077 Gottingen,
Germany.
We have characterized the MPH1 gene from Saccharomyces cerevisiae. mph1 mutants
display a spontaneous mutator phenotype. Homologs were found in archaea and in
the EST libraries of Drosophila, mouse, and man. Mph1 carries the signature
motifs of the DEAH family of helicases. Selected motifs were shown to be
necessary for MPH1 function by introducing missense mutations. Possible indirect
effects on translation and splicing were excluded by demonstrating nuclear
localization of the protein and splicing proficiency of the mutant. A mutation
spectrum did not show any conspicuous deviations from wild type except for an
underrepresentation of frameshift mutations. The mutator phenotype was dependent
on REV3 and RAD6. The mutant was sensitive to MMS, EMS, 4-NQO, and camptothecin,
but not to UV light and X rays. Epistasis analyses were carried out with
representative mutants from various repair pathways (msh6, mag1, apn1, rad14,
rad52, rad6, mms2, and rev3). No epistatic interactions were found, either for
the spontaneous mutator phenotype or for MMS, EMS, and 4-NQO sensitivity. mph1
slightly increased the UV sensitivity of mms2, rad6, and rad14 mutants, but no
effect on X-ray sensitivity was observed. These data suggest that MPH1 is not
part of a hitherto known repair pathway. Possible functions are discussed.
PMID: 10880470 [PubMed - indexed for MEDLINE]
437: Genetics 2000 Jul;155(3):1033-44
Isolation and characterization of HRT1 using a genetic screen for mutants unable
to degrade Gic2p in saccharomyces cerevisiae.
Blondel M, Galan JM, Peter M.
Swiss Institute for Experimental Cancer Research (ISREC), 1066 Epalinges/VD,
Switzerland.
Skp1p-cullin-F-box (SCF) protein complexes are ubiquitin ligases required for
degradation of many regulatory proteins involved in cell cycle progression,
morphogenesis, and signal transduction. Using a genetic screen, we have isolated
a novel allele of the HRT1/RBX1 gene in budding yeast (hrt1-C81Y). hrt1-C81Y
mutant cells exhibited an aberrant morphology but were viable at all
temperatures. The cells displayed multiple genetic interactions with mutations
in known SCF components and were defective for the degradation of several SCF
targets including Gic2p, Far1p, Sic1p, and Cln2p. In addition, they also failed
to degrade the F-box proteins Grr1p, Cdc4p, and Met30p. Wild-type Hrt1p but not
Hrt1p-C81Y was able to bind multiple F-box proteins in an F-box-dependent
manner. Hrt1p-C81Y harbors a single mutation in its ring-finger domain, which is
conserved in subunits of distinct E3 ligases. Finally, Hrt1p was localized in
both nucleus and cytoplasm and despite a short half-life was expressed
constitutively throughout the cell cycle. Taken together, these results suggest
that Hrt1p is a core subunit of multiple SCF complexes.
PMID: 10880467 [PubMed - indexed for MEDLINE]
438: EMBO J 2000 Jul 3;19(13):3215-22
The yeast prion [URE3] can be greatly induced by a functional mutated URE2
allele.
Fernandez-Bellot E, Guillemet E, Cullin C.
Centre de Genetique Moleculaire, Centre National de la Recherche Scientifique,
Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
The non-Mendelian element [URE3] of yeast is considered to be a prion form of
the Ure2 protein. The [URE3] phenotype occurs at a frequency of 10(-5) in
haploid yeast strains, is reversible, and its frequency is increased by
overexpressing the URE2 gene. We created a new mutant of the Ure2 protein,
called H2p, which results in a 1000-fold increase in the rate of [URE3]
occurrence. To date, only the overexpression of various C-terminal truncated
mutants of Ure2p gives rise to a comparable level. The h2 allele is, thus, the
first characterized URE2 allele that induces prion formation when expressed at a
low level. By shuffling mutated and wild-type domains of URE2, we also created
the first mutant Ure2 protein that is functional and induces prion formation. We
demonstrate that the domains of URE2 function synergistically in cis to induce
[URE3] formation, which highlights the importance of intramolecular interactions
in Ure2p folding. Additionally, we show using a green fluorescent protein (GFP)
fusion protein that the h2 allele exhibits numerous filiform structures that are
not generated by the wild-type protein.
PMID: 10880435 [PubMed - indexed for MEDLINE]
439: J Biol Chem 2000 Sep 15;275(37):28816-25
Subunit interactions within the Saccharomyces cerevisiae DNA polymerase epsilon
(pol epsilon ) complex. Demonstration of a dimeric pol epsilon.
Dua R, Edwards S, Levy DL, Campbell JL.
Braun Laboratories, California Institute of Technology, Pasadena, California
91125, USA.
Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon) is essential for
chromosomal replication. A major form of pol epsilon purified from yeast
consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have
investigated the protein/protein interactions between these polypeptides by
using expression of individual subunits in baculovirus-infected Sf9 insect cells
and by using the yeast two-hybrid assay. The essential subunits, Pol2p and
Dpb2p, interact directly in the absence of the other two subunits, and the
C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for
interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact
directly with each other in the absence of the other two subunits. We propose
that Pol2p.Dpb2p and Dpb3p.Dpb4p complexes interact with each other and document
several interactions between individual members of the two respective complexes.
We present biochemical evidence to support the proposal that pol epsilon may be
dimeric in vivo. Gel filtration of the Pol2p.Dpb2p complexes reveals a novel
heterotetrameric form, consisting of two heterodimers of Pol2p.Dpb2p. Dpb2p, but
not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be
mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication
defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce
dimerization of Pol2p. This suggests, but does not prove, that dimerization may
also occur in vivo and be essential for DNA replication.
PMID: 10878005 [PubMed - indexed for MEDLINE]
440: J Biol Chem 2000 Jul 7;275(27):20527-32
Influence of FAD structure on its binding and activity with the C406A mutant of
recombinant human liver monoamine oxidase A.
Nandigama RK, Edmondson DE.
Departments of Biochemistry and Chemistry, Emory University School of Medicine,
Atlanta, Georgia 30322, USA.
The FAD binding site of human liver monoamine oxidase A (MAO A) has been
investigated by mutagenesis of the amino acid site of covalent FAD attachment
(Cys-406) to an alanyl residue. Expression of the C406A mutant in Saccharomyces
cerevisiae results in the formation of an active enzyme, as found previously
with the rat liver enzyme. The activity of this mutant enzyme is labile to
solubilization, thus requiring all experiments to be done with membrane
preparations. C406A MAO A was expressed in a rib 5(-) strain of S. cerevisiae in
the presence of 16 different riboflavin analogues. Inactive apoC406A MAO A is
formed by induction of the enzyme in the absence of riboflavin. FAD but not FMN
or riboflavin restores catalytic activity with an apparent K(d) of 62 +/- 5 nm.
The results from both in vivo and in vitro reconstitution experiments show
increased activity levels (up to approximately 7-fold higher) with those
analogues exhibiting higher oxidation-reduction potentials than normal flavin
and decreased activity levels with analogues exhibiting lower potentials.
Analogues with substituents on the pyrimidine ring bind to C406A MAO A more
weakly than normal FAD, suggesting specific interactions with the N(3) and N(1)
positions. Analogues with substituents in the 7 and 8 positions bind to C406A
MAO A with affinities comparable with that of normal FAD. These results are
discussed in regard to functional significance of 8alpha-covalent binding of
flavins to proteins.
PMID: 10877844 [PubMed - indexed for MEDLINE]
441: Curr Biol 2000 Jun 15;10(12):727-30
A myosin light chain mediates the localization of the budding yeast IQGAP-like
protein during contractile ring formation.
Shannon KB, Li R.
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115,
USA.
Cytokinesis in animal cells is accomplished through constriction of an
actomyosin ring [1] [2] [3], which must assemble at the correct time and place
in order to ensure proper division of genetic material and organelles. Budding
yeast is a useful model system for determining the biochemical pathway of
contractile ring assembly. The budding yeast IQGAP-like protein, Cyk1/Iqg1p, has
multiple roles in the assembly and contraction of the actomyosin ring [4] [5]
[6]. Previously, the IQ motifs of Cyk1/Iqg1p were shown to be required for the
localization of this protein at the bud neck [6]. We have investigated the
binding partner of the IQ motifs, which are predicted to interact with
calmodulin-like proteins. Mlc1p was originally identified as a light chain for a
type V myosin, Myo2p; however, a cytokinesis defect associated with disruption
of the MLC1 gene suggested that the essential function of Mlc1p may involve
interactions with other proteins [7]. We show that Mlc1p binds the IQ motifs of
Cyk1/Iqg1p and present evidence that this interaction recruits Cyk1/Iqg1p to the
bud neck. Immunofluorescence staining shows that Mlc1p is localized to sites of
polarized cell growth as well as the bud neck before and independently of Cyk1p.
These results demonstrate that Mlc1p is important for the assembly of the
actomyosin ring in budding yeast and that this function is mediated through
interaction with Cyk1/Iqg1p.
PMID: 10873803 [PubMed - indexed for MEDLINE]
442: Annu Rev Biochem 1999;68:649-86
MCM proteins in DNA replication.
Tye BK.
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca,
New York 14853-2703, USA.
The MCM proteins are essential replication initiation factors originally
identified as proteins required for minichromosome maintenance in Saccharomyces
cerevisiae. The best known among them are a family of six structurally related
proteins, MCM2-7, which are evolutionally conserved in all eukaryotes. The
MCM2-7 proteins form a hexameric complex. This complex is a key component of the
prereplication complex that assembles at replication origins during early G1
phase. New evidence suggests that the MCM2-7 proteins may be involved not only
in the initiation but also in the elongation of DNA replication. Orchestration
of the functional interactions between the MCM2-7 proteins and other components
of the prereplication complex by cell cycle-dependent protein kinases results in
initiation of DNA synthesis once every cell cycle.
Publication Types:
Review
Review, Academic
PMID: 10872463 [PubMed - indexed for MEDLINE]
443: Proc Natl Acad Sci U S A 2000 Jul 5;97(14):7916-20
A regulatory shortcut between the Snf1 protein kinase and RNA polymerase II
holoenzyme.
Kuchin S, Treich I, Carlson M.
Department of Genetics and Development and Department of Microbiology, Columbia
University, New York, NY 10032, USA.
RNA polymerase II holoenzymes respond to activators and repressors that are
regulated by signaling pathways. Here we present evidence for a "shortcut"
mechanism in which the Snf1 protein kinase of the glucose signaling pathway
directly regulates transcription by the yeast holoenzyme. In response to glucose
limitation, the Snf1 kinase stimulates transcription by holoenzyme that has been
artificially recruited to a reporter by a LexA fusion to a holoenzyme component.
We show that Snf1 interacts physically with the Srb/mediator proteins of the
holoenzyme in both two-hybrid and coimmunoprecipitation assays. We also show
that a catalytically hyperactive Snf1, when bound to a promoter as a LexA fusion
protein, activates transcription in a glucose-regulated manner; moreover, this
activation depends on the integrity of the Srb/mediator complex. These results
suggest that direct regulatory interactions between signal transduction pathways
and RNA polymerase II holoenzyme provide a mechanism for transcriptional control
in response to important signals.
PMID: 10869433 [PubMed - indexed for MEDLINE]
444: Mol Cell Biol 2000 Jul;20(14):5321-9
The C terminus of the Saccharomyces cerevisiae alpha-factor receptor contributes
to the formation of preactivation complexes with its cognate G protein.
Dosil M, Schandel KA, Gupta E, Jenness DD, Konopka JB.
Department of Molecular Genetics and Microbiology, State University of New York,
Stony Brook, New York 11794-5222, USA.
Binding of the alpha-factor pheromone to its G-protein-coupled receptor (encoded
by STE2) activates the mating pathway in MATa yeast cells. To investigate
whether specific interactions between the receptor and the G protein occur prior
to ligand binding, we analyzed dominant-negative mutant receptors that compete
with wild-type receptors for G proteins, and we analyzed the ability of
receptors to suppress the constitutive signaling activity of mutant Galpha
subunits in an alpha-factor-independent manner. Although the amino acid
substitution L236H in the third intracellular loop of the receptor impairs
G-protein activation, this substitution had no influence on the ability of the
dominant-negative receptors to sequester G proteins or on the ability of
receptors to suppress the GPA1-A345T mutant Galpha subunit. In contrast, removal
of the cytoplasmic C-terminal domain of the receptor eliminated both of these
activities even though the C-terminal domain is unnecessary for G-protein
activation. Moreover, the alpha-factor-independent signaling activity of
ste2-P258L mutant receptors was inhibited by the coexpression of wild-type
receptors but not by coexpression of truncated receptors lacking the C-terminal
domain. Deletion analysis suggested that the distal half of the C-terminal
domain is critical for sequestration of G proteins. The C-terminal domain was
also found to influence the affinity of the receptor for alpha-factor in cells
lacking G proteins. These results suggest that the C-terminal cytoplasmic domain
of the alpha-factor receptor, in addition to its role in receptor
downregulation, promotes the formation of receptor-G-protein preactivation
complexes.
PMID: 10866688 [PubMed - indexed for MEDLINE]
445: J Biol Chem 2000 Sep 1;275(35):26925-34
The TATA-binding protein-associated factor yTafII19p functionally interacts with
components of the global transcriptional regulator Ccr4-Not complex and
physically interacts with the Not5 subunit.
Lemaire M, Collart MA.
Departement de Biochimie Medicale, Centre Medical Universitaire, 1 rue Michel
Servet, 1211 Geneva 4, Switzerland.
The Saccharomyces cerevisiae HIS3 gene is a model system to characterize
transcription initiation from different types of core promoters. The NOT genes
were identified by mutations that preferentially increased transcription of the
HIS3 promoter lacking a canonical TATA sequence. They encode proteins associated
in a complex that also contains the Caf1 and Ccr4 proteins. It has been
suggested that the Ccr4-Not complex represses transcription by inhibiting
factors more specifically required for promoters lacking a TATA sequence. A
potential target is the yTaf(II)19 subunit of TFIID, which, when depleted, leads
to a preferential decrease of HIS3 TATA-less transcription. We isolated
conditional taf19 alleles that display synthetic growth phenotypes when combined
with not4 or specific not5 alleles. Inactivation of yTaf(II)19p by shifting
these mutants to the restrictive temperature led to a more rapid and striking
decrease in transcription from promoters that do not contain a canonical TATA
sequence. We demonstrated by the two-hybrid assay and directly in vitro that
yTaf(II)19p and Not5p could interact. Finally, we found by the two-hybrid assay
that yTaf(II)19p also interacted with many components of the Ccr4-Not complex.
Taken together, our results provide evidence that interactions between Not5p and
yTaf(II)19p may be involved in transcriptional regulation by the Ccr4-Not
complex.
PMID: 10864925 [PubMed - indexed for MEDLINE]
446: J Mol Biol 2000 Jun 30;300(1):11-6
A compact monomeric intermediate identified by NMR in the denaturation of
dimeric triose phosphate isomerase.
Morgan CJ, Wilkins DK, Smith LJ, Kawata Y, Dobson CM.
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of
Oxford, South Parks Road, Oxford, OX1 3QT, UK.
The denaturation of triose phosphate isomerase (TIM) from Saccharomyces
cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR
spectroscopy in conjunction with optical spectroscopy. In the absence of
denaturant, the hydrodynamic radius of 29.6(+/-0.25) A and the substantial
chemical shift dispersion evident in the NMR spectrum are consistent with the
highly structured dimeric native state of the protein. On the addition of 2. 2 M
guanidine hydrochloride the effective hydrodynamic radius increases to
51.4(+/-0.43) A, consistent with that anticipated for the polypeptide chain in a
highly unstructured random coil state. In 1.1 M guanidine hydrochloride,
however, the effective hydrodynamic radius is 24.0(+/-0.25) A, a value
substantially decreased relative to that of the native dimeric state but very
close to that anticipated for a monomeric species with native-like compaction
(23. 5 A). The lack of chemical shift dispersion indicates, however, that few
tertiary interactions persist within this species. Far UV CD and intrinsic
fluorescence measurements show that this compact intermediate retains
significant secondary structure and that on average the fluorophores are
partially excluded from solvent. Such a species could be important in the
formation of dimeric TIM from its unfolded state. Copyright 2000 Academic Press.
PMID: 10864494 [PubMed - indexed for MEDLINE]
447: J Clin Invest 2000 Jun;105(12):1711-21
Inhibition of cystic fibrosis transmembrane conductance regulator by novel
interaction with the metabolic sensor AMP-activated protein kinase.
Hallows KR, Raghuram V, Kemp BE, Witters LA, Foskett JK.
Renal-Electrolyte and Hypertension Division, Department of Medicine, University
of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated
Cl(-) channel that regulates other epithelial transport proteins by
uncharacterized mechanisms. We employed a yeast two-hybrid screen using the
COOH-terminal 70 residues of CFTR to identify proteins that might be involved in
such interactions. The alpha1 (catalytic) subunit of AMP-activated protein
kinase (AMPK) was identified as a dominant and novel interacting protein. The
interaction is mediated by residues 1420-1457 in CFTR and by the COOH-terminal
regulatory domain of alpha1-AMPK. Mutations of two protein trafficking motifs
within the 38-amino acid region in CFTR each disrupted the interaction.
GST-fusion protein pull-down assays in vitro and in transfected cells confirmed
the CFTR-alpha1-AMPK interaction and also identified alpha2-AMPK as an
interactor with CFTR. AMPK is coexpressed in CFTR-expressing cell lines and
shares an apical distribution with CFTR in rat nasal epithelium. AMPK
phosphorylated full-length CFTR in vitro, and AMPK coexpression with CFTR in
XENOPUS: oocytes inhibited cAMP-activated CFTR whole-cell Cl(-) conductance by
approximately 35-50%. Because AMPK is a metabolic sensor in cells and responds
to changes in cellular ATP, regulation of CFTR by AMPK may be important in
inhibiting CFTR under conditions of metabolic stress, thereby linking
transepithelial transport to cell metabolic state.
PMID: 10862786 [PubMed - indexed for MEDLINE]
448: Yeast 2000 Jun 30;16(9):811-27
Mutational analysis of the karmellae-inducing signal in Hmg1p, a yeast HMG-CoA
reductase isozyme.
Profant DA, Roberts CJ, Wright RL.
Department of Zoology, Box 351800, University of Washington, Seattle, WA 98195,
USA.
In response to elevated levels of HMG-CoA reductase, an integral endoplasmic
reticulum (ER) membrane protein, cells assemble novel ER arrays. These membranes
provide useful models for exploration of ER structure and function, as well as
general features of membrane biogenesis and turnover. Yeast express two
functional HMG-CoA reductase isozymes, Hmg1p and Hmg2p, each of which induces
morphologically different ER arrays. Hmg1p induces stacks of paired
nuclear-associated membranes called karmellae. In contrast, Hmg2p induces
peripheral ER membrane arrays and short nuclear-associated membrane stacks. In
spite of their ability to induce different cellular responses, both Hmg1p and
Hmg2p have similar structures, including a polytopic membrane domain containing
eight predicted transmembrane helices. By examining a series of recombinant
HMG-CoA reductase proteins, our laboratory previously demonstrated that the last
ER-lumenal loop (Loop G) of the Hmg1p membrane domain contains a signal needed
for proper karmellae assembly. Our goal was to examine the primary sequence
requirements within Loop G that were critical for proper function of this
signal. To this end, we randomly mutagenized the Loop G sequence, expressed the
mutagenized Hmg1p in yeast, and screened for inability to generate karmellae at
wild-type levels. Out of approximately 4000 strains with Loop G mutations, we
isolated 57 that were unable to induce wild-type levels of karmellae assembly.
Twenty-nine of these mutants contained one or more point mutations in the Loop G
sequence, including nine single point mutants, four of which had severe defects
in karmellae assembly. Comparison of these mutations to single point mutations
that did not affect karmellae assembly did not reveal obvious patterns of
sequence requirements. For example, both conservative and non-conservative
changes were present in both groups and changes that altered the total charge of
the Loop G region were observed in both groups. Our hypothesis is that Loop G
serves as a karmellae-inducing signal by mediating protein-protein or
protein-lipid interactions and that amino acids revealed by this analysis may be
important for maintaining the proper secondary structure needed for these
interactions. Copyright 2000 John Wiley & Sons, Ltd.
PMID: 10861905 [PubMed - indexed for MEDLINE]
449: EMBO J 2000 Jun 15;19(12):3016-27
Structure of the C-terminal domain of Tup1, a corepressor of transcription in
yeast.
Sprague ER, Redd MJ, Johnson AD, Wolberger C.
Department of Biophysics and Biophysical Chemistry and Howard Hughes Medical
Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205,
USA.
The Tup1-Ssn6 corepressor complex regulates the expression of several sets of
genes, including genes that specify mating type in the yeast Saccharomyces
cerevisiae. Repression of mating-type genes occurs when Tup1-Ssn6 is brought to
the DNA by the Matalpha2 DNA-binding protein and assembled upstream of a- and
haploid-specific genes. We have determined the 2.3 A X-ray crystal structure of
the C-terminal domain of Tup1 (accesion No. 1ERJ), a 43 kDa fragment that
contains seven copies of the WD40 sequence motif and binds to the Matalpha2
protein. Moreover, this portion of the protein can partially substitute for
full-length Tup1 in bringing about transcriptional repression. The structure
reveals a seven-bladed beta propeller with an N-terminal subdomain that is
anchored to the side of the propeller and extends the beta sheet of one of the
blades. Point mutations in Tup1 that specifically affect the Tup1-Matalpha2
interaction cluster on one surface of the propeller. We identified regions of
Tup1 that are conserved among the fungal Tup1 homologs and may be important in
protein-protein interactions with additional components of the Tup1-mediated
repression pathways.
PMID: 10856245 [PubMed - indexed for MEDLINE]
450: Chromosoma 2000;109(1-2):86-93
Meiotic recombination in RAD54 mutants of Saccharomyces cerevisiae.
Schmuckli-Maurer J, Heyer WD.
Institute of General Microbiology, University of Bern, Switzerland.
The Rad54 protein is an important component of the recombinational DNA repair
pathway in vegetative Saccharomyces cerevisiae cells. Unlike those in other
members of the RAD52 group, the meiotic defect in rad54 is rather mild, reducing
spore viability only to 26%-65%. A consistently greater requirement for Rad54p
during meiosis was observed in hybrid strains, suggesting that Rad54p has a
certain role in interhomolog interactions. Such a role is probably minor as no
recombination defects were found in the surviving gametes in three genetic
intervals on chromosome V. Also, the spore viability pattern in tetrads did not
reflect an increase in nondisjunction at meiosis I indicative of a meiotic
recombination defect. We suggest that the meiotic defect of rad54 cells lies in
the failure to repair meiosis-specific double-strand breaks outside the context
of the highly differentiated pathway leading to interhomolog joint molecules and
meiotic crossovers that ensure accurate segregation at meiosis I.
PMID: 10855498 [PubMed - indexed for MEDLINE]
451: Gene 2000 May 30;250(1-2):1-14
The continued evolution of two-hybrid screening approaches in yeast: how to
outwit different preys with different baits.
Fashena SJ, Serebriiskii I, Golemis EA.
Division of Basic Science, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
The original two-hybrid system, an experimental approach designed to detect
protein interactions, exploited the modular nature of many transcription
factors. It has provided the intellectual and technical seed for the evolution
of an array of innovative approaches, the application of which broadens the
scope of experimentally feasible questions to include the interaction of
proteins with diverse binding partners. The available array of modified and
alternative approaches facilitates the analysis of complex cellular machinery
and signaling networks that rely on multiple protein interactions. Such advances
have facilitated the functional analysis of proteins on the genome level, a feat
considered untenable a decade ago.
Publication Types:
Review
Review, Tutorial
PMID: 10854774 [PubMed - indexed for MEDLINE]
452: Curr Opin Microbiol 2000 Jun;3(3):303-8
Systematic and large-scale two-hybrid screens.
Uetz P, Hughes RE.
Department of Genetics, University of Washington, Box 357360, Seattle, WA
98195-7360, USA. uetz@u.washington.edu
The increasing rate at which complete genome sequences become available
necessitates rapid and robust methods for investigating the functions of their
encoded proteins. Efforts have been made to study protein function by
systematically screening large sets of proteins using the two-hybrid method.
Analyses of the complete proteomes of baceriophage T7, the mammalian viruses
hepatitis C and vaccinia, as well as of several protein complexes including RNA
splicing proteins and RNA polymerase III from yeast, have been undertaken.
Saccharomyces cerevisiae has been studied extensively by two-hybrid methods,
with more than 2500 protein-protein interactions described. Systematic studies
on metazoan proteomes are, however, still in their infancy.
Publication Types:
Review
Review, Tutorial
PMID: 10851163 [PubMed - indexed for MEDLINE]
453: Plant Cell Physiol 2000 Apr;41(4):523-33
Modulation of 14-3-3 protein interactions with target polypeptides by physical
and metabolic effectors.
Athwal GS, Lombardo CR, Huber JL, Masters SC, Fu H, Huber SC.
US Department of Agriculture, Department of Horticultural Science, North
Carolina State University, Raleigh 27695, USA.
The proteins commonly referred to as 14-3-3s have recently come to prominence in
the study of protein:protein interactions, having been shown to act as
allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3
proteins to the regulatory phosphorylation site of nitrate reductase (NR) was
studied in real-time by surface plasmon resonance, using primarily an
immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and
yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a
Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an
increase in steady-state binding level (Req). As shown previously for plant
14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly
with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s
to the phosphopeptide ligand occurred in the absence of divalent cations when
the pH was reduced below neutral, and the basis for enhanced binding was a
reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to
the NR based peptide ligand. The binding of AMP to 14-3-3s was directly
demonstrated by equilibrium dialysis (plant), and from the observation that
recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.
PMID: 10845467 [PubMed - indexed for MEDLINE]
454: Mol Cells 2000 Apr 30;10(2):232-5
Random changes of amino acid residues with expected frequency by saturated point
mutagenesis.
Kim SJ, Park H, Kim JK, Lee JY, Ahn K, Choe M, Choi YJ, Kim J.
Graduate School of Biotechnology, Korea University, Seoul.
The yeast transcriptional activator protein, Gcn4p from Saccharomyces cerevisiae
binds to the specific sequence in the promoters of many amino acid biosynthetic
genes for general control. A new random saturation mutagenesis method was
developed to isolate Gcn4p derivatives with only one or two mutations in the DNA
binding domain without using radioactive isotope. This will be used to identify
the amino acids of Gcn4p involved in protein-protein interactions. Saturation
mutagenesis in the DNA binding domain of Gcn4p was performed using spiked
degenerate oligonucleotides containing randomized codon bases designed
specifically for only one or two base changes in the mutagenized area. These
oligonucleotides were synthesized to have two flanking restriction enzyme sites
for cloning to the appropriate vector. The 3' ends were mutually primed after
hybridization via the palindromic sequences of the restriction enzyme sites.
These molecules were then converted to double stranded DNA upon treatment with
DNA polymerase. Here, a library collection of 100,680 in an altered Gcn4p pool
was generated by cloning a mixed-base oligonucleotide in the place of the
sequence coding for the DNA binding domains. The quality of the library was
examined by DNA sequencing and found to be in good agreement with the expected
statistical values. Calculated mutation frequency was 66% of mutant nucleotide
rate and actual sequencing data revealed 68% mutant nucleotide rates from the
sequenced library. Thus, among 21 mutants, 16 had one point mutations and 5 had
two point mutations. This approach appears to be an effective and general tool
for creating proteins with one or two amino acid change(s) in their molecules.
PMID: 10850667 [PubMed - indexed for MEDLINE]
455: Mol Cell Biol 2000 Jul;20(13):4838-48
Bypass of a meiotic checkpoint by overproduction of meiotic chromosomal
proteins.
Bailis JM, Smith AV, Roeder GS.
Department of Molecular, Cellular, and Developmental Biology, Yale University,
New Haven, CT 06520-8103, USA.
The Saccharomyces cerevisiae zip1 mutant, which exhibits defects in synaptonemal
complex formation and meiotic recombination, triggers a checkpoint that causes
cells to arrest at the pachytene stage of meiotic prophase. Overproduction of
either the meiotic chromosomal protein Red1 or the meiotic kinase Mek1 bypasses
this checkpoint, allowing zip1 cells to sporulate. Red1 or Mek1 overproduction
also promotes sporulation of other mutants (zip2, dmc1, hop2) that undergo
checkpoint-mediated arrest at pachytene. In addition, Red1 overproduction
antagonizes interhomolog interactions in the zip1 mutant, substantially
decreasing double-strand break formation, meiotic recombination, and homologous
chromosome pairing. Mek1 overproduction, in contrast, suppresses
checkpoint-induced arrest without significantly decreasing meiotic
recombination. Cooverproduction of Red1 and Mek1 fails to bypass the checkpoint;
moreover, overproduction of the meiotic chromosomal protein Hop1 blocks the Red1
and Mek1 overproduction phenotypes. These results suggest that meiotic
chromosomal proteins function in the signaling of meiotic prophase defects and
that the correct stoichiometry of Red1, Mek1, and Hop1 is needed to achieve
checkpoint-mediated cell cycle arrest at pachytene.
PMID: 10848609 [PubMed - indexed for MEDLINE]
456: Mol Cell Biol 2000 Jul;20(13):4806-13
Identification of amino acid residues in the Caenorhabditis elegans POU protein
UNC-86 that mediate UNC-86-MEC-3-DNA ternary complex formation.
Rockelein I, Rohrig S, Donhauser R, Eimer S, Baumeister R.
Genzentrum, Ludwig-Maximilians-Universitat, D-81377 Munich, Germany.
The POU homeodomain protein UNC-86 and the LIM homeodomain protein MEC-3 are
essential for the differentiation of the six mechanoreceptor neurons in the
nematode Caenorhabditis elegans. Previous studies have indicated that UNC-86 and
MEC-3 bind cooperatively to at least three sites in the mec-3 promoter and
synergistically activate transcription. However, the molecular details of the
interactions of UNC-86 with MEC-3 and DNA have not been investigated so far.
Here we used a yeast system to identify the functional domains in UNC-86
required for transcriptional activation and to characterize the interaction of
UNC-86 with MEC-3 in vivo. Our results suggest that transcriptional activation
is mediated by the amino terminus of UNC-86, whereas amino acids in the POU
domain mediate DNA binding and interaction with MEC-3. By random mutagenesis, we
identified mutations that only affect the DNA binding properties of UNC-86, as
well as mutations that prevent coactivation by MEC-3. We demonstrated that both
the POU-specific domain and the homeodomain of UNC-86, as well as DNA bases
adjacent to the proposed UNC-86 binding site, are involved in the formation of a
transcriptionally active complex with MEC-3. These data suggest that some
residues involved in the contact of UNC-86 with MEC-3 also contribute to the
interaction of the functionally nonrelated POU protein Oct-1 with Oca-B, whereas
other positions have different roles.
PMID: 10848606 [PubMed - indexed for MEDLINE]
457: Mol Endocrinol 2000 Jun;14(6):889-99
Characterization of transactivational property and coactivator mediation of rat
mineralocorticoid receptor activation function-1 (AF-1).
Fuse H, Kitagawa H, Kato S.
Pharmacological Research Department, Teikoku Hormone Manufacturing Company,
Ltd., Tokyo, Japan.
The autonomous activation function-2 (AF-2) in the mineralocorticoid receptor
(MR) E/F domain is known to play a major role in the ligand-induced
transactivation function of MR; however, it remained unclear about the
transactivation function of its A/B domain. We therefore tried to characterize
the MR A/B domain as the AF-1 and further studied the actions of known
coactivators for AF-2 in the E/F ligand-binding domain in the function of the MR
A/B domain. Deletion analyses of rat and human MRs revealed that the A/B domains
harbor a transactivation function acting as AF-1. The MR mutant (E959Q) with a
point mutation in helix 12, which causes a complete loss of MR AF-2 activity,
still retained ligand-induced transactivation function, indicating a significant
role for AF-1 in the full activity of the ligand-induced MR function. Among the
coactivators tested to potentiate the MR AF-2, TIF2 and p300 potentiated the MR
AF-1 through two different core regions [amino acids (a.a.) 1-169, a.a. 451-603]
and exhibited functional interactions with the MR A/B domain in the cultured
cells. However, such interactions were undetectable in a yeast and in an in
vitro glutathione-S-transferase pull-down assay, indicating that the functional
interaction of TIF2 and p300 with the MR A/B domain to support the MR AF-1
activity require some unknown nuclear factor(s) or a proper modification of the
A/B domain in the cells.
PMID: 10847590 [PubMed - indexed for MEDLINE]
458: J Cell Biochem 2000 May;78(2):179-85
Recruitment of chromatin remodeling machines.
Peterson CL, Logie C.
Program in Molecular Medicine and Department of Biochemistry and Molecular
Biology, University of Massachusetts Medical School, Worcester, Massachusetts
01605, USA. craig.peterson@umassmed.edu
The assembly of eukaryotic DNA into folded nucleosomal arrays has drastic
consequences for many nuclear processes that require access to the DNA sequence,
including RNA transcription, DNA replication, recombination, and repair. Two
types of highly conserved chromatin remodeling enzymes have been implicated as
regulators of the repressive nature of chromatin structure: ATP-dependent
remodeling complexes and nuclear histone acetyltransferases (HATs). Recent
studies indicate that both types of enzymes can be recruited to chromosomal loci
through either physical interactions with transcriptional activators or via the
global accessibility of chromatin during S phase of the cell cycle. Here we
review these recent observations and discuss the implications for gene-specific
regulation by chromatin remodeling machines. Copyright 2000 Wiley-Liss, Inc.
Publication Types:
Review
Review, Tutorial
PMID: 10842313 [PubMed - indexed for MEDLINE]
459: Bioessays 2000 Jun;22(6):503-6
Building a protein interaction map: research in the post-genome era.
Chen Z, Han M.
Howard Hughes Medical Institute, University of Colorado at Boulder, Boulder,
Colorado.
With the extensive amount of information generated by genome-wide sequencing,
the entire set of gene products in an organism can now be predicted. The
challenge of understanding the function of each gene in the genome has led to
the development of many large-scale and high-throughput experimental techniques.
Recently, two papers, Walhout et al.(1) and Uetz et al.,(2) have described
studies that add a new functional dimension to research conducted on a
genome-wide scale. These two groups have utilized the yeast two-hybrid system to
identify interactions among the entire complement of proteins encoded by the
Caenorhabditis elegans and the Saccharomyces cerevisiae genomes, respectively.
Using a set of 29 genes that have been previously characterized, Walhout et al.
demonstrated the feasibility and efficiency of this technique by building an
interaction matrix among a large number of proteins. On an even larger scale,
Uetz et al. conducted two-hybrid analyses using proteins that represent over 87%
of the total gene products in yeast and identified interactions for about 15% of
the total yeast proteins. BioEssays 22:503-506, 2000. Copyright 2000 John Wiley
& Sons, Inc.
Publication Types:
Review
Review, Tutorial
PMID: 10842303 [PubMed - indexed for MEDLINE]
460: J Biol Chem 2000 Sep 22;275(38):29368-76
Rap1p-binding sites in the saccharomyces cerevisiae GPD1 promoter are involved
in its response to NaCl.
Eriksson P, Alipour H, Adler L, Blomberg A.
Department of Cell and Molecular Biology-Microbiology, Lundberg Laboratory,
Goteborg University, Medicinaregatan 9C, S-413 90 Goteborg, Sweden.
Mechanisms involved in transcriptional regulation of the osmotically controlled
GPD1 gene in Saccharomyces cerevisiae were investigated by promoter analysis.
The GPD1 gene encodes NAD(+)-dependent glycerol-3-phosphate dehydrogenase, a key
enzyme in the production of the compatible solute glycerol. By analysis of
promoter deletions, we identified a region at nucleotides -478 to -324, in
relation to start of translation, to be of great importance for both basal
activity and osmotic induction of GPD1. Electrophoretic mobility shift and DNase
I footprint analyses demonstrated protein binding to parts of this region that
contain three consensus sequences for Rap1p (repressor activator protein
1)-binding sites. Actual binding of Rap1p to this region was confirmed by
demonstrating enhanced electrophoretic mobility of the protein-DNA complex with
extracts containing an N-terminally truncated version of Rap1p. The detected
Rap1p-DNA interactions were not affected by changes in the osmolarity of the
growth medium. Specific inactivation of the Rap1p-binding sites by a C-to-A
point mutation in the core of the consensus showed that this factor is a major
determinant of GPD1 expression since mutations in all three putative binding
sites for Rap1p strongly hampered osmotic induction and drastically lowered
basal activity. We also show that the Rap1p-binding sites appear functionally
distinct; the most distal site (core of the consensus at position -386)
exhibited the highest affinity for Rap1p and was strictly required for low salt
induction (< or =0.6 m NaCl), but not for the response at higher salinities (>
or =0.8 m NaCl). This indicates tha different molecular mechanisms might be
operational for low and high salt responses of the GPD1 promoter.
PMID: 10842169 [PubMed - indexed for MEDLINE]
461: Biochemistry 2000 Jun 13;39(23):6910-7
Prenyl-flavonoids as potent inhibitors of the Pdr5p multidrug ABC transporter
from Saccharomyces cerevisiae.
Conseil G, Decottignies A, Jault JM, Comte G, Barron D, Goffeau A, Di Pietro A.
Laboratoire de Biochimie Structurale et Fonctionnelle, Institut de Biologie et
Chimie des Protinverted question markeines, UPR 412 du Centre National de la
Recherche Scientifique, Lyon, France.
The Pdr5p multidrug ABC ("ATP-binding cassette) transporter was highly
overexpressed in plasma membranes from a yeast strain exhibiting both pdr1-3
gain-of-function mutation in the transcription factor-encoding gene PDR1 and
disruption of genes encoding other plasma membrane ABC transporters. Solubilized
and purified Pdr5p displayed a tryptophan-characteristic intrinsic fluorescence,
whose quenching was used to monitor interactions with substrates and effectors.
The transporter exhibited a magnesium-dependent binding affinity for ATP and its
fluorescent analogue 2'(3')-N-methylanthraniloyl-ATP, producing a marked
fluorescence resonance-energy transfer. It also bound a series of known drug
substrates and modulators. Interestingly, yeast Pdr5p interacted with flavonoids
recently found to bind to cancer cell P-glycoprotein and to the protozoan
parasite multidrug transporter. The extent of high-affinity binding of
prenyl-flavonoids to purified Pdr5p was correlated to their efficiency to
inhibit energy-dependent quenching of rhodamine 6G fluorescence catalyzed by
Pdr5p-enriched plasma membranes. The hydrophobic flavonoid derivative 6-(3,
3-dimethylallyl)galangin was the most efficient, with a K(i) of 0.18 microM for
competitive inhibition of the MgATP-dependent quenching of rhodamine 6G
fluorescence. In contrast, inhibition of either ATP or UTP hydrolysis occurred
at much higher concentrations and appeared to be noncompetitive.
Prenyl-flavonoids therefore behave as potent inhibitors of drug binding to the
yeast Pdr5p ABC transporter.
PMID: 10841772 [PubMed - indexed for MEDLINE]
462: Proc Natl Acad Sci U S A 2000 Jun 6;97(12):6306-10
Crystal structure of RPB5, a universal eukaryotic RNA polymerase subunit and
transcription factor interaction target.
Todone F, Weinzierl RO, Brick P, Onesti S.
Blackett Laboratory and Department of Biochemistry, Imperial College, Exhibition
Road, London SW7 2AZ, United Kingdom.
Eukaryotic nuclei contain three different types of RNA polymerases (RNAPs), each
consisting of 12-18 different subunits. The evolutionarily highly conserved RNAP
subunit RPB5 is shared by all three enzymes and therefore represents a key
structural/functional component of all eukaryotic RNAPs. Here we present the
crystal structure of the RPB5 subunit from Saccharomyces cerevisiae. The
bipartite structure includes a eukaryote-specific N-terminal domain and a
C-terminal domain resembling the archaeal RNAP subunit H. RPB5 has been
implicated in direct protein-protein contacts with transcription factor IIB, one
of the components of the RNAP(II) basal transcriptional machinery, and
gene-specific activator proteins, such as the hepatitis B virus transactivator
protein X. The experimentally mapped regions of RPB5 involved in these
interactions correspond to distinct and surface-exposed alpha-helical
structures.
PMID: 10841537 [PubMed - indexed for MEDLINE]
463: Biochim Biophys Acta 2000 May 31;1458(2-3):443-56
Organisation of the yeast ATP synthase F(0):a study based on cysteine mutants,
thiol modification and cross-linking reagents.
Velours J, Paumard P, Soubannier V, Spannagel C, Vaillier J, Arselin G, Graves
PV.
Institut de Biochimie et Genetique Cellulaires du CNRS, 1 rue Camille Saint
Saens, 33077, cedex, Bordeaux, France. john.velours@ibgc.u-bordeaux2.fr
A topological study of the yeast ATP synthase membranous domain was undertaken
by means of chemical modifications and cross-linking experiments on the
wild-type complex and on mutated enzymes obtained by site-directed mutagenesis
of genes encoding ATP synthase subunits. The modification by non-permeant
maleimide reagents of the Cys-54 of mutated subunit 4 (subunit b), of the Cys-23
in the N-terminus of subunit 6 (subunit a) and of the Cys-91 in the C-terminus
of mutated subunit f demonstrated their location in the mitochondrial
intermembrane space. Near-neighbour relationships between subunits of the
complex were demonstrated by means of homobifunctional and heterobifunctional
reagents. Our data suggest interactions between the first transmembranous
alpha-helix of subunit 6, the two hydrophobic segments of subunit 4 and the
unique membrane-spanning segments of subunits i and f. The amino acid residue
174 of subunit 4 is close to both oscp and the beta-subunit, and the residue 209
is close to oscp. The dimerisation of subunit 4 in the membrane revealed that
this component is located in the periphery of the enzyme and interacts with
other ATP synthase complexes.
Publication Types:
Review
Review, Tutorial
PMID: 10838057 [PubMed - indexed for MEDLINE]
464: Biochim Biophys Acta 2000 May 31;1458(2-3):428-42
Insights into ATP synthase assembly and function through the molecular genetic
manipulation of subunits of the yeast mitochondrial enzyme complex.
Devenish RJ, Prescott M, Roucou X, Nagley P.
Department of Biochemistry and Molecular Biology, Monash University, P.O. Box
13D, Vic. 3800, Australia.
Development of an increasingly detailed understanding of the eucaryotic
mitochondrial ATP synthase requires a detailed knowledge of the stoichiometry,
structure and function of F(0) sector subunits in the contexts of the proton
channel and the stator stalk. Still to be resolved are the precise locations and
roles of other supernumerary subunits present in mitochondrial ATP synthase
complexes, but not found in the bacterial or chloroplast enzymes. The highly
developed system of molecular genetic manipulation available in the yeast
Saccharomyces cerevisiae, a unicellular eucaryote, permits testing for gene
function based on the effects of gene disruption or deletion. In addition, the
genes encoding ATP synthase subunits can be manipulated to introduce specific
amino acids at desired positions within a subunit, or to add epitope or affinity
tags at the C-terminus, enabling questions of stoichiometry, structure and
function to be addressed. Newly emerging technologies, such as fusions of
subunits with GFP are being applied to probe the dynamic interactions within
mitochondrial ATP synthase, between ATP synthase complexes, and between ATP
synthase and other mitochondrial enzyme complexes.
Publication Types:
Review
Review, Tutorial
PMID: 10838056 [PubMed - indexed for MEDLINE]
465: Curr Biol 2000 Jun 1;10(11):675-8
Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle
checkpoint function.
Brady DM, Hardwick KG.
Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology,
University of Edinburgh, UK.
The spindle checkpoint delays the metaphase to anaphase transition in response
to defects in kinetochore-microtubule interactions in the mitotic apparatus (see
[1] [2] [3] [4] for reviews). The Mad and Bub proteins were identified as key
components of the spindle checkpoint through budding yeast genetics [5] [6] and
are highly conserved [3]. Most of the spindle checkpoint proteins have been
localised to kinetochores, yet almost nothing is known about the molecular
events which take place there. Mad1p forms a tight complex with Mad2p [7], and
has been shown to recruit Mad2p to kinetochores [8]. Similarly, Bub3p binds to
Bub1p [9] and may target it to kinetochores [10]. Here, we show that budding
yeast Mad1p has a regulated association with Bub1p and Bub3p during a normal
cell cycle and that this complex is found at significantly higher levels once
the spindle checkpoint is activated. We find that formation of this complex
requires Mad2p and Mps1p but not Mad3p or Bub2p. In addition, we identify a
conserved motif within Mad1p that is essential for Mad1p-Bub1p-Bub3p complex
formation. Mutation of this motif abolishes checkpoint function, indicating that
formation of the Mad1p-Bub1p-Bub3p complex is a crucial step in the spindle
checkpoint mechanism.
PMID: 10837255 [PubMed - indexed for MEDLINE]
466: EMBO J 2000 Jun 1;19(11):2515-24
Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a
highly conserved small protein of yeast plasma membrane.
Navarre C, Goffeau A.
Unite de Biochimie Physiologique, Universite Catholique de Louvain, Croix du Sud
2-20, 1348 Louvain-la-Neuve, Belgium.
Yeast plasma membranes contain a small 55 amino acid hydrophobic polypeptide,
Pmp3p, which has high sequence similarity to a novel family of plant
polypeptides that are overexpressed under high salt concentration or low
temperature treatment. The PMP3 gene is not essential under normal growth
conditions. However, its deletion increases the plasma membrane potential and
confers sensitivity to cytotoxic cations, such as Na(+) and hygromycin B.
Interestingly, the disruption of PMP3 exacerbates the NaCl sensitivity phenotype
of a mutant strain lacking the Pmr2p/Enap Na(+)-ATPases and the Nha1p Na(+)/H(+)
antiporter, and suppresses the potassium dependency of a strain lacking the K(+)
transporters, Trk1p and Trk2p. All these phenotypes could be reversed by the
addition of high Ca(2+) concentration to the medium. These genetic interactions
indicate that the major effect of the PMP3 deletion is a hyperpolarization of
the plasma membrane potential that probably promotes a non-specific influx of
monovalent cations. Expression of plant RCI2A in yeast could substitute for the
loss of Pmp3p, indicating a common role for Pmp3p and the plant homologue.
PMID: 10835350 [PubMed - indexed for MEDLINE]
467: Mol Cell Biol 2000 Jun;20(12):4199-209
Two regulators of Ste12p inhibit pheromone-responsive transcription by separate
mechanisms.
Olson KA, Nelson C, Tai G, Hung W, Yong C, Astell C, Sadowski I.
Department of Biochemistry and Molecular Biology, University of British
Columbia, Vancouver, Canada.
The yeast Saccharomyces cerevisiae transcription factor Ste12p is responsible
for activating genes in response to MAP kinase cascades controlling mating and
filamentous growth. Ste12p is negatively regulated by two inhibitor proteins,
Dig1p (also called Rst1p) and Dig2p (also called Rst2p). The expression of a
C-terminal Ste12p fragment (residues 216 to 688) [Ste12p(216-688)] from a GAL
promoter causes FUS1 induction in a strain expressing wild-type STE12,
suggesting that this region can cause the activation of endogenous Ste12p.
Residues 262 to 594 are sufficient to cause STE12-dependent FUS1 induction when
overexpressed, and this region of Ste12p was found to bind Dig1p but not Dig2p
in yeast extracts. In contrast, recombinant glutathione S-transferase-Dig2p
binds to the Ste12p DNA-binding domain (DBD). Expression of DIG2, but not DIG1,
from a GAL promoter inhibits transcriptional activation by an Ste12p DBD-VP16
fusion. Furthermore, disruption of dig1, but not dig2, causes elevated
transcriptional activation by a LexA-Ste12p(216-688) fusion. Ste12p has multiple
regions within the C terminus (flanking residue 474) that can promote
multimerization in vitro, and we demonstrate that these interactions can
contribute to the activation of endogenous Ste12p by overproduced C-terminal
fragments. These results demonstrate that Dig1p and Dig2p do not function by
redundant mechanisms but rather inhibit pheromone-responsive transcription
through interactions with separate regions of Ste12p.
PMID: 10825185 [PubMed - indexed for MEDLINE]
468: J Biol Chem 2000 Aug 11;275(32):24928-34
The yeast histone acetyltransferase A2 complex, but not free Gcn5p, binds stably
to nucleosomal arrays.
Sendra R, Tse C, Hansen JC.
Departament de Bioquimica i Biologia Molecular, Universitat de Valencia, E-46100
Valencia, Spain.
We have investigated the structural basis for the differential catalytic
function of the yeast Gcn5p-containing histone acetyltransferase (HAT) A2
complex and free recombinant yeast Gcn5p (rGcn5p). HAT A2 is shown to be a
unique complex that contains Gcn5p, Ada2p, and Ada3p, but not proteins specific
to other related HAT A complexes, e.g. ADA, SAGA. Nevertheless, HAT A2 produces
the same unique polyacetylation pattern of nucleosomal substrates reported
previously for ADA and SAGA, demonstrating that proteins specific to the ADA and
SAGA complexes do not influence the enzymatic activity of Gcn5p within the HAT
A2 complex. To investigate the role of substrate interactions in the
differential behavior of free and complexed Gcn5p, sucrose density gradient
centrifugation was used to characterize the binding of HAT A2 and free rGcn5p to
intact and trypsinized nucleosomal arrays, H3/H4 tetramer arrays, and nucleosome
core particles. We find that HAT A2 forms stable complexes with all nucleosomal
substrates tested. In distinct contrast, rGcn5p does not interact stably with
nucleosomal arrays, despite being able to specifically monoacetylate the H3 N
terminus of nucleosomal substrates. Our data suggest that the ability of the HAT
A2 complex to bind stably to nucleosomal arrays is functionally related to both
local and global acetylation by the complexed and free forms of Gcn5p.
PMID: 10825174 [PubMed - indexed for MEDLINE]
469: J Biol Chem 2000 Aug 4;275(31):23500-8
Replication protein A physically interacts with the Bloom's syndrome protein and
stimulates its helicase activity.
Brosh RM Jr, Li JL, Kenny MK, Karow JK, Cooper MP, Kureekattil RP, Hickson ID,
Bohr VA.
Laboratory of Molecular Genetics, NIA, National Institutes of Health, Baltimore,
Maryland 21224, USA.
Bloom's syndrome is a rare autosomal recessive disorder characterized by genomic
instability and predisposition to cancer. BLM, the gene defective in Bloom's
syndrome, encodes a 159-kDa protein possessing DNA-stimulated ATPase and
ATP-dependent DNA helicase activities. We have examined mechanistic aspects of
the catalytic functions of purified recombinant BLM protein. Through analyzing
the effects of different lengths of DNA cofactor on ATPase activity, we provide
evidence to suggest that BLM translocates along single-stranded DNA in a
processive manner. The helicase reaction catalyzed by BLM protein was examined
as a function of duplex DNA length. We show that BLM catalyzes unwinding of
short DNA duplexes (=71 base pairs (bp)) but is severely compromised on longer
DNA duplexes (>/=259-bp). The presence of the human single-stranded DNA-binding
protein (human replication protein A (hRPA)) stimulates the BLM unwinding
reaction on the 259-bp partial duplex DNA substrate. Heterologous
single-stranded DNA-binding proteins fail to stimulate similarly the helicase
activity of BLM protein. This is the first demonstration of a functional
interaction between BLM and another protein. Consistent with a functional
interaction between hRPA and the BLM helicase, we demonstrate a direct physical
interaction between the two proteins mediated by the 70-kDa subunit of RPA. The
interactions between BLM and hRPA suggest that the two proteins function
together in vivo to unwind DNA duplexes during replication, recombination, or
repair.
PMID: 10825162 [PubMed - indexed for MEDLINE]
470: Science 2000 May 19;288(5469):1242-4
Distinct classes of yeast promoters revealed by differential TAF recruitment.
Li XY, Bhaumik SR, Green MR.
Howard Hughes Medical Institute, Program in Molecular Medicine, University of
Massachusetts Medical School, Worcester, MA 01605, USA.
The transcription factor TFIID contains the TATA box binding protein (TBP) and
multiple TBP-associated factors (TAFs). Here, the association of TFIID
components with promoters that either are dependent on multiple TAFs (TAFdep) or
have no apparent TAF requirement (TAFind) is analyzed in yeast. At TAFdep
promoters, TAFs are present at levels comparable to that of TBP, whereas at
TAFind promoters, TAFs are present at levels that approximate background. After
inactivation of several general transcription factors, including TBP, TAFs are
still recruited by activators to TAFdep promoters. The results reveal two
classes of promoters: at TAFind promoters, TBP is recruited in the apparent
absence of TAFs, whereas at TAFdep promoters, TAFs are co-recruited with TBP in
a manner consistent with direct activator-TAF interactions.
PMID: 10817999 [PubMed - indexed for MEDLINE]
471: J Cell Biol 2000 May 15;149(4):863-74
Microtubule interactions with the cell cortex causing nuclear movements in
Saccharomyces cerevisiae.
Adames NR, Cooper JA.
Department of Cell Biology and Physiology, Washington University School of
Medicine, St. Louis, Missouri 63110, USA.
During mitosis in budding yeast the nucleus first moves to the mother-bud neck
and then into the neck. Both movements depend on interactions of cytoplasmic
microtubules with the cortex. We investigated the mechanism of these movements
in living cells using video analysis of GFP-labeled microtubules in wild-type
cells and in EB1 and Arp1 mutants, which are defective in the first and second
steps, respectively. We found that nuclear movement to the neck is largely
mediated by the capture of microtubule ends at one cortical region at the
incipient bud site or bud tip, followed by microtubule depolymerization.
Efficient microtubule interactions with the capture site require that
microtubules be sufficiently long and dynamic to probe the cortex. In contrast,
spindle movement into the neck is mediated by microtubule sliding along the bud
cortex, which requires dynein and dynactin. Free microtubules can also slide
along the cortex of both bud and mother. Capture/shrinkage of microtubule ends
also contributes to nuclear movement into the neck and can serve as a backup
mechanism to move the nucleus into the neck when microtubule sliding is
impaired. Conversely, microtubule sliding can move the nucleus into the neck
even when capture/shrinkage is impaired.
PMID: 10811827 [PubMed - indexed for MEDLINE]
472: EMBO J 2000 May 15;19(10):2323-31
The chromo domain protein chd1p from budding yeast is an ATP-dependent
chromatin-modifying factor.
Tran HG, Steger DJ, Iyer VR, Johnson AD.
Department of Biochemistry and Biophysics and Department of Microbiology and
Immunology, University of California at San Francisco, San Francisco, CA 94143,
USA.
CHD proteins are members of the chromo domain family, a class of proteins
involved in transcription, DNA degradation and chromatin structure. In higher
eukaryotes, there are two distinct subfamilies of CHD proteins: CHD1 and CHD3/4.
Analyses carried out in vitro indicate that the CHD3/4 proteins may regulate
transcription via alteration of chromatin structure. However, little is known
about the role of CHD proteins in vivo, particularly the CHD1 subfamily. To
understand better the cellular function of CHD proteins, we initiated a study on
the Chd1p protein from budding yeast. Using genomic DNA arrays, we identified
genes whose expression is affected by the absence of Chd1p. A synthetic-lethal
screen uncovered genetic interactions between SWI/SNF genes and CHD1.
Biochemical experiments using Chd1p purified from yeast showed that it
reconfigures the structure of nucleosome core particles in a manner distinct
from the SWI-SNF complex. Taken together, these results suggest that Chd1p
functions as a nucleosome remodeling factor, and that Chd1p may share
overlapping roles with the SWI-SNF complex to regulate transcription.
PMID: 10811623 [PubMed - indexed for MEDLINE]
473: Biochem Pharmacol 2000 Jan 15;59(2):177-85
Agonistic and synergistic activity of tamoxifen in a yeast model system.
Graumann K, Jungbauer A.
Institute for Applied Microbiology, University of the Agricultural Sciences,
Vienna, Austria.
The background of agonist/antagonist behaviour of the non-steroidal antiestrogen
tamoxifen is still not fully understood. Depending on cell type, its activities
range from full agonistic to antagonistic in different tissues. We investigated
the transactivational properties of tamoxifen in a basic yeast model system
which reconstitutes ligand-dependent human estrogen receptor-alpha (hER alpha)
gene activation. Tamoxifen exerted low agonist activity in this system compared
to 17 beta-estradiol (E2). Efficiencies and potencies of several isomers were
calculated by fitting experimental data with a logistic dose-response function.
Cis-, trans- and cis-transtamoxifen and trans-4-hydroxytamoxifen (4-OHT) showed
comparable efficiencies and potencies in yeast. When subeffective doses of
trans-, cis-/trans-, or trans-4-OH tamoxifen were combined with increasing 17
beta-estradiol concentrations, even a synergistic increase in efficiencies could
be observed. Interestingly, the cis-isomer did not show this synergistic effect.
Mutation of the N-terminus of the estrogen receptor changed the
transactivational behaviour of tamoxifen and abolished the synergistic action
with 17 beta-estradiol. Except for 4-OHT, the potencies of the investigated
isomers, defined as ligand concentrations with half-maximal response, highly
correlated with the binding affinities to hER alpha. Therefore, cis-, trans-,
and cis-/trans-tamoxifen could be regarded as full agonists in yeast, while
4-OHT was regarded as a partial antagonist in yeast. Furthermore, these results
indicate that the functional difference between trans-tamoxifen and trans-4-OHT
is not due to their different affinities for the receptor protein.
PMID: 10810452 [PubMed - indexed for MEDLINE]
474: J Biol Chem 2000 May 19;275(20):15350-6
Intramolecular interactions between the juxtamembrane domain and phosphatase
domains of receptor protein-tyrosine phosphatase RPTPmu. Regulation of catalytic
activity.
Feiken E, van Etten I, Gebbink MF, Moolenaar WH, Zondag GC.
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan
121, 1066 CX Amsterdam, The Netherlands.
RPTPmu is a receptor-like protein-tyrosine phosphatase (RPTP) whose ectodomain
mediates homotypic cell-cell interactions. The intracellular part of RPTPmu
contains a relatively long juxtamembrane domain (158 amino acids; aa) and two
conserved phosphatase domains (C1 and C2). The membrane-proximal C1 domain is
responsible for the catalytic activity of RPTPmu, whereas the membrane-distal C2
domain serves an unknown function. The regulation of RPTP activity remains
poorly understood, although dimerization has been proposed as a general
mechanism of inactivation. Using the yeast two-hybrid system, we find that the
C1 domain binds to an N-terminal noncatalytic region in RPTPmu, termed JM (aa
803-955), consisting of a large part of the juxtamembrane domain (120 aa) and a
small part of the C1 domain (33 aa). When co-expressed in COS cells, the JM
polypeptide binds to both the C1 and the C2 domain. Strikingly, the isolated JM
polypeptide fails to interact with either full-length RPTPmu or with truncated
versions of RPTPmu that contain the JM region, consistent with the JM-C1 and
JM-C2 interactions being intramolecular rather than intermolecular. Furthermore,
we find that large part of the juxtamembrane domain (aa 814-922) is essential
for C1 to be catalytically active. Our findings suggest a model in which RPTPmu
activity is regulated by the juxtamembrane domain undergoing intramolecular
interactions with both the C1 and C2 domain.
PMID: 10809770 [PubMed - indexed for MEDLINE]
475: J Biol Chem 2000 May 19;275(20):14979-84
Peptides selected to bind the Gal80 repressor are potent transcriptional
activation domains in yeast.
Han Y, Kodadek T.
Departments of Internal Medicine and Biochemistry, Center for Biomedical
Inventions, Ryburn Center for Molecular Cardiology, University of Texas
Southwestern Medical Center, Dallas, Texas 75235-8573, USA.
The activation domain of the yeast Gal4 protein binds specifically to the Gal80
repressor and is also thought to associate with one or more coactivators in the
RNA polymerase II holoenzyme and chromatin remodeling machines. This is a
specific example of a common situation in biochemistry where a single protein
domain can interact with multiple partners. Are these different interactions
related chemically? To probe this point, phage display was employed to isolate
peptides from a library based solely on their ability to bind Gal80 protein in
vitro. Peptide-Gal80 protein association is shown to be highly specific and of
moderate affinity. The Gal80 protein-binding peptides compete with the native
activation domain for the repressor, suggesting that they bind to the same site.
It was then asked if these peptides could function as activation domains in
yeast when tethered to a DNA binding domain. Indeed, this is the case.
Furthermore, one of the Gal80-binding peptides binds directly to a domain of the
Gal11 protein, a known coactivator. The fact that Gal80-binding peptides are
functional activation domains argues that repressor binding and
activation/coactivator binding are intimately related properties. This peptide
library-based approach should be generally useful for probing the chemical
relationship of different binding interactions or functions of a given native
domain.
PMID: 10809742 [PubMed - indexed for MEDLINE]
476: Mol Endocrinol 2000 May;14(5):718-32
GCN5 and ADA adaptor proteins regulate triiodothyronine/GRIP1 and SRC-1
coactivator-dependent gene activation by the human thyroid hormone receptor.
Anafi M, Yang YF, Barlev NA, Govindan MV, Berger SL, Butt TR, Walfish PG.
Samuel Lunenfeld Research Institute, University of Toronto Medical School, Mount
Sinai Hospital, Ontario, Canada.
We have used yeast genetics and in vitro protein-protein interaction experiments
to explore the possibility that GCN5 (general control nonrepressed protein 5)
and several other ADA (alteration/deficiency in activation) adaptor proteins of
the multimeric SAGA complex can regulate T3/GRIP1 (glucocorticoid receptor
interacting protein 1) and SRC-1 (steroid receptor coactivator-1)
coactivator-dependent activation of transcription by the human T3 receptor beta1
(hTRbeta1). Here, we show that in vivo activation of a T3/GRIP1 or SRC-1
coactivator-dependent T3 hormone response element by hTRbeta1 is dependent upon
the presence of yeast GCN5, ADA2, ADA1, or ADA3 adaptor proteins and that the
histone acetyltransferase (HAT) domains and bromodomain (BrD) of yGCN5 must be
intact for maximal activation of transcription. We also observed that hTRbeta1
can bind directly to yeast or human GCN5 as well as hADA2, and that the
hGCN5(387-837) sequence could bind directly to either GRIP1 or SRC-1
coactivator. Importantly, the T3-dependent binding of hTRbeta1 to hGCN5(387-837)
could be markedly increased by the presence of GRIP1 or SRC1. Mutagenesis of
GRIP1 nuclear receptor (NR) Box II and III LXXLL motifs also substantially
decreased both in vivo activation of transcription and in vitro T3-dependent
binding of hTRbeta1 to hGCN5. Taken together, these experiments support a
multistep model of transcriptional initiation wherein the binding of T3 to
hTRbeta1 initiates the recruitment of p160 coactivators and GCN5 to form a
trimeric transcriptional complex that activates target genes through
interactions with ADA/SAGA adaptor proteins and nucleosomal histones.
PMID: 10809234 [PubMed - indexed for MEDLINE]
477: Proc Natl Acad Sci U S A 2000 May 9;97(10):5267-72
Distinct roles of the NH2- and COOH-terminal domains of the protein inhibitor of
activated signal transducer and activator of transcription (STAT) 1 (PIAS1) in
cytokine-induced PIAS1-Stat1 interaction.
Liao J, Fu Y, Shuai K.
Division of Hematology-Oncology, Department of Medicine, University of
California, Los Angeles, CA 90095, USA.
STATs are activated by tyrosine phosphorylation on cytokine stimulation. A
tyrosine-phosphorylated STAT forms a functional dimer through reciprocal Src
homology 2 domain (SH2)-phosphotyrosyl peptide interactions. IFN treatment
induces the association of PIAS1 and Stat1, which results in the inhibition of
Stat1-mediated gene activation. The molecular basis of the cytokine-dependent
PIAS1-Stat1 interaction has not been understood. We report here that a region
near the COOH terminus of PIAS1 (amino acids 392-541) directly interacts with
the NH(2)-terminal domain of Stat1 (amino acids 1-191). A mutant PIAS1 lacking
the Stat1-interacting domain failed to inhibit Stat1-mediated gene activation.
By using a modified yeast two-hybrid assay, we demonstrated that PIAS1
specifically interacts with the Stat1 dimer, but not tyrosine-phosphorylated or
-unphosphorylated Stat1 monomer. In addition, whereas the NH(2)-terminal region
of PIAS1 does not interact with Stat1, it serves as a modulatory domain by
preventing the interaction of the COOH-terminal domain of PIAS1 with the Stat1
monomer. Thus, the cytokine-induced PIAS1-Stat1 interaction is mediated through
the specific recognition of the dimeric form of Stat1 by PIAS1.
PMID: 10805787 [PubMed - indexed for MEDLINE]
478: Mol Cell Biol 2000 Jun;20(11):3965-76
Identification of domains and residues within the epsilon subunit of eukaryotic
translation initiation factor 2B (eIF2Bepsilon) required for guanine nucleotide
exchange reveals a novel activation function promoted by eIF2B complex
formation.
Gomez E, Pavitt GD.
Department of Anatomy and Physiology, Medical Sciences Institute, University of
Dundee, Dundee, United Kingdom.
Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide
exchange factor for protein synthesis initiation factor 2 (eIF2). Composed of
five subunits, it converts eIF2 from a GDP-bound form to the active eIF2-GTP
complex. This is a regulatory step of translation initiation. In vitro, eIF2B
catalytic function can be provided by the largest (epsilon) subunit alone
(eIF2Bepsilon). This activity is stimulated by complex formation with the other
eIF2B subunits. We have analyzed the roles of different regions of eIF2Bepsilon
in catalysis, in eIF2B complex formation, and in binding to eIF2 by
characterizing mutations in the Saccharomyces cerevisiae gene encoding
eIF2Bepsilon (GCD6) that impair the essential function of eIF2B. Our analysis of
nonsense mutations indicates that the C terminus of eIF2Bepsilon (residues 518
to 712) is required for both catalytic activity and interaction with eIF2. In
addition, missense mutations within this region impair the catalytic activity of
eIF2Bepsilon without affecting its ability to bind eIF2. Internal, in-frame
deletions within the N-terminal half of eIF2Bepsilon disrupt eIF2B complex
formation without affecting the nucleotide exchange activity of eIF2Bepsilon
alone. Finally, missense mutations identified within this region do not affect
the catalytic activity of eIF2Bepsilon alone or its interactions with the other
eIF2B subunits or with eIF2. Instead, these missense mutations act indirectly by
impairing the enhancement of the rate of nucleotide exchange that results from
complex formation between eIF2Bepsilon and the other eIF2B subunits. This
suggests that the N-terminal region of eIF2Bepsilon is an activation domain that
responds to eIF2B complex formation.
PMID: 10805739 [PubMed - indexed for MEDLINE]
479: Nat Struct Biol 2000 May;7(5):375-9
Structural analysis of WW domains and design of a WW prototype.
Macias MJ, Gervais V, Civera C, Oschkinat H.
Forschungsinstitut fur Molekulare Pharmakologie, Alfred-Kowalke-Str. 4, 10315
Berlin, Germany. macias@EMBL-Heidelberg.de
Two new NMR structures of WW domains, the mouse formin binding protein and a
putative 84.5 kDa protein from Saccharomyces cerevisiae, show that this domain,
only 35 amino acids in length, defines the smallest monomeric triple-stranded
antiparallel beta-sheet protein domain that is stable in the absence of
disulfide bonds, tightly bound ions or ligands. The structural roles of
conserved residues have been studied using site-directed mutagenesis of both
wild type domains. Crucial interactions responsible for the stability of the WW
structure have been identified. Based on a network of highly conserved long
range interactions across the beta-sheet structure that supports the WW fold and
on a systematic analysis of conserved residues in the WW family, we have
designed a folded prototype WW sequence.
PMID: 10802733 [PubMed - indexed for MEDLINE]
480: J Biol Chem 2000 Jul 21;275(29):22470-8
An actin subdomain 2 mutation that impairs thin filament regulation by troponin
and tropomyosin.
Korman VL, Hatch V, Dixon KY, Craig R, Lehman W, Tobacman LS.
Departments of Biochemistry and Internal Medicine, University of Iowa, College
of Medicine, Iowa City, Iowa 52242, USA.
Striated muscle thin filaments adopt different quaternary structures, depending
upon calcium binding to troponin and myosin binding to actin. Modification of
actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting
that this region of actin may contain important protein-protein interaction
sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation
increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+)
to mutant actin filaments containing troponin-tropomyosin produced little
increase in the thin filament-myosin S1 MgATPase rate. Despite this,
three-dimensional reconstruction of electron microscope images of filaments in
the presence of troponin and Ca(2+) showed tropomyosin to be in a position
similar to that found for muscle actin filaments, where most of the myosin
binding site is exposed. Troponin-tropomyosin bound with comparable affinity to
mutant and wild type actin in the absence and presence of calcium, and in the
presence of myosin S1, tropomyosin bound very tightly to both types of actin.
The mutation decreased actin-myosin S1 affinity 13-fold in the presence of
troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The
results suggest the importance of negatively charged actin subdomain 2 residues
56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and
for regulatory conformational changes in the actin-troponin-tropomyosin complex.
PMID: 10801864 [PubMed - indexed for MEDLINE]
481: Curr Opin Cell Biol 2000 Jun;12(3):361-71
The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm.
Ryan KJ, Wente SR.
Department of Cell Biology and Physiology, Washington University School of
Medicine, Box 8228, St Louis, MO 63110, USA. kryan@cellbio. wustl.edu
Compositional analysis ofnuclear pore complexes (NPCs) is nearing completion,
and efforts are now focused on understanding how these protein machines work.
Recent analysis of soluble transport factor interactions with NPC proteins
reveals distinct and overlapping pathways for movement between the nucleus and
cytoplasm. New fluorescence- and microscopy-based strategies have been used to
monitor the pathway of NPC assembly and to reveal the dynamics of the NPC during
transport.
Publication Types:
Review
Review, Tutorial
PMID: 10801463 [PubMed - indexed for MEDLINE]
482: Biochem Biophys Res Commun 2000 May 10;271(2):464-8
Purification and polymerization properties of two lethal yeast actin mutants.
Frieden C, Du J, Schriefer L, Buzan J.
Department of Biochemistry and Molecular Biophysics, Washington University
School of Medicine, St. Louis, Missouri, 63110, USA. frieden@biochem.wustl.edu
The budding yeast Saccharomyces cerevisiae contains a single actin gene and the
gene product, actin, is essential for growth. Two mutants of yeast actin that do
not support yeast growth were prepared from yeast by coexpressing the mutant and
a 6-histidine-tagged wild-type actin followed by separation of the wild-type and
mutant actin using Ni-NTA chromatography as described elsewhere [Buzan, J., Du,
J., Karpova, T., and Frieden, C. (1999) Proc. Natl. Acad. Sci. USA 96,
2823-2827]. The mutations, in muscle actin numbering, were at positions 334
(Glu334Lys) and 168 (Gly168Arg) and were chosen based on phenotypic changes
observed in the behavior of actin mutants of Caenorhabditis elegans. Glu334 is
located on the surface of actin between subdomains 1 and 3 while Gly168 is
located in a region near actin-actin contacts in the actin filament. The
Glu334Lys mutant polymerized slightly faster than wild-type yeast actin,
suggesting that loss of interactions with some actin binding protein, rather
than loss of actin-actin contacts, was responsible for its inability to support
yeast growth. The Gly168Arg mutant polymerized at a rate similar to wild-type
but the extent was considerably less, kinetic characteristics suggesting a high
critical concentration (ca. 4 microM) without a large change in the ability to
form nuclei for the nucleation-elongation process. Copyright 2000 Academic
Press.
PMID: 10799320 [PubMed - indexed for MEDLINE]
483: Mol Biol Cell 2000 May;11(5):1753-64
The yeast heat shock transcription factor changes conformation in response to
superoxide and temperature.
Lee S, Carlson T, Christian N, Lea K, Kedzie J, Reilly JP, Bonner JJ.
Departments of Biology and Chemistry, Indiana University, Bloomington, Indiana
47405-3700, USA.
In vitro DNA-binding assays demonstrate that the heat shock transcription factor
(HSF) from the yeast Saccharomyces cerevisiae can adopt an altered conformation
when stressed. This conformation, reflected in a change in electrophoretic
mobility, requires that two HSF trimers be bound to DNA. Single trimers do not
show this change, which appears to represent an alteration in the cooperative
interactions between trimers. HSF isolated from stressed cells displays a higher
propensity to adopt this altered conformation. Purified HSF can be stimulated in
vitro to undergo the conformational change by elevating the temperature or by
exposing HSF to superoxide anion. Mutational analysis maps a region critical for
this conformational change to the flexible loop between the minimal DNA-binding
domain and the flexible linker that joins the DNA-binding domain to the
trimerization domain. The significance of these findings is discussed in the
context of the induction of the heat shock response by ischemic stroke, hypoxia,
and recovery from anoxia, all known to stimulate the production of superoxide.
PMID: 10793149 [PubMed - indexed for MEDLINE]
484: Cell Struct Funct 2000 Feb;25(1):11-20
Overexpression of PRA2, a Rab/Ypt-family small GTPase from Pea Pisum sativum,
aggravates the growth defect of yeast ypt mutants.
Matsuda N, Ueda T, Sasaki Y, Nakano A.
Molecular Membrane Biology Laboratory, RIKEN, Wako, Saitama, Japan.
A large number of Rab/Ypt-family small GTPases have been identified from higher
plants. While some of them can complement yeast ypt mutants, the expression of
Arabidopsis Ara4 protein aggravated the growth defect of a subset of ypt
mutants, probably because of the titration of common regulator(s) of yeast Ypt
proteins [Ueda, T. et al. (1996) Plant Cell, 8: 2079-20911. PRA2 from pea Pisum
sativum encodes an interesting Rab GTPase whose expression is regulated by light
[Yoshida, K. et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 6636-6640]. We
examined whether PRA2 complements any of the yeast ypt mutants and found again
that PRA2 does not complement but rather confers the growth defect to some of
the ypt mutants. No growth defect was observed when PRA2 was expressed in the
wild-type yeast cells. Unlike the case of Ara4, neither Arabidopsis nor yeast
GDI remedied the growth defect by Pra2, indicating that the mechanism of the
exacerbation is different. Mutational analysis of PRA2 suggests that the growth
inhibition can be ascribed to unidentified factor(s) which prefers the GTP-bound
form of Pra2. This yeast system will be useful for identifying such putative
regulatory factor(s) from yeast and plants and analyzing their interactions with
Pra2.
PMID: 10791890 [PubMed - indexed for MEDLINE]
485: Genetics 2000 May;155(1):69-83
Genetic interactions between GLC7, PPZ1 and PPZ2 in saccharomyces cerevisiae.
Venturi GM, Bloecher A, Williams-Hart T, Tatchell K.
Department of Biochemistry and Molecular Biology, Louisiana State University
Medical Center, Shreveport, Louisiana 71130, USA.
GLC7 encodes an essential serine/threonine protein type I phosphatase in
Saccharomyces cerevisiae. Three other phosphatases (Ppz1p, Ppz2p, and Sal6p)
share >59% identity in their catalytic region with Glc7p. ppz1 ppz2 null mutants
have no apparent growth defect on rich media. However, null alleles of PPZ1 and
PPZ2, in combination with mutant alleles of GLC7, confer a range of growth
defects varying from slow growth to lethality. These results indicate that
Glc7p, Ppz1p, and Ppz2p may have overlapping functions. To determine if this
overlap extends to interaction with targeting subunits, Glc7p-binding proteins
were tested for interaction in the two-hybrid system with the functional
catalytic domain of Ppz1p. Ppz1p interacts strongly with a number of Glc7p
regulatory subunits, including Glc8p, a protein that shares homology with
mammalian PP1 inhibitor I2. Genetic data suggest that Glc8p positively affects
both Glc7p and Ppz1p functions. Together our data suggest that Ppz1p and Ppz2p
may have overlapping functions with Glc7p and that all three phosphatases may
act through common regulatory proteins.
PMID: 10790385 [PubMed - indexed for MEDLINE]
486: RNA 2000 Apr;6(4):638-50
REF, an evolutionary conserved family of hnRNP-like proteins, interacts with
TAP/Mex67p and participates in mRNA nuclear export.
Stutz F, Bachi A, Doerks T, Braun IC, Seraphin B, Wilm M, Bork P, Izaurralde E.
Institute of Microbiology, Lausanne, Switzerland.
Vertebrate TAP and its yeast ortholog Mex67p are involved in the export of
messenger RNAs from the nucleus. TAP has also been implicated in the export of
simian type D viral RNAs bearing the constitutive transport element (CTE).
Although TAP directly interacts with CTE-bearing RNAs, the mode of interaction
of TAP/Mex67p with cellular mRNAs is different from that with the CTE RNA and is
likely to be mediated by protein-protein interactions. Here we show that Mex67p
directly interacts with Yra1p, an essential yeast hnRNP-like protein. This
interaction is evolutionarily conserved as Yra1p also interacts with TAP.
Conditional expression in yeast cells implicates Yra1 p in the export of
cellular mRNAs. Database searches revealed that Yra1p belongs to an
evolutionarily conserved family of hnRNP-like proteins having more than one
member in Mus musculus, Xenopus laevis, Caenorhabditis elegans, and
Schizosaccharomyces pombe and at least one member in several species including
plants. The murine members of the family directly interact with TAP. Because
members of this protein family are characterized by the presence of one
RNP-motif RNA-binding domain and exhibit RNA-binding activity, we called these
proteins REF-bps for RNA and export factor binding proteins. Thus, Yra1p and
members of the REF family of hnRNP-like proteins may facilitate the interaction
of TAP/Mex67p with cellular mRNAs.
PMID: 10786854 [PubMed - indexed for MEDLINE]
487: Cell 2000 Apr 14;101(2):223-33
A kaiC-interacting sensory histidine kinase, SasA, necessary to sustain robust
circadian oscillation in cyanobacteria.
Iwasaki H, Williams SB, Kitayama Y, Ishiura M, Golden SS, Kondo T.
Division of Biological Science, Graduate School of Science, Nagoya University,
Japan.
Both regulated expression of the clock genes kaiA, kaiB, and kaiC and
interactions among the Kai proteins are proposed to be important for circadian
function in the cyanobacterium Synechococcus sp. strain PCC 7942. We have
identified the histidine kinase SasA as a KaiC-interacting protein. SasA
contains a KaiB-like sensory domain, which appears sufficient for interaction
with KaiC. Disruption of the sasA gene lowered kaiBC expression and dramatically
reduced amplitude of the kai expression rhythms while shortening the period.
Accordingly, sasA disruption attenuated circadian expression patterns of all
tested genes, some of which became arrhythmic. Continuous sasA overexpression
eliminated circadian rhythms, whereas temporal overexpression changed the phase
of kaiBC expression rhythm. Thus, SasA is a close associate of the
cyanobacterial clock that is necessary to sustain robust circadian rhythms.
PMID: 10786837 [PubMed - indexed for MEDLINE]
488: Eur J Biochem 2000 May;267(9):2680-7
Uncoupling proteins 2 and 3 interact with members of the 14.3.3 family.
Pierrat B, Ito M, Hinz W, Simonen M, Erdmann D, Chiesi M, Heim J.
Novartis Pharma Inc., Basle, Switzerland.
Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial
anion carrier proteins (MATP). Localized in the inner membrane of the organelle,
they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may
play an important role in the regulation of thermogenesis and, thus, on the
resting metabolic rate in humans. To identify interacting proteins that may be
involved in the regulation of the activity of UCPs, the yeast two-hybrid system
was applied. Segments of hUCP2 containing the hydrophilic loops facing the
intermembrane space, or combinations of these, were used to screen an adipocyte
activation domain (AD) fusion library. The 14.3.3 protein isoforms theta, beta,
zeta were identified as possible interacting partners of hUCP2. Screening of a
human skeletal muscle AD fusion library, on the other hand, yielded several
clones all of them encoding the gamma isoform of the 14.3.3 family. Mapping
experiments further revealed that all these 14.3.3 proteins interact
specifically with the C-terminal intermembrane space domain of both hUCP2 and
hUCP3 whereas no interactions could be detected with the C-terminal part of
hUCP1. Direct interaction between UCP3 and 14.3.3 theta could be demonstrated
after in vitro translation by coimmunoprecipitation. When coexpressed in a
heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of
UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins
could be involved in the targeting of UCPs to the mitochondria.
PMID: 10785390 [PubMed - indexed for MEDLINE]
489: J Biol Chem 2000 Jul 7;275(27):20562-71
Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA
polymerase II.
Chang A, Cheang S, Espanel X, Sudol M.
Department of Biochemistry and Molecular Biology, New York University/Mount
Sinai School of Medicine, New York, New York 10029, USA.
RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to
form a physical and functional complex with RNA polymerase II (RNA pol II). The
amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds
membrane phospholipids; and three WW domains, which are protein interaction
modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5
contains a HECT (homologous to E6-AP carboxyl terminus) domain that
catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as
an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as
membrane localization and substrate recognition modules, respectively. We report
that the second (and possibly third) Rsp5 WW domain mediates binding to the
carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD
comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical
for interaction with a specific group of WW domains. An analysis of synthetic
peptides revealed a minimal CTD sequence that is sufficient to bind to the
second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays.
Furthermore, we found that specific "imperfect" CTD repeats can form a complex
with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal
CTD sequence on serine, threonine and tyrosine residues acts as a negative
regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining
to phosphorylation-driven interactions between the RNA pol II CTD and the WW
domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite
for targeted RNA pol II degradation.
PMID: 10781604 [PubMed - indexed for MEDLINE]
490: Mol Cell Biol 2000 May;20(10):3597-607
Phospholipase C is involved in kinetochore function in Saccharomyces cerevisiae.
Lin H, Choi JH, Hasek J, DeLillo N, Lou W, Vancura A.
Department of Biological Sciences, St. John's University, Jamaica, New York
11439, USA.
The budding yeast PLC1 gene encodes a homolog of the delta isoform of mammalian
phosphoinositide-specific phospholipase C. Here, we present evidence that Plc1p
associates with the kinetochore complex CBF3. This association is mediated
through interactions with two established kinetochore proteins, Ndc10p and
Cep3p. We show by chromatin immunoprecipitation experiments that Plc1p resides
at centromeric loci in vivo. Deletion of PLC1, as well as plc1 mutations which
abrogate the interaction of Plc1p with the CBF3 complex, results in a higher
frequency of minichromosome loss, nocodazole sensitivity, and mitotic delay.
Overexpression of Ndc10p suppresses the nocodazole sensitivity of plc1 mutants,
implying that the association of Plc1p with CBF3 is important for optimal
kinetochore function. Chromatin extracts from plc1Delta cells exhibit reduced
microtubule binding to minichromosomes. These results suggest that Plc1p
associates with kinetochores and regulates some aspect of kinetochore function
and demonstrate an intranuclear function of phospholipase C in eukaryotic cells.
PMID: 10779349 [PubMed - indexed for MEDLINE]
491: Mol Cell Biol 2000 May;20(10):3538-49
Deletion of the PAT1 gene affects translation initiation and suppresses a PAB1
gene deletion in yeast.
Wyers F, Minet M, Dufour ME, Vo LT, Lacroute F.
Centre de Genetique Moleculaire, C.N.R.S., 91198 Gif sur Yvette, France.
wyers@cgm.cnrs-gif.fr
The yeast poly(A) binding protein Pab1p mediates the interactions between the 5'
cap structure and the 3' poly(A) tail of mRNA, whose structures synergistically
activate translation in vivo and in vitro. We found that deletion of the PAT1
(YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for
the normal initiation of translation. A fraction of Pat1p cosediments with free
40S ribosomal subunits on sucrose gradients. The PAT1 gene is not essential for
viability, although disruption of the gene severely impairs translation
initiation in vivo, resulting in the accumulation of 80S ribosomes and in a
large decrease in the amounts of heavier polysomes. Pat1p contributes to the
efficiency of translation in a yeast cell-free system. However, the synergy
between the cap structure and the poly(A) tail is maintained in vitro in the
absence of Pat1p. Analysis of translation initiation intermediates on gradients
indicates that Pat1p acts at a step before or during the recruitment of the 40S
ribosomal subunit by the mRNA, a step which may be independent of that involving
Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and
that Pat1p might be required for promoting the formation or the stabilization of
the preinitiation translation complexes.
PMID: 10779343 [PubMed - indexed for MEDLINE]
492: J Biol Chem 2000 Apr 28;275(17):12926-33
Reversible transdominant inhibition of a metabolic pathway. In vivo evidence of
interaction between two sequential tricarboxylic acid cycle enzymes in yeast.
Velot C, Srere PA.
Research Service of the Department of Veterans Affairs Medical Center, Dallas,
Texas 75216, USA. poffenb1@airmail.net
The enzymes of the Krebs tricarboxylic acid cycle in mitochondria are proposed
to form a supramolecular complex, in which there is channeling of intermediates
between enzyme active sites. While interactions have been demonstrated in vitro
between most of the sequential tricarboxylic acid cycle enzymes, no direct
evidence has been obtained in vivo for such interactions. We have isolated, in
the Saccharomyces cerevisiae gene encoding the tricarboxylic acid cycle enzyme
citrate synthase Cit1p, an "assembly mutation," i.e. a mutation that causes a
tricarboxylic acid cycle deficiency without affecting the citrate synthase
activity. We have shown that a 15-amino acid peptide from wild type Cit1p
encompassing the mutation point inhibits the tricarboxylic acid cycle in a
dominant manner, and that the inhibitory phenotype is overcome by a
co-overexpression of Mdh1p, the mitochondrial malate dehydrogenase. These data
provide the first direct in vivo evidence of interaction between two sequential
tricarboxylic acid cycle enzymes, Cit1p and Mdh1p, and indicate that the
characterization of assembly mutations by the reversible transdominant
inhibition method may be a powerful way to study multienzyme complexes in their
physiological context.
PMID: 10777592 [PubMed - indexed for MEDLINE]
493: Nucleic Acids Res 2000 May 15;28(10):2060-8
DNA repair in a yeast origin of replication: contributions of photolyase and
nucleotide excision repair.
Suter B, Wellinger RE, Thoma F.
Institut fur Zellbiologie, ETH-Zurich, Honggerberg, CH-8093 Zurich, Switzerland.
DNA damage formation and repair are tightly linked to protein-DNA interactions
in chromatin. We have used minichromosomes in yeast as chromatin substrates in
vivo to investigate how nucleotide excision repair (NER) and repair by
DNA-photolyase (photoreactivation) remove pyrimidine dimers from an origin of
replication ( ARS1 ). The ARS1 region is nuclease sensitive and flanked by
nucleosomes on both sides. Photoreactivation was generally faster than NER at
all sites. Site-specific heterogeneity of repair was observed for both pathways.
This heterogeneity was different for NER and photoreactivation and it was
altered in a minichromosome where ARS1 was transcribed. The results indicate
distinct inter-actions of the repair systems with protein complexes bound in the
ARS region (ORC, Abf1) and a predominant role of photolyase in CPD repair of an
origin of replication.
PMID: 10773073 [PubMed - indexed for MEDLINE]
494: Nucleic Acids Res 2000 May 15;28(10):2049-59
Domain specific interaction in the XRCC1-DNA polymerase beta complex.
Marintchev A, Robertson A, Dimitriadis EK, Prasad R, Wilson SH, Mullen GP.
Department of Biochemistry, University of Connecticut Health Center, 263
Farmington Avenue, Farmington, CT 06032, USA.
XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms
complexes with DNA polymerase beta (beta-Pol), DNA ligase III and
poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The
domains in XRCC1 have been determined, and characterization of the domain-domain
interaction in the XRCC1-beta-Pol complex has provided information on the
specificity and mechanism of binding. The domain structure of XRCC1, determined
using limited proteolysis, was found to include an N-terminal domain (NTD), a
central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal
BRCT-II domain. The BRCT-I-linker-BRCT-II C-terminal fragment and the
linker-BRCT-II C-terminal fragment were relatively stable to proteolysis
suggestive of a non-random conformation of the linker. A predicted inner domain
was found not to be stable to proteolysis. Using cross-linking experiments,
XRCC1 was found to bind intact beta-Pol and the beta-Pol 31 kDa domain. The
XRCC1-NTD(1-183)(residues 1-183) was found to bind beta-Pol, the beta-Pol 31 kDa
domain and the beta-Pol C-terminal palm-thumb (residues 140-335), and the
interaction was further localized to XRCC1-NTD(1-157)(residues 1-157). The
XRCC1-NTD(1-183)-beta-Pol 31 kDa domain complex was stable at high salt (1 M
NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen,
polypeptides expressed from two XRCC1 constructs, which included residues 36-355
and residues 1-159, were found to interact with beta-Pol, the beta-Pol 31 kDa
domain, and the beta-Pol C-terminal thumb-only domain polypeptides expressed
from the respective beta-Pol constructs. Neither the XRCC1-NTD(1-159), nor the
XRCC1(36-355)polypeptide was found to interact with a beta-Pol thumbless
polypeptide. A third XRCC1 polypeptide (residues 75-212) showed no interaction
with beta-Pol. In quantitative gel filtration and analytical ultracentrifugation
experiments, the XRCC1-NTD(1-183)was found to bind beta-Pol and its 31 kDa
domain in a 1:1 complex with high affinity (K(d) of 0.4-2.4 microM). The
combined results indicate a thumb-domain specific 1:1 interaction between the
XRCC1-NTD(1-159)and beta-Pol that is of an affinity comparable to other binding
interactions involving beta-Pol.
PMID: 10773072 [PubMed - indexed for MEDLINE]
495: Biochemistry 2000 Apr 25;39(16):4869-80
Participation of the amino-terminal domain in the self-association of the
full-length yeast TATA binding protein.
Daugherty MA, Brenowitz M, Fried MG.
Department of Biochemistry, The Pennsylvania State University College of
Medicine, Hershey, Pennsylvania 17033, USA.
The association of monomeric TATA binding protein with promoter DNA is an
essential first step in many current models of eukaryotic transcription
initiation. This step is followed by others in which additional transcription
factors, and finally RNA polymerase, assemble at the promoter. Here we
characterize the quaternary interactions of the Saccharomyces cerevisiae
TATA-binding protein (yTBP), in the absence of other proteins or DNA. The data
reveal a robust pattern in which yTBP monomers equilibrate with tetramers and
octamers over a broad span of temperatures (4 degrees C = T = 37 degrees C)
and salt concentrations (60 mM = [KCl] = 1 M), that includes the
physiological range. Association is highly cooperative, with octamer formation
favored by approximately 9 kcal/mol over tetramer formation. Changes in
association constant with [KCl] are consistent with an assembly-linked release
of ions at low salt and an assembly-linked uptake of ions at high salt, for both
monomer right arrow over left arrow tetramer and tetramer right arrow over left
arrow octamer reaction steps. Fluorescence emission spectra and steady-state
anisotropies reveal that the amino-terminal domain changes conformation and
dynamics at both association steps and that the polarity of the environment near
tryptophan 26 is sensitive to changes in [KCl] in the monomeric and tetrameric
states but not the octameric state. These results are consistent with a
[salt]-dependent change in the assembly mechanism near 300 mM KCl and suggest
that the amino-terminal domain may modulate the self-association of the
full-length protein. TBP self-association may regulate many of its cellular
functions, including transit of the nuclear membrane and participation in
transcription initiation.
PMID: 10769145 [PubMed - indexed for MEDLINE]
496: J Cell Biol 2000 Apr 17;149(2):249-54
The transcriptional coactivator CBP interacts with beta-catenin to activate gene
expression.
Takemaru KI, Moon RT.
Howard Hughes Medical Institute, Department of Pharmacology, and Center for
Developmental Biology, University of Washington School of Medicine, Seattle,
Washington 98195, USA.
Beta-catenin plays a pivotal role in the transcriptional activation of
Wnt-responsive genes by binding to TCF/LEF transcription factors. Although it
has been suggested that the COOH-terminal region of beta-catenin functions as an
activation domain, the mechanisms of activation remain unclear. To screen for
potential transcriptional coactivators that bind to the COOH-terminal region of
beta-catenin, we used a novel yeast two-hybrid system, the Ras recruitment
system (RRS) that detects protein-protein interactions at the inner surface of
the plasma membrane. Using this system, we isolated the CREB-binding protein
(CBP). Armadillo (Arm) repeat 10 to the COOH terminus of beta-catenin is
involved in binding to CBP, whereas beta-catenin interacts directly with the
CREB-binding domain of CBP. Beta-catenin synergizes with CBP to stimulate the
activity of a synthetic reporter in vivo. Conversely, beta-catenin-dependent
transcriptional activation is repressed by E1A, an antagonist of CBP function,
but not by an E1A mutant that does not bind to CBP. The activation of Wnt target
genes such as siamois and Xnr3 in Xenopus embryos is also sensitive to E1A.
These findings suggest that CBP provides a link between beta-catenin and the
transcriptional machinery, and possibly mediates the oncogenic function of
beta-catenin.
PMID: 10769018 [PubMed - indexed for MEDLINE]
497: J Biol Chem 2000 Jun 23;275(25):19288-96
Structure-function analysis of the dolichyl phosphate-mannose: protein
O-mannosyltransferase ScPmt1p.
Girrbach V, Zeller T, Priesmeier M, Strahl-Bolsinger S.
Lehrstuhl fur Zellbiologie und Pflanzenphysiologie, Universitat Regensburg,
93040 Regensburg, Germany.
Protein O-mannosylation is an essential protein modification. It is initiated at
the endoplasmic reticulum by a family of dolichyl phosphate-mannose:protein
O-mannosyltransferases (Pmts), which is evolutionarily conserved from yeast to
humans. Saccharomyces cerevisiae Pmt1p is an integral membrane protein of the
endoplasmic reticulum. ScPmt1p forms a complex with ScPmt2p that is required for
maximum transferase activity. Recently, we proposed a seven-transmembrane
structural model for ScPmt1p. A large, hydrophilic, endoplasmic
reticulum-oriented segment is flanked by five amino-terminal and two
carboxyl-terminal membrane-spanning domains. Based on this model, a
structure-function analysis of ScPmt1p was performed. Deletion mutagenesis
identified the N-terminal third of the transferase as being essential for the
formation of a functional ScPmt1p-ScPmt2p complex. Deletion of the central
hydrophilic loop eliminates mannosyltransferase activity, but not
ScPmt1p-ScPmt2p interactions. Alignment of all fully characterized PMT family
members revealed that this central loop region contains three highly conserved
peptide motifs, which can be considered as signatures of the PMT family. In
addition, a number of invariant amino acid residues were identified throughout
the entire protein sequence. In order to evaluate the functional significance of
these conserved residues site-directed mutagenesis was performed. We show that
several amino acid substitutions in the conserved motifs significantly reduce
ScPmt1p activity. Further, the invariant residues Arg-64, Glu-78, Arg-138, and
Leu-408 are essential for ScPmt1p function. In particular, Arg-138 is crucial
for ScPmt1p-ScPmt2p complex formation.
PMID: 10764776 [PubMed - indexed for MEDLINE]
498: Nature 2000 Mar 30;404(6777):515-8
Yeast Sm-like proteins function in mRNA decapping and decay.
Tharun S, He W, Mayes AE, Lennertz P, Beggs JD, Parker R.
Department of Molecular and Cellular Biology and Howard Hughes Medical
Institute, University of Arizona, Tucson 85721, USA.
One of the main mechanisms of messenger RNA degradation in eukaryotes occurs by
deadenylation-dependent decapping which leads to 5'-to-3' decay. A family of
Sm-like (Lsm) proteins has been identified, members of which contain the 'Sm'
sequence motif, form a complex with U6 small nuclear RNA and are required for
pre-mRNA splicing. Here we show that mutations in seven yeast Lsm proteins
(Lsm1-Lsm7) also lead to inhibition of mRNA decapping. In addition, the
Lsm1-Lsm7 proteins co-immunoprecipitate with the mRNA decapping enzyme (Dcp1), a
decapping activator (Pat1/Mrt1) and with mRNA. This indicates that the Lsm
proteins may promote decapping by interactions with the mRNA and the decapping
machinery. In addition, the Lsm complex that functions in mRNA decay appears to
be distinct from the U6-associated Lsm complex, indicating that Lsm proteins
form specific complexes that affect different aspects of mRNA metabolism.
PMID: 10761922 [PubMed - indexed for MEDLINE]
499: Biochim Biophys Acta 2000 Apr 12;1484(2-3):93-106
Recent advances in the study of prenylated proteins.
Sinensky M.
Department of Biochemistry and Molecular Biology, James H. Quillen College of
Medicine, East Tennessee State University, Johnson City, TN 37614-0581, USA.
sinensky@etsu.edu
Post-translational modification of proteins with isoprenoids was first
recognized as a general phenomenon in 1984. In recent years, our understanding,
including mechanistic studies, of the enzymatic reactions associated with these
modifications and their physiological functions has increased dramatically. Of
particular functional interest is the role of prenylation in facilitating
protein-protein interactions and membrane-associated protein trafficking. The
loss of proper localization of Ras proteins when their farnesylation is
inhibited has also permitted a new target for anti-malignancy pharmaceuticals.
Recent advances in the enzymology and function of protein prenylation are
reviewed in this article.
Publication Types:
Review
Review, Tutorial
PMID: 10760460 [PubMed - indexed for MEDLINE]
500: Eur J Biochem 2000 Apr;267(8):2409-18
Complementation of deletion mutants in the genes encoding the F1-ATPase by
expression of the corresponding bovine subunits in yeast S. cerevisiae.
Lai-Zhang J, Mueller DM.
Department of Biochemistry and Molecular Biology, The Chicago Medical School,
Chicago, IL 60064, USA.
The F1F0 ATP synthase is composed of the F1-ATPase which is bound to F0, in the
inner membrane of the mitochondrion. Assembly and function of the enzyme is a
complicated task requiring the interactions of many proteins for the folding,
import, assembly, and function of the enzyme. The F1-ATPase is a multimeric
enzyme composed of five subunits in the stoichiometry of
alpha3beta3gammadeltaepsilon. This study demonstrates that four of the five
bovine subunits of the F1-ATPase can be imported and function in an otherwise
yeast enzyme effectively complementing mutations in the genes encoding the
corresponding yeast ATPase subunits. In order to demonstrate this, the coding
regions of each of the five genes were separately deleted in yeast providing
five null mutant strains. All of the strains displayed negative or a slow growth
phenotype on medium containing glycerol as the carbon source and strains with a
null mutation in the gene encoding the gamma-, delta- or epsilon-gene became
completely, or at a high frequency, cytoplasmically petite. The subunits of
bovine F1 were expressed individually in the yeast strains with the
corresponding null mutations and targeted to the mitochondrion using a yeast
mitochondrial leader peptide. Expression of the bovine alpha-, beta-, gamma-,
and epsilon-, but not the delta-, subunit complemented the corresponding null
mutations in yeast correcting the corresponding negative phenotypes. These
results indicate that yeast is able to import, assemble subunits of bovine
F1-ATPase in mitochondria and form a functional chimeric yeast/bovine enzyme
complex.
PMID: 10759867 [PubMed - indexed for MEDLINE]
501: Plant J 2000 Feb;21(4):379-85
The Arabidopsis Cdc2a-interacting protein ICK2 is structurally related to ICK1
and is a potent inhibitor of cyclin-dependent kinase activity in vitro.
Lui H, Wang H, Delong C, Fowke LC, Crosby WL, Fobert PR.
Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan,
Canada S7N 5E2.
Cyclin-dependent kinases (CDKs) are important regulators of the eukaryotic cell
division cycle. To study protein-protein interactions involving plant CDKs, the
Arabidopsis thaliana Cdc2aAt was used as bait in the yeast two-hybrid system.
Here we report on the isolation of ICK2, and show that it interacts with
Cdc2aAt, but not with a second CDK from Arabidopsis, Cdc2bAt. ICK2 contains a
carboxy-terminal domain related to that of ICK1, a previously described CDK
inhibitor from Arabidopsis, and to the CDK-binding domain of the mammalian
inhibitor p27Kip1. Outside of this domain, ICK2 is distinct from ICK1, p27Kip1,
and other proteins. At nanogram levels (8 nM), purified recombinant ICK2
inhibits p13Suc1-associated histone H1 kinase activity from Arabidopsis tissue
extracts, demonstrating that it is a potent inhibitor of plant CDK activity in
vitro. ICK2 mRNA was present in all tissues analysed by Northern hybridization,
and its distribution was distinct from that of ICK1. These results demonstrate
that plants possess a family of differentially regulated CDK inhibitors that
contain a conserved carboxy terminal but with distinct amino terminal regions.
PMID: 10758489 [PubMed - indexed for MEDLINE]
502: Plant J 2000 Feb;21(4):341-9
Modes of interaction between the Arabidopsis Rab protein, Ara4, and its putative
regulator molecules revealed by a yeast expression system.
Ueda T, Matsuda N, Uchimiya H, Nakano A.
Molecular Membrane Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama
351-0198, Japan.
Ara4, a member of the Rab/Ypt GTPase family derived from Arabidopsis thaliana,
causes severe growth inhibition when expressed in several yeast ypt mutants.
Mutational analysis of ARA4 indicated that the Ara4 protein titrates at least
three factors in yeast, including the GDP dissociation inhibitor (GDI). The
coexpression of AtGDI1 (Arabidopsis GDI) suppressed the growth defect caused by
Ara4 in yeast ypt1, suggesting that Ara4 and AtGDI1 interact in yeast to
compensate for the titration of yeast GDI. We screened an Arabidopsis cDNA
library for other suppressors that may also interact with Ara4 physiologically.
A novel suppressor, SAY1, encoded a hydrophilic protein with two putative
coiled-coil regions, which showed partial similarity to the yeast Vps27 protein.
To understand the structural requirements of Ara4 for interacting with these
molecules, we examined whether AtGDI1 and SAY1 could suppress the growth defect
of ypt1 caused by various mutant versions of ARA4. The results indicated that
the interaction between Ara4 and AtGDI1 depends on the conserved C-terminal
Cys-motif and Thr44 in the effector domain of Ara4. In contrast, neither of
these motifs is necessary for the interaction between Say1 and Ara4. This
approach provides a powerful method to dissect complex interactions between a
GTPase and its regulators.
PMID: 10758485 [PubMed - indexed for MEDLINE]
503: J Immunol Methods 2000 Apr 21;238(1-2):29-43
Phagocytosis of yeast: a method for concurrent quantification of binding and
internalization using differential interference contrast microscopy.
Bos H, de Souza W.
Laboratorio de Biologia Celular e Tecidual, Centro de Biociencias e
Biotecnologia, Universidade Estadual do Norte Fluminense, Campos, Brazil.
In studies of phagocytosis there is a need to distinguish targets that are
internalized by the cell from those that are bound to the cell surface. The
present work describes a simple method by which internalized and surface-bound
yeast particles can be identified by differential interference contrast
microscopy, using trypan blue to stain surface-bound yeast particles. The method
has the advantage that both internalized and surface-bound particles can be
visualized without the need to switch the illumination source and/or filter
sets, thus facilitating concurrent quantitation of binding and internalization.
The method was evaluated with the phagocytosis-modulating agents horseradish
peroxidase (HRP) and cytochalasin D, using adherent resident macrophages as
phagocytic cells. When macrophages are challenged with a particular type of
target, they usually bind many more targets than they ingest. It was shown that
yeast particles were arrested in the initial binding phase of phagocytosis
depending on the region of macrophage plasma membrane where binding sites were
formed. Failure of surface-bound yeast particles to trigger internalization was
not due to modifications of the yeast particle surface. Nor was it due to
binding to non-phagocytic receptors, or low-affinity receptor-ligand
interactions. The glycoprotein HRP inhibited only the binding stage of
phagocytosis, whereas cytochalasin D, a drug that affects actin polymerization,
inhibited both binding and internalization. However, when the yeast particles
were pre-incubated in fresh mouse serum, cytochalasin D inhibited only the
internalization step. The assay described here may be useful in studies
concerned with the function and expression of phagocytosis-mediating surface
lectins.
PMID: 10758233 [PubMed - indexed for MEDLINE]
504: J Chromatogr A 2000 Mar 24;873(2):195-208
The influence of cell adsorbent interactions on protein adsorption in expanded
beds.
Fernandez-Lahore HM, Geilenkirchen S, Boldt K, Nagel A, Kula MR, Thommes J.
Institut fur Enzymtechnologie, Heinrich-Heine Universitat Dusseldorf, Julich,
Germany.
Expanded bed adsorption (EBA) is a primary recovery operation allowing the
adsorption of proteins directly from unclarified feedstock, e.g. culture
suspensions, homogenates or crude extracts. Thus solid-liquid separation is
combined with adsorptive purification in a single step. The concept of
integration requires that the solid components of the feed solution are regarded
as a part of the process, which influences stability, reproducibility, and
overall performance. This aspect is investigated here at the example of the
influence of presence and concentration of intact yeast cells (S. cerevisiae) on
the adsorption of model proteins (hen egg white lysozyme and bovine serum
albumin) to various stationary phases (cation and anion-exchange, hydrophobic
interaction, immobilised metal affinity). The interaction of the cells with the
adsorbents is determined qualitatively and quantitatively by a pulse response
method as well as by a finite bath technique under different operating
conditions. The consequence of these interactions for the stability of expanded
beds in suspensions of varying cell concentration is measured by residence time
distributions (RTDs) after tracer pulse injection (NaBr, LiCl). Analysis of the
measured RTD by the PDE model allows the calculation of the fraction of
perfectly fluidised bed (phi), a parameter which may be regarded as a critical
quantity for the estimation of the quality of fluidisation of adsorbents in cell
containing suspensions. The correlation between bed stability and performance is
made by analysing the breakthrough of model proteins during adsorption from
unclarified yeast culture broth. A clear relationship is found between the
degree of cell/adsorbent interaction, bed stability in terms of the phi
parameter, and the sorption efficiency. Only beds characterised by a phi value
larger than 0.8 in the presence of cells will show a conserved performance
compared to adsorption from cell free solutions. A drop in phi, which is due to
interactions of the fluidised adsorbent particles with cells from the feed, will
directly result in a reduced breakthrough efficiency. The data presented
highlight the importance of including the potential interaction of solid
feedstock components and the expanded adsorbents into the design of EBA
processes, as the interrelation found here is a key factor for the overall
performance of EBA as a truly integrated operation.
PMID: 10757297 [PubMed - indexed for MEDLINE]
505: J Mol Biol 2000 Apr 21;298(1):111-21
High-resolution crystal structure of S. cerevisiae Ypt51(DeltaC15)-GppNHp, a
small GTP-binding protein involved in regulation of endocytosis.
Esters H, Alexandrov K, Constantinescu AT, Goody RS, Scheidig AJ.
Abteilung fur Physikalische Biochemie, Max-Planck Institut fur molekulare
Physiologie, Otto-Hahn-Strasse 11, Dortmund, 44227, Germany.
Ypt/Rab proteins are membrane-associated small GTP-binding proteins which play a
central role in the coordination, activation and regulation of vesicle-mediated
transport in eukaryotic cells. We present the 1.5 A high-resolution crystal
structure of Ypt51 in its active, GppNHp-bound conformation. Ypt51 is an
important regulator involved in the endocytic membrane traffic of Saccharomyces
cerevisiae. The structure reveals small but significant structural differences
compared with H-Ras p21. The effector loop and the catalytic loop are well
defined and stabilized by extensive hydrophobic interactions. The switch I and
switch II regions form a well-defined epitope for hypothetical effector protein
binding. Sequence comparisons between the different isoforms Ypt51, Ypt52 and
Ypt53 provide the first insights into determinants for specific effector binding
and for fine-tuning of the intrinsic GTP-hydrolysis rate. Copyright 2000
Academic Press.
PMID: 10756108 [PubMed - indexed for MEDLINE]
506: J Biol Chem 2000 Jun 2;275(22):16443-9
Evidence for simultaneous protein interactions between human Rad51 paralogs.
Schild D, Lio Y, Collins DW, Tsomondo T, Chen DJ.
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley,
California 94720, USA. dschild@lbl.gov
In yeast, the Rad51-related proteins include Rad55 and Rad57, which form a
heterodimer that interacts with Rad51. Five human Rad51 paralogs have been
identified (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2, and Rad51D/Rad51L3),
and each interacts with one or more of the others. Previously we reported that
HsRad51 interacts with XRCC3, and Rad51C interacts with XRCC3, Rad51B, and
HsRad51. Here we report that in the yeast two-hybrid system, Rad51D interacts
with XRCC2 and Rad51C. No other interactions, including self-interactions, were
found, indicating that the observed interactions are specific. The yeast Rad51
interacts with human Rad51 and XRCC3, suggesting Rad51 conservation since the
human yeast divergence. Data from yeast three-hybrid experiments indicate that a
number of the pairs of interactions between human Rad51 paralogs can occur
simultaneously. For example, Rad51B expression enhances the binding of Rad51C to
XRCC3 and to HsRad51D, and Rad51C expression allows the indirect interaction of
Rad51B with Rad51D. Experiments using 6xHis-tagged proteins in the baculovirus
system confirm several of our yeast results, including Rad51B interaction with
Rad51D only when Rad51C is simultaneously expressed and Rad51C interaction with
XRCC2 only when Rad51D is present. These results suggest that these proteins may
participate in one complex or multiple smaller ones.
PMID: 10749867 [PubMed - indexed for MEDLINE]
507: J Biol Chem 2000 Jun 9;275(23):17241-8
Functional characterization of yeast mitochondrial release factor 1.
Askarian-Amiri ME, Pel HJ, Guevremont D, McCaughan KK, Poole ES, Sumpter VG,
Tate WP.
Department of Biochemistry and Centre for Gene Research, University of Otago, P.
O. Box 56, 9015 Dunedin, New Zealand.
The yeast Saccharomyces cerevisiae mitochondrial release factor was expressed
from the cloned MRF1 gene, purified from inclusion bodies, and refolded to give
functional activity. The gene encoded a factor with release activity that
recognized cognate stop codons in a termination assay with mitochondrial
ribosomes and in an assay with Escherichia coli ribosomes. The noncognate stop
codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the
heterologous assay. The mitochondrial release factor 1 protein bound to
bacterial ribosomes and formed a cross-link with the stop codon within a mRNA
bound in a termination complex. The affinity was strongly dependent on the
identity of stop signal. Two alleles of MRF1 that contained point mutations in a
release factor 1 specific region of the primary structure and that in vivo
compensated for mutations in the decoding site rRNA of mitochondrial ribosomes
were cloned, and the expressed proteins were purified and refolded. The variant
proteins showed impaired binding to the ribosome compared with mitochondrial
release factor 1. This structural region in release factors is likely to be
involved in codon-dependent specific ribosomal interactions.
PMID: 10748224 [PubMed - indexed for MEDLINE]
508: J Biol Chem 2000 May 19;275(20):15157-65
Structural and functional characterization of interaction between hepatitis B
virus X protein and the proteasome complex.
Zhang Z, Torii N, Furusaka A, Malayaman N, Hu Z, Liang TJ.
Liver Diseases Section, NIDDK, National Institutes of Health, Bethesda, Maryland
20892, USA.
Hepatitis B virus (HBV) has a unique fourth open reading frame coding for a
16.5-kDa protein known as hepatitis B virus X protein (HBX). The importance of
HBX in the life cycle of HBV has been well established, but the underlying
molecular function of HBX remains controversial. We previously identified a
proteasome subunit PSMA7 that interacts specifically with HBX in the
Saccharomyces cerevisiae two-hybrid system. Here we demonstrate that PSMC1, an
ATPase-like subunit of the 19 S proteasome component, also interacts with HBX
and PSMA7. Analysis of the interacting domains among PSMA7, PSMC1, and HBX by
deletion and site-directed mutagenesis suggested a mutually competitive
structural relationship among these polypeptides. The competitive nature of
these interactions is further demonstrated using a modified yeast two-hybrid
dissociator system. The crucial HBX sequences involved in interaction with PSMA7
and PSMC1 are important for its function as a transcriptional coactivator. HBX,
while functioning as a coactivator of AP-1 and acidic activator VP-16 in
mammalian cells, had no effect on the transactivation function of their
functional orthologs GCN4 and Gal4 in yeast. Overexpression of PSMC1 seemed to
suppress the expression of various reporters in mammalian cells; this effect,
however, was overcome by coexpression of HBX. In addition, HBX expression
inhibited the cellular turnover of c-Jun and ubiquitin-Arg-beta-galactosidase,
two well known substrates of the ubiquitin-proteasome pathway. Thus, interaction
of HBX with the proteasome complex in metazoan cells may underlie the functional
basis of proteasome as a cellular target of HBX.
PMID: 10748218 [PubMed - indexed for MEDLINE]
509: J Biol Chem 2000 Jul 21;275(29):22255-67
DNA recognition, strand selectivity, and cleavage mode during integrase family
site-specific recombination.
Tribble G, Ahn YT, Lee J, Dandekar T, Jayaram M.
Department of Microbiology, University of Texas, Austin, Texas 78712, Faculty of
Applied Marine Sciences, Cheju University, Cheju City 690756, South Korea.
We have probed the association of Flp recombinase with its DNA target using
protein footprinting assays. The results are consistent with the domain
organization of the Flp protein and with the general features of the protein-DNA
interactions revealed by the crystal structures of the recombination
intermediates formed by Cre, the Flp-related recombinase. The similarity in the
organization of the Flp and Cre target sites and in their recognition by the
respective recombinases implies that the overall DNA-protein geometry during
strand cleavage in the two systems must also be similar. Within the functional
recombinase dimer, it is the interaction between two recombinase monomers bound
on either side of the strand exchange region (or spacer) that provides the
allosteric activation of a single active site. Whereas Cre utilizes the cleavage
nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one
monomer donating the tyrosine to its partner). By using synthetic Cre and Flp
DNA substrates that are geometrically restricted in similar ways, we have mapped
the positioning of the active and inactive tyrosine residues during cis and
trans cleavage events. We find that, for a fixed substrate geometry, Flp and Cre
cleave the labile phosphodiester bond at the same spacer end, not at opposite
ends. Our results provide a model that accommodates local heterogeneities in
peptide orientations in the two systems while preserving the global functional
architecture of the reaction complex.
PMID: 10748094 [PubMed - indexed for MEDLINE]
510: J Biol Chem 2000 Jun 2;275(22):16632-7
Regulation of phospholipase C-beta 3 activity by Na+/H+ exchanger regulatory
factor 2.
Hwang JI, Heo K, Shin KJ, Kim E, Yun C, Ryu SH, Shin HS, Suh PG.
Department of Life Science, National Creative Research Initiative Center for
Calcium and Learning, Division of Molecular and Life Science and School of
Environmental Engineering, Pohang University of Science and Technology, Pohang
790-784, South Korea.
Among the phospholipase C that catalyzes the hydrolysis of phosphatidylinositol
4,5-bisphosphate, four mammalian phospholipase C-beta (PLC-beta) isotypes
(isotypes 1-4) are activated through G protein-coupled receptors (GPCRs).
Although the regulation of the PLC-betas by GPCRs and heterotrimeric G proteins
has been extensively studied, little is known about the molecular determinants
that regulate their activity. The PLC-beta isozymes carry a putative
PSD-95/Dlg/ZO-1 (PDZ) binding motif (X(S/T)X(V/L)COOH) at their carboxyl
terminus, which is implicated in specific interactions with anchor proteins.
Using the yeast two-hybrid system, we identified Na(+)/H(+) exchanger regulatory
factor 2 (NHERF2) as a protein that interacted with a C-terminal heptapeptide of
PLC-beta3. Immunoprecipitation studies revealed that NHERF2 interacts
specifically with PLC-beta3, but not with other PLC-beta isotypes. Furthermore,
PLC-beta3 interacted with NHERF2 rather than with other PDZ-containing proteins.
This interaction required the COOH-terminal NTQL sequence of PLC-beta3 and the
second PDZ domain of NHERF2. Interestingly, NHERF2 potentiated the PLC-beta
activation by carbachol in COS7 and HeLa cells, while mutant NHERF2, lacking the
second PDZ domain, had no such effect. Taken together, the data suggest that
NHERF2 may act as a modulator underlying the process of PLC-beta3-mediated
signaling.
PMID: 10748023 [PubMed - indexed for MEDLINE]
511: J Biol Chem 2000 May 19;275(20):15014-8
Architectural principles for the structure and function of the glucocorticoid
receptor tau 1 core activation domain.
Warnmark A, Gustafsson JA, Wright AP.
Department of Biosciences, Karolinska Institutet, Novum, Huddinge S-141 57,
Sweden. anette.warnmark@cbt.ki.se
A 58-amino acid region mediates the core transactivation activity of the
glucocorticoid receptor tau1 activation domain. This tau1 core domain is
unstructured in aqueous buffers, but in the presence of trifluoroethanol three
alpha-helical segments are induced. Two of these putative structural modules
have been tested in different combinations with regard to transactivation
potential in vivo and binding capacity to the coactivators in vitro. The results
show that whereas single modules are not transcriptionally active, any
combination of two or three modules is sufficient, with trimodular constructs
having the highest activity. However, proteins containing one, two, or three
segments bind Ada2 and cAMP-response element-binding protein with similar
affinity. A single segment is thus able to bind a target factor but cannot
transactivate target genes significantly. The results are consistent with models
in which activation domains are comprised of short activation modules that allow
multiple interactions with coactivators. Our results also suggest that an
increased number of modules may not result in correspondingly higher affinity
but instead that the concentration of binding sites is increased, which gives
rise to a higher association rate. This is consistent with a model where the
association rate for activator-target factor interactions rather than the
equilibrium constant is the most relevant measure of activator potency.
PMID: 10747977 [PubMed - indexed for MEDLINE]
512: Biochemistry 2000 Apr 11;39(14):4199-205
Environmental study of subunit i, a F(o) component of the yeast ATP synthase.
Paumard P, Vaillier J, Napias C, Arselin G, Brethes D, Graves PV, Velours J.
Institut de Biochimie et Genetique Cellulaires du CNRS, Universite Victor
Segalen, Bordeaux 2,1 rue Camille Saint-Saens, 33077 Bordeaux Cedex, France.
The topology of subunit i, a component of the yeast F(o)F(1)-ATP synthase, was
determined by the use of cysteine-substituted mutants. The N(in)-C(out)
orientation of this intrinsic subunit was confirmed by chemical modification of
unique cysteine residues with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic
acid. Near-neighbor relationships between subunit i and subunits 6, f, g, and d
were demonstrated by cross-link formation following sulfhydryl oxidation or
reaction with homobifunctional and heterobifunctional reagents. Our data suggest
interactions between the unique membrane-spanning segment of subunit i and the
first transmembranous alpha-helix of subunit 6 and a stoichiometry of 1 subunit
i per complex. Cross-linked products between mutant subunits i and proteins
loosely bound to the F(o)F(1)-ATP synthase suggest that subunit i is located at
the periphery of the enzyme and interacts with proteins of the inner
mitochondrial membrane that are not involved in the structure of the yeast ATP
synthase.
PMID: 10747812 [PubMed - indexed for MEDLINE]
513: Biochemistry 2000 Apr 11;39(14):3943-54
Characterization of the DNA-binding domains from the yeast cell-cycle
transcription factors Mbp1 and Swi4.
Taylor IA, McIntosh PB, Pala P, Treiber MK, Howell S, Lane AN, Smerdon SJ.
Divisions of Protein Structure and Molecular Structure, National Institute for
Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
The minimal DNA-binding domains of the Saccharomyces cerevisiae transcription
factors Mbp1 and Swi4 have been identified and their DNA binding properties have
been investigated by a combination of methods. An approximately 100 residue
region of sequence homology at the N-termini of Mbp1 and Swi4 is necessary but
not sufficient for full DNA binding activity. Unexpectedly, nonconserved
residues C-terminal to the core domain are essential for DNA binding.
Proteolysis of Mbp1 and Swi4 DNA-protein complexes has revealed the extent of
these sequences, and C-terminally extended molecules with substantially enhanced
DNA binding activity compared to the core domains alone have been produced. The
extended Mbp1 and Swi4 proteins bind to their cognate sites with similar
affinity [K(A) approximately (1-4) x 10(6) M(-)(1)] and with a 1:1
stoichiometry. However, alanine substitution of two lysine residues (116 and
122) within the C-terminal extension (tail) of Mbp1 considerably reduces the
apparent affinity for an MCB (MluI cell-cycle box) containing oligonucleotide.
Both Mbp1 and Swi4 are specific for their cognate sites with respect to
nonspecific DNA but exhibit similar affinities for the SCB (Swi4/Swi6 cell-cycle
box) and MCB consensus elements. Circular dichroism and (1)H NMR spectroscopy
reveal that complex formation results in substantial perturbations of base
stacking interactions upon DNA binding. These are localized to a central
5'-d(C-A/G-CG)-3' region common to both MCB and SCB sequences consistent with
the observed pattern of specificity. Changes in the backbone amide proton and
nitrogen chemical shifts upon DNA binding have enabled us to experimentally
define a DNA-binding surface on the core N-terminal domain of Mbp1 that is
associated with a putative winged helix-turn-helix motif. Furthermore,
significant chemical shift differences occur within the C-terminal tail of Mbp1,
supporting the notion of two structurally distinct DNA-binding regions within
these proteins.
PMID: 10747782 [PubMed - indexed for MEDLINE]
514: Genetics 2000 Apr;154(4):1473-84
A role for the noncatalytic N terminus in the function of Cdc25, a Saccharomyces
cerevisiae Ras-guanine nucleotide exchange factor.
Chen RA, Michaeli T, Van Aelst L, Ballester R.
Department of Molecular, Cellular and Developmental Biology, University of
California, Santa Barbara, CA 93106, USA.
The Saccharomyces cerevisiae CDC25 gene encodes a guanine nucleotide exchange
factor (GEF) for Ras proteins. Its catalytic domain is highly homologous to
Ras-GEFs from all eukaryotes. Even though Cdc25 is the first Ras-GEF identified
in any organism, we still know very little about how its function is regulated
in yeast. In this work we provide evidence for the involvement of the N terminus
of Cdc25 in the regulation of its activity. A truncated CDC25 lacking the
noncatalytic C-terminal coding sequence was identified in a screen of high-copy
suppressors of the heat-shock-sensitive phenotype of strains in which the Ras
pathway is hyper-activated. The truncated gene acts as a dominant-negative
mutant because it only suppresses the heat-shock sensitivity of strains that
require the function of CDC25. Our two-hybrid assays and immunoprecipitation
analyses show interactions between the N terminus of Cdc25 and itself, the C
terminus, and the full-length protein. These results suggest that the
dominant-negative effect may be a result of oligomerization with endogenous
Cdc25. Further evidence of the role of the N terminus of Cdc25 in the regulation
of its activity is provided by the mapping of the activating mutation of
CDC25HS20 to the serine residue at position 365 in the noncatalytic N-terminal
domain. This mutation induces a phenotype similar to activating mutants of other
genes in the Ras pathway in yeast. Hence, the N terminus may exert a negative
control on the catalytic activity of the protein. Taken together these results
suggest that the N terminus plays a crucial role in regulating Cdc25 and
consequently Ras activity, which in S. cerevisiae is essential for cell cycle
progression.
PMID: 10747046 [PubMed - indexed for MEDLINE]
515: EMBO J 2000 Apr 3;19(7):1598-612
Histone H2A is required for normal centromere function in Saccharomyces
cerevisiae.
Pinto I, Winston F.
Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA
02115, USA.
Histones are structural and functional components of the eukaryotic chromosome,
and their function is essential for normal cell cycle progression. In this work,
we describe the characterization of two Saccharomyces cerevisiae cold-sensitive
histone H2A mutants. Both mutants contain single amino acid replacements of
residues predicted to be on the surface of the nucleosome and in close contact
with DNA. We show that these H2A mutations cause an increase-in-ploidy
phenotype, an increased rate of chromosome loss, and a defect in traversing the
G(2)-M phase of the cell cycle. Moreover, these H2A mutations show genetic
interactions with mutations in genes encoding kinetochore components. Finally,
chromatin analysis of these H2A mutants has revealed an altered centromeric
chromatin structure. Taken together, these results strongly suggest that histone
H2A is required for proper centromere-kinetochore function during chromosome
segregation.
PMID: 10747028 [PubMed - indexed for MEDLINE]
516: RNA 2000 Mar;6(3):352-68
Splicing enhancement in the yeast rp51b intron.
Libri D, Lescure A, Rosbash M.
Centre National de la Recherche Scientifique, Centre de Genetique Moleculaire,
Gif-sur-Yvette, France. Libri@cgm.cnrs-gif.fr
Splicing enhancement in higher eukaryotes has been linked to SR proteins, to U1
snRNP, and to communication between splice sites across introns or exons
mediated by protein-protein interactions. It has been previously shown that, in
yeast, communication mediated by RNA-RNA interactions between the two ends of
introns is a basis for splicing enhancement. We designed experiments of
randomization-selection to isolate splicing enhancers that would work
independently from RNA secondary structures. Surprisingly, one of the two
families of sequences selected was essentially composed of 5' splice site
variants. We show that this sequence enhances splicing independently of
secondary structure, is exportable to heterologous contexts, and works in
multiple copies with additive effects. The data argue in favor of an early role
for splicing enhancement, possibly coincident with commitment complex formation.
Genetic compensation experiments with U1 snRNA mutants suggest that U1 snRNP
binding to noncanonical locations is required for splicing enhancement.
PMID: 10744020 [PubMed - indexed for MEDLINE]
517: Plant J 2000 Jan;21(2):143-55
Interactions of the developmental regulator ABI3 with proteins identified from
developing Arabidopsis seeds.
Kurup S, Jones HD, Holdsworth MJ.
IACR-Long Ashton Research Station, Department of Agricultural Sciences,
University of Bristol, Long Ashton, UK.
The ABI3 locus is a major regulator of embryo development in Arabidopsis and is
essential for the simultaneous activation of the maturation pathway, as well as
repression of germination and seedling development. We used a two-hybrid screen
in yeast in order to identify proteins that interact with ABI3. Four
ABI3-interacting proteins (AIPs) were identified which showed specific in vivo
and in vitro interactions with the C-terminal region of ABI3 that contains the
B2 and B3 domains, previously shown to have DNA binding activity. The expression
characteristics of the genes encoding the AIPs have also been analysed in
wild-type and abi3, lec1 and fus3 embryo mutants. This analysis demonstrated
differential expression of these genes during normal embryo development and in
the mutant lines. All the AIPs show homology to existing transcription factors
and therefore they may function with ABI3 within the network of transcriptional
regulators that control embryo development in Arabidopsis.
PMID: 10743655 [PubMed - indexed for MEDLINE]
518: Bioseparation 1999;8(1-5):99-109
Cell/adsorbent interactions in expanded bed adsorption of proteins.
Feuser J, Walter J, Kula MR, Thommes J.
Institut fur Enzymtechnologie, Heinrich-Heine Universitat Dusseldorf, Julich,
Germany.
Expanded bed adsorption (EBA) is an integrated technology for the primary
recovery of proteins from unclarified feedstock. A method is presented which
allows a qualitative and quantitative understanding of the main mechanisms
governing the interaction of biomass with fluidized resins. A pulse response
technique was used to determine the adsorption of various cell types (yeast,
Gram positive and Gram negative bacteria, mammalian cells and yeast homogenate)
to a range of commercially available matrices for EBA. Cells and cell debris
were found to interact with the ligands of agarose based resins mainly by
electrostatic forces. From the adsorbents investigated the anion exchange matrix
showed the most severe interactions, while cation exchange and affinity
adsorbents appeared to be less affected. Within the range of biologic systems
under study E. coli cells had the lowest tendency of binding to all matrices
while hybridoma cells attached to all the adsorbents except the protein A
affinity matrix. The method presented may be employed for screening of suitable
biomass/adsorbent combinations, which yield a robust and reliable initial
capture step by expanded bed adsorption from unclarified feedstock.
PMID: 10734561 [PubMed - indexed for MEDLINE]
519: Mol Gen Genet 2000 Feb;263(1):60-72
Mutations in CDC14 result in high sensitivity to cyclin gene dosage in
Saccharomyces cerevisiae.
Yuste-Rojas M, Cross FR.
Rockefeller University, New York, NY 10021, USA.
We screened for mutations that resulted in lethality when the G1 cyclin Cln2p
was overexpressed throughout the cell cycle in Saccharomyces cerevisiae.
Mutations in five complementation groups were found to give this phenotype, and
three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations
in CDC14 may have been recovered in the screen because Cdc14p may reduce the
cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may
normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement
with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion
of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with
cdc14-1, as did the deletion of HCT1, which is required for proteolysis of
Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2,
also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14
strains arrested with replicated but unseparated DNA and unseparated spindle
pole bodies; this phenotype is distinct from the late mitotic arrest of the
sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14
CLN2 overexpressor. We found genetic interactions between CDC14 and the
replication initiator gene CDC6, extending previous observations of interactions
between the late mitotic function of Cdc14p and control of DNA replication. We
also describe genetic interactions between CDC28 and CDC14.
PMID: 10732674 [PubMed - indexed for MEDLINE]
520: Mol Gen Genet 2000 Feb;263(1):12-21
HD-Zip proteins of families I and II from rice: interactions and functional
properties.
Meijer AH, de Kam RJ, d'Erfurth I, Shen W, Hoge JH.
Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory,
The Netherlands. meijer@rulbim.leidenuniv.nl
Proteins of the closely related homeodomain-leucine zipper (HD-Zip) families I
and II in plants are putative transcription factors that interact with similar
pseudopalindromic DNA recognition sites. We have previously described the Oshox1
gene from rice, which encodes an HD-Zip II protein. To identify further rice
HD-Zip proteins, one-hybrid screens were performed in yeast strains containing a
HIS3 reporter gene with upstream HD-Zip recognition sites. This resulted in the
isolation of six new cDNAs encoding HD-Zip proteins belonging to family I
(Oshox4, -5, -6) or family II (Oshox2, -3, -7). In transient assays, using rice
suspension-cultured cells transformed by particle bombardment, we showed
previously that Oshox1 can transcriptionally repress the activity of reporter
gene constructs with upstream HD-Zip binding sites. Here, we confirm the
repression properties of Oshox1 by showing that the repression function can be
conferred on a heterologous DNA-binding domain. This portable functional domain
(residues 1-155) is located proximal to the HD-Zip domain. In yeast, the same
region of the Oshox1 protein was found to confer transcriptional activation
instead of repression, pointing to the possibility that cell type-specific
factors may determine the functional properties of the Oshox1 protein in rice.
Like Oshox1, another HD-Zip family II protein (Oshox3) was also found to
function as a transcriptional repressor in rice cells. In contrast, two HD-Zip I
family proteins (Oshox4 and -5) appeared to act as activators in both rice and
yeast cells. Results of two-hybrid assays and electrophoretic mobility shift
assays strongly suggest that all HD-Zip proteins of families I and II can form
homodimers and also heterodimers with all HD-Zip proteins of the same family.
Heterodimerization across the HD-Zip families I and II apparently does not to
occur.
PMID: 10732669 [PubMed - indexed for MEDLINE]
521: Gene 2000 Jan 25;242(1-2):369-79
A genomic approach of the hepatitis C virus generates a protein interaction map.
Flajolet M, Rotondo G, Daviet L, Bergametti F, Inchauspe G, Tiollais P, Transy
C, Legrain P.
INSERM U163, Institut Pasteur, Paris, France.
The hepatitis C virus (HCV) causes severe liver disease, including liver cancer.
A vaccine preventing HCV infection has not yet been developed, and, given the
increasing number of infected people, this virus is now considered a major
public-health problem. The HCV genome is a plus-stranded RNA that encodes a
single polyprotein processed into at least 10 mature polypeptides. So far, only
the interaction between the protease NS3 and its cofactor, NS4A, which is
involved in the processing of the non-structural region, has been extensively
studied. Our work was aimed at constructing a protein interaction map of HCV. A
classical two-hybrid system failed to detect any interactions between mature HCV
polypeptides, suggesting incorrect folding, expression or targetting of these
proteins. We therefore developed a two-hybrid strategy, based on exhaustive
screens of a random genomic HCV library. Using this method, we found known
interactions, such as the capsid homodimer and the protease dimer, NS3-NS4A, as
well as several novel interactions such as NS4A-NS2. Thus, our results are
consistent with the idea that the use of a random genomic HCV library allows the
selection of correctly folded viral protein fragments. Interacting domains of
the viral polyprotein are identified, opening the possibility of developing
specific anti-viral agents, based on their ability to modulate these
interactions.
PMID: 10721731 [PubMed - indexed for MEDLINE]
522: Biochimie 2000 Jan;82(1):71-8
Characterization of genetic interactions with RFA1: the role of RPA in DNA
replication and telomere maintenance.
Smith J, Zou H, Rothstein R.
Department of Genetics & Development, Columbia University College of Physicians
& Surgeons, New York, NY 10032-2704, USA.
Replication protein A (RPA) is a heterotrimeric single-stranded DNA binding
protein whose role in DNA replication, recombination and repair has been mainly
elucidated through in vitro biochemical studies utilizing the mammalian complex.
However, the identification of homologs of all three subunits in Saccharomyces
cerevisiae offers the opportunity of examining the in vivo role of RPA. In our
laboratory, we have previously isolated a missense allele of the RFA1 gene,
encoding the p70 subunit of the RPA complex. Strains containing this mutant
allele, rfa1-D228Y, display increased levels of direct-repeat recombination,
decreased levels of heteroallelic recombination, UV sensitivity and a S-phase
delay. In this study, we have characterized further the role of RPA by screening
other replication and repair mutants for a synthetic lethal phenotype in
combination with the rfa1-D228Y allele. Among the replication mutants examined,
only one displayed a synthetic lethal phenotype, pol12-100, a conditional allele
of the B subunit of pol alpha-primase. In addition, a delayed senescence
phenotype was observed in raf1-D228Y strains containing a null mutation of HDF1,
the S. cerevisiae homolog of the 70 kDa subunit of Ku. Interestingly, a
synergistic reduction in telomere length observed in the double mutants suggests
that the shortening of telomeres may be the cause of the decreased viability in
these strains. Furthermore, this result represents the first evidence of a role
for RPA in telomere maintenance.
PMID: 10717390 [PubMed - indexed for MEDLINE]
523: Biochimie 2000 Jan;82(1):5-17
Mechanisms and consequences of replication fork arrest.
Hyrien O.
Ecole Normale Superieure, Paris, France.
Chromosome replication is not a uniform and continuous process. Replication
forks can be slowed down or arrested by DNA secondary structures, specific
protein-DNA complexes, specific DNA-RNA hybrids, or interactions between the
replication and transcription machineries. Replication arrest has important
implications for the topology of replication intermediates and can trigger
homologous and illegitimate recombination. Thus, replication arrest may be a key
factor in genome instability. Several examples of these phenomena are reviewed
here.
Publication Types:
Review
Review, Tutorial
PMID: 10717381 [PubMed - indexed for MEDLINE]
524: Genes Dev 2000 Mar 1;14(5):559-73
Rules for DNA target-site recognition by a lactococcal group II intron enable
retargeting of the intron to specific DNA sequences.
Mohr G, Smith D, Belfort M, Lambowitz AM.
Institute for Cellular and Molecular Biology, Department of Chemistry and
Biochemistry, and Section of Molecular Genetics and Microbiology, School of
Biological Sciences, University of Texas at Austin, Austin, Texas 78712, USA.
Group II intron homing occurs primarily by a mechanism in which the intron RNA
reverse splices into a DNA target site and is then reverse transcribed by the
intron-encoded protein. The DNA target site is recognized by an RNP complex
containing the intron-encoded protein and the excised intron RNA. Here, we
analyzed DNA target-site requirements for the Lactococcus lactis Ll.LtrB group
II intron in vitro and in vivo. Our results suggest a model similar to yeast
mtDNA introns, in which the intron-encoded protein first recognizes a small
number of nucleotide residues in double-stranded DNA and causes DNA unwinding,
enabling the intron RNA to base-pair with the DNA for reverse splicing.
Antisense-strand cleavage requires additional interactions between the protein
and 3' exon. Key nucleotide residues are recognized directly by the
intron-encoded protein independent of sequence context, and there is a stringent
requirement for fixed spacing between target site elements recognized by the
protein and RNA components of the endonuclease. Experiments with DNA substrates
containing GC-clamps or "bubbles" indicate a requirement for DNA unwinding in
the 3' exon but not the distal 5' exon region. Finally, by applying the
target-site recognition rules, we show that the L1.LtrB intron can be modified
to insert at new sites in a plasmid-borne thyA gene in Escherichia coli. This
strategy should be generally applicable to retargeting group II introns and to
delivering foreign sequences to specific sites in heterologous genomes.
PMID: 10716944 [PubMed - indexed for MEDLINE]
525: J Biol Chem 2000 Mar 17;275(11):7925-34
Cell wall biogenesis of Blastomyces dermatitidis. Evidence for a novel mechanism
of cell surface localization of a virulence-associated adhesin via extracellular
release and reassociation with cell wall chitin.
Brandhorst T, Klein B.
Departments of Pediatrics, Internal Medicine, and Medical Microbiology and
Immunology, and the Comprehensive Cancer Center, University of Wisconsin Medical
School, Madison, Wisconsin 53792, USA.
Pathogenic yeast of Blastomyces dermatitidis express a surface protein adhesin,
WI-1. Due to the crucial role of WI-1 in adherence and disease pathogenesis, we
investigated how the protein localizes to the surface of B. dermatitidis. WI-1
released extracellularly by wild-type yeast coated the surfaces of co-cultured
knockout yeast within 3 h of incubation, implying that secreted WI-1 provides a
pathway for loading the protein onto the yeast cell wall. In radioligand binding
assays, purified WI-1 bound saturably, specifically, and with high affinity
(K(d) = 8.3 x 10(-9)) to the cell surface of knockout yeast devoid of WI-1. WI-1
added exogenously, in vitro, to knockout yeast was indistinguishable from native
cell surface WI-1 by fluorescence staining and restored adhesivity to the
knockout yeast in macrophage binding and phagocytosis assays. Analysis of
interactions between WI-1 and elements of the yeast cell wall identified chitin
as the anchor point for WI-1. This interaction was shown to hinge on the
24-amino acid tandem repeat sequence of WI-1. Efforts to extract surface WI-1
from the yeast demonstrated that it is fastened to the wall by non-covalent
interactions and covalent links between cysteine residues. We conclude that the
yeast cell surface adhesin WI-1 localizes to the cell wall, in part, through
extracellular release followed by high affinity binding back onto exposed chitin
fibrils. These findings point to a novel pathway of cell wall biogenesis in
yeast and an unanticipated role for chitin in anchoring and displaying a surface
adhesin and virulence determinant.
PMID: 10713109 [PubMed - indexed for MEDLINE]
526: J Biol Chem 2000 Mar 17;275(11):7887-93
HS1 interacts with Lyn and is critical for erythropoietin-induced
differentiation of erythroid cells.
Ingley E, Sarna MK, Beaumont JG, Tilbrook PA, Tsai S, Takemoto Y, Williams JH,
Klinken SP.
Laboratory for Cancer Medicine, Department of Biochemistry, the University of
Western Australia and Royal Perth Hospital, WA 6001, Western Australia,
Australia.
Erythroid cells terminally differentiate in response to erythropoietin binding
its cognate receptor. Previously we have shown that the tyrosine kinase Lyn
associates with the erythropoietin receptor and is essential for hemoglobin
synthesis in three erythroleukemic cell lines. To understand Lyn signaling
events in erythroid cells, the yeast two-hybrid system was used to analyze
interactions with other proteins. Here we show that the hemopoietic-specific
protein HS1 interacted directly with the SH3 domain of Lyn, via its proline-rich
region. A truncated HS1, bearing the Lyn-binding domain, was introduced into J2E
erythroleukemic cells to determine the impact upon responsiveness to
erythropoietin. Truncated HS1 had a striking effect on the phenotype of the J2E
line-the cells were smaller, more basophilic than the parental proerythoblastoid
cells and had fewer surface erythropoietin receptors. Moreover, basal and
erythropoietin-induced proliferation and differentiation were markedly
suppressed. The inability of cells containing the truncated HS1 to differentiate
may be a consequence of markedly reduced levels of Lyn and GATA-1. In addition,
erythropoietin stimulation of these cells resulted in rapid, endosome-mediated
degradation of endogenous HS1. The truncated HS1 also suppressed the development
of erythroid colonies from fetal liver cells. These data show that disrupting
HS1 has profoundly influenced the ability of erythroid cells to terminally
differentiate.
PMID: 10713104 [PubMed - indexed for MEDLINE]
527: Mol Biol Cell 2000 Mar;11(3):983-98
Sec24p and Iss1p function interchangeably in transport vesicle formation from
the endoplasmic reticulum in Saccharomyces cerevisiae.
Kurihara T, Hamamoto S, Gimeno RE, Kaiser CA, Schekman R, Yoshihisa T.
Department of Molecular and Cell Biology, Howard Hughes Medical Institute,
University of California, Berkeley, Berkeley, California 94720, USA.
The Sec23p/Sec24p complex functions as a component of the COPII coat in vesicle
transport from the endoplasmic reticulum. Here we characterize Saccharomyces
cerevisiae SEC24, which encodes a protein of 926 amino acids (YIL109C), and a
close homologue, ISS1 (YNL049C), which is 55% identical to SEC24. SEC24 is
essential for vesicular transport in vivo because depletion of Sec24p is lethal,
causing exaggeration of the endoplasmic reticulum and a block in the maturation
of carboxypeptidase Y. Overproduction of Sec24p suppressed the temperature
sensitivity of sec23-2, and overproduction of both Sec24p and Sec23p suppressed
the temperature sensitivity of sec16-2. SEC24 gene disruption could be
complemented by overexpression of ISS1, indicating functional redundancy between
the two homologous proteins. Deletion of ISS1 had no significant effect on
growth or secretion; however, iss1Delta mutants were found to be synthetically
lethal with mutations in the v-SNARE genes SEC22 and BET1. Moreover,
overexpression of ISS1 could suppress mutations in SEC22. These genetic
interactions suggest that Iss1p may be specialized for the packaging or the
function of COPII v-SNAREs. Iss1p tagged with His(6) at its C terminus
copurified with Sec23p. Pure Sec23p/Iss1p could replace Sec23p/Sec24p in the
packaging of a soluble cargo molecule (alpha-factor) and v-SNAREs (Sec22p and
Bet1p) into COPII vesicles. Abundant proteins in the purified vesicles produced
with Sec23p/Iss1p were indistinguishable from those in the regular COPII
vesicles produced with Sec23p/Sec24p.
PMID: 10712514 [PubMed - indexed for MEDLINE]
528: Mol Cell 2000 Jan;5(1):133-40
Mapping interactions between nuclear transport factors in living cells reveals
pathways through the nuclear pore complex.
Damelin M, Silver PA.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts, USA.
The interactions between transport receptors and proteins of the nuclear pore
complex (NPC) are fundamental to understanding nucleocytoplasmic transport. In
order to delineate the path that a particular transport receptor takes through
the NPC, we have employed fluorescence resonance energy transfer (FRET) between
enhanced cyan and yellow fluorescent proteins (ECFP, EYFP) in living cells. A
panel of yeast strains expressing functional receptor--ECFP and
nucleoporin--EYFP fusions has been analyzed with a FRET assay. With this
approach, we define points of contact in the NPC for the related importin
Pse1/Kap121 and exportin Msn5. These data demonstrate the utility of FRET in
mapping dynamic protein interactions in a genetic system. Furthermore, the data
indicate that an importin and exportin have overlapping pathways through the
NPC.
PMID: 10678175 [PubMed - indexed for MEDLINE]
529: Nucleic Acids Res 2000 Apr 1;28(7):1576-84
Scp160p, a multiple KH-domain protein, is a component of mRNP complexes in
yeast.
Lang BD, Fridovich-Keil JL.
Graduate Program in Biochemistry, Cell and Developmental Biology, Emory
University, Atlanta, GA, USA.
Scp160p is a 160 kDa protein in the yeast Saccharomyces cerevisiae that contains
14 repeats of the hnRNP K-homology (KH) domain, and demonstrates significant
sequence homology to a family of proteins collectively known as vigilins. As a
first step towards defining the function of Scp160p, we have characterized the
subcellular distribution and in vivo interactions of this protein. Using sucrose
gradient fractionation studies we have demonstrated that Scp160p in cytoplasmic
lysates is predominantly associated with polyribosomes. Furthermore, we have
found that Scp160p is released from polyribosomes by EDTA in the form of a large
complex of> or =1300 kDa that is sensitive both to RNase and NaCl. Using
affinity-chromatography to isolate these complexes, we have identified two
protein components other than Scp160p: poly(A) binding protein, Pab1p, and
Bfr1p. The presence of Pab1p confirms these complexes to be mRNPs. The presence
of Bfr1p is intriguing because the null phenotype for this gene is essentially
the same as that reported for scp160 -null cells: increased cell size and
aberrant DNA content. These results demonstrate that Scp160p associates with
polyribosome-bound mRNP complexes in vivo, implicating a role for this protein
in one or more levels of mRNA metabolism in yeast.
PMID: 10710424 [PubMed - indexed for MEDLINE]
530: Bioinformatics 1999 Oct;15(10):776-84
Genes regulated cooperatively by one or more transcription factors and their
identification in whole eukaryotic genomes.
Wagner A.
Department of Biology, University of New Mexico, Albuquerque, USA.
wagnera@unm.edu
MOTIVATION: The question addressed here is how cooperative interactions among
transcription factors (TFs), a very frequent phenomenon in eukaryotic
transcriptional regulation, can be used to identify genes that are regulated by
one or more TFs with known DNA binding specificities. Cooperativity may be
homotypic, involving binding of only one transcription factor to multiple sites
in a gene's regulatory region. It may also be heterotypic, involving binding of
more than one TF. Both types of cooperativity have in common that the binding
sites for the respective TFs form tightly linked 'clusters', groups of binding
sites often more closely associated than expected by chance alone. RESULTS: A
statistical technique suitable for the identification of statistically
significant homotypic or heterotypic TF binding site clusters in whole
eukaryotic genomes is presented. It can be used to identify genes likely to be
regulated by the TFs. Application of the technique is illustrated with two
transcription factors involved in the cell cycle and mating control of the yeast
Saccharomyces cerevisiae, indicating that the results obtained are biologically
meaningful. This rapid and inexpensive computational method of generating
hypotheses about gene regulation thus generates information that may be used to
guide subsequent costly and laborious experimental approaches, and that may aid
in the assignment of biological functions to putative open reading frames.
PMID: 10705431 [PubMed - indexed for MEDLINE]
531: Proc Natl Acad Sci U S A 2000 Feb 15;97(4):1516-20
Studies on the role of the hydrophobic domain of Ost4p in interactions with
other subunits of yeast oligosaccharyl transferase.
Kim H, Park H, Montalvo L, Lennarz WJ.
Department of Biochemistry, Institute for Cell and Developmental Biology, State
University of New York, Stony Brook, NY 11794-5215, USA.
In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT), which
catalyzes the transfer of dolichol-linked oligosaccharide chains to nascent
polypeptides in the endoplasmic reticulum, consists of nine nonidentical
membrane protein subunits. Genetic and biochemical evidence indicated these nine
proteins exist in three subcomplexes. Three of the OT subunits (Ost4p, Ost3p,
and Stt3p) have been proposed to exist in one subcomplex. To investigate the
interaction of these three membrane proteins, initially we carried out a
mutational analysis of Ost4p, which is an extraordinarily small membrane protein
containing only 36 amino acid residues. This analysis indicated that when single
amino acid residues in a region close to the luminal face of the putative
transmembrane domain of Ost4p were changed into an ionizable amino acid such as
Lys or Asp, growth at 37 degrees C and OT activity measured in vitro were
impaired. In addition, using immunoprecipitation techniques and Western blot
analysis, we found that with these mutations the interaction between Ost4p,
Ost3p, and Stt3p was disrupted. Introduction of Lys or Asp residues at other
positions in the putative transmembrane domain or at the N or C terminus of
Ost4p had no effect on disrupting subunit interactions or impairing the activity
of OT. These findings suggest that a localized region of the putative
transmembrane domain of Ost4p mediates in stabilization of the interaction with
the two other OT subunits (Ost3p and Stt3p) in a subcomplex in the endoplasmic
reticulum membrane.
PMID: 10677492 [PubMed - indexed for MEDLINE]
532: Proc Natl Acad Sci U S A 2000 Mar 14;97(6):2491-6
The interaction of nitric oxide (NO) with the yeast transcription factor Ace1: A
model system for NO-protein thiol interactions with implications to metal
metabolism.
Shinyashiki M, Chiang KT, Switzer CH, Gralla EB, Valentine JS, Thiele DJ, Fukuto
JM.
Department of Pharmacology, University of California at Los Angeles Medical
School, Center for the Health Sciences, Los Angeles, CA 90095-1735, USA.
Nitric oxide (NO) was found to inhibit the copper-dependent induction of the
yeast CUP1 gene. This effect is attributable to an inhibition of the
copper-responsive CUP1 transcriptional activator Ace1. A mechanism is proposed
whereby the metal binding thiols of Ace1 are chemically modified via NO- and
O(2)-dependent chemistry, thereby diminishing the ability of Ace1 to bind and
respond to copper. Moreover, it is proposed that demetallated Ace1 is
proteolytically degraded in the cell, resulting in a prolonged inhibition of
copper-dependent CUP1 induction. These findings indicate that NO may serve as a
disrupter of yeast copper metabolism. More importantly, considering the
similarity of Ace1 to other mammalian metal-binding proteins, this work lends
support to the hypothesis that NO may regulate/disrupt metal homeostasis under
both normal physiological and pathophysiological circumstances.
PMID: 10694579 [PubMed - indexed for MEDLINE]
533: Genes Dev 2000 Feb 15;14(4):493-503
Progression of meiotic DNA replication is modulated by interchromosomal
interaction proteins, negatively by Spo11p and positively by Rec8p.
Cha RS, Weiner BM, Keeney S, Dekker J, Kleckner N.
Department of Molecular Biology, Harvard University, Cambridge, Massachusetts
02138 USA.
Spo11p is a key mediator of interhomolog interactions during meiosis. Deletion
of the SPO11 gene decreases the length of S phase by approximately 25%. Rec8p is
a key coordinator of meiotic interhomolog and intersister interactions. Deletion
of the REC8 gene increases S-phase length, by approximately 10% in wild-type and
approximately 30% in a spo11Delta background. Thus, the progression of DNA
replication is modulated by interchromosomal interaction proteins. The
spo11-Y135F DSB (double strand break) catalysis-defective mutant is normal for
S-phase modulation and DSB-independent homolog pairing but is defective for
later events, formation of DSBs, and synaptonemal complexes. Thus, earlier and
later functions of Spo11 are defined. We propose that meiotic S-phase
progression is linked directly to development of specific chromosomal features
required for meiotic interhomolog interactions and that this feedback process is
built upon a more fundamental mechanism, common to all cell types, by which
S-phase progression is coupled to development of nascent intersister connections
and/or related aspects of chromosome morphogenesis. Roles for Rec8 and/or Spo11
in progression through other stages of meiosis are also revealed.
PMID: 10691741 [PubMed - indexed for MEDLINE]
534: Proc Natl Acad Sci U S A 2000 Feb 29;97(5):2373-8
The movement protein NSm of tomato spotted wilt tospovirus (TSWV): RNA binding,
interaction with the TSWV N protein, and identification of interacting plant
proteins.
Soellick T, Uhrig JF, Bucher GL, Kellmann JW, Schreier PH.
Max-Planck-Institut fur Zuchtungsforschung, Carl-von-Linne-Weg 10, D-50829 Koln,
Germany.
The nonstructural NSm protein of tomato spotted wilt tospovirus (TSWV)
represents a putative viral movement protein involved in cell-to-cell movement
of nonenveloped ribonucleocapsid structures. To study the molecular basis of NSm
function, we expressed the protein in Escherichia coli and investigated
protein-protein and protein-RNA interactions of NSm protein in vitro. NSm
specifically interacts with TSWV N protein and binds single-stranded RNA in a
sequence-nonspecific manner. Using NSm as a bait in a yeast two-hybrid screen,
we identified two homologous NSm-binding proteins of the DnaJ family from
Nicotiana tabacum and Arabidopsis thaliana.
PMID: 10688879 [PubMed - indexed for MEDLINE]
535: Biochemistry 2000 Feb 22;39(7):1716-24
Catalytic and DNA binding properties of the ogg1 protein of Saccharomyces
cerevisiae: comparison between the wild type and the K241R and K241Q active-site
mutant proteins.
Guibourt N, Castaing B, Van Der Kemp PA, Boiteux S.
Departement de Radiobiologie et Radiopathologie, UMR 217 CNRS-CEA "Radiobiologie
Moleculaire et Cellulaire", Commissariat a l'Energie Atomique, DSV, BP6,
92265-Fontenay aux Roses, France.
The Ogg1 protein of Saccharomyces cerevisiae belongs to a family of DNA
glycosylases and apurinic/apyrimidinic site (AP) lyases, the signature of which
is the alpha-helix-hairpin-alpha-helix-Gly/Pro-Asp (HhH-GPD) active site motif
together with a conserved catalytic lysine residue, to which we refer as the
HhH-GPD/K family. In the yeast Ogg1 protein, yOgg1, the HhH-GPD/K motif spans
residues 225-260 and the conserved lysine is K241. In this study, we have
purified the K241R and K241Q mutant proteins and compared their catalytic and
DNA binding properties to that of the wild-type yOgg1. The results show that the
K241R mutation greatly impairs both the DNA glycosylase and the AP lyase
activities of yOgg1. Specificity constants for cleavage of a 34mer
oligodeoxyribonucleotide containing a 7,8-dihydro-8-oxoguanine (8-OxoG) paired
with a cytosine, [8-OxoG.C], are 56 x 10(-)(3) and 5 x 10(-)(3) min(-)(1)
nM(-)(1) for the wild-type and the K241R protein, respectively. On the other
hand, the K241Q mutation abolishes the DNA glycosylase and AP lyase activities
of yOgg1. In contrast, the K241R and K241Q proteins have conserved wild-type DNA
binding properties. K(dapp) values for binding of [8-OxoG.C] are 6.9, 7.4, and
4.8 nM for the wild-type, K241R, and K241Q proteins, respectively. The results
also show that AP site analogues such as 1, 3-propanediol (Pr), tetrahydrofuran
(F), or cyclopentanol (Cy) are not substrates but constitute good inhibitors of
the wild-type yOgg1. Therefore, we have used a 59mer [Pr.C] duplex to further
analyze the DNA binding properties of the wild-type, K241R, and K241Q proteins.
Hydroxyl radical footprints of the wild-type yOgg1 show strong protection of six
nucleotides centered around the Pr lesion in the damaged strand. On the
complementary strand, only the cytosine placed opposite Pr was strongly
protected. The same footprints were observed with the K241R and K241Q proteins,
confirming their wild-type DNA binding properties. These results indicate that
the K241Q mutant protein can be used to study interactions between yOgg1 and DNA
containing metabolizable substrates such as 8-OxoG or an AP site.
PMID: 10677220 [PubMed - indexed for MEDLINE]
536: Mol Cell Biol 2000 Mar;20(6):2209-17
Fourteen residues of the U1 snRNP-specific U1A protein are required for
homodimerization, cooperative RNA binding, and inhibition of polyadenylation.
Klein Gunnewiek JM, Hussein RI, van Aarssen Y, Palacios D, de Jong R, van
Venrooij WJ, Gunderson SI.
Department of Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The
Netherlands.
It was previously shown that the human U1A protein, one of three U1 small
nuclear ribonucleoprotein-specific proteins, autoregulates its own production by
binding to and inhibiting the polyadenylation of its own pre-mRNA. The U1A
autoregulatory complex requires two molecules of U1A protein to cooperatively
bind a 50-nucleotide polyadenylation-inhibitory element (PIE) RNA located in the
U1A 3' untranslated region. Based on both biochemical and nuclear magnetic
resonance structural data, it was predicted that protein-protein interactions
between the N-terminal regions (amino acids [aa] 1 to 115) of the two U1A
proteins would form the basis for cooperative binding to PIE RNA and for
inhibition of polyadenylation. In this study, we not only experimentally
confirmed these predictions but discovered some unexpected features of how the
U1A autoregulatory complex functions. We found that the U1A protein
homodimerizes in the yeast two-hybrid system even when its ability to bind RNA
is incapacitated. U1A dimerization requires two separate regions, both located
in the N-terminal 115 residues. Using both coselection and gel mobility shift
assays, U1A dimerization was also observed in vitro and found to depend on the
same two regions that were found in vivo. Mutation of the second
homodimerization region (aa 103 to 115) also resulted in loss of inhibition of
polyadenylation and loss of cooperative binding of two U1A protein molecules to
PIE RNA. This same mutation had no effect on the binding of one U1A protein
molecule to PIE RNA. A peptide containing two copies of aa 103 to 115 is a
potent inhibitor of polyadenylation. Based on these data, a model of the U1A
autoregulatory complex is presented.
PMID: 10688667 [PubMed - indexed for MEDLINE]
537: Nature 2000 Feb 10;403(6770):623-7
Comment in:
Nature. 2000 Feb 10;403(6770):601-3.
A comprehensive analysis of protein-protein interactions in Saccharomyces
cerevisiae.
Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D,
Narayan V, Srinivasan M, Pochart P, Qureshi-Emili A, Li Y, Godwin B, Conover D,
Kalbfleisch T, Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg JM.
Department of Genetics, University of Washington, Seattle 98195-7360, USA.
Two large-scale yeast two-hybrid screens were undertaken to identify
protein-protein interactions between full-length open reading frames predicted
from the Saccharomyces cerevisiae genome sequence. In one approach, we
constructed a protein array of about 6,000 yeast transformants, with each
transformant expressing one of the open reading frames as a fusion to an
activation domain. This array was screened by a simple and automated procedure
for 192 yeast proteins, with positive responses identified by their positions in
the array. In a second approach, we pooled cells expressing one of about 6,000
activation domain fusions to generate a library. We used a high-throughput
screening procedure to screen nearly all of the 6,000 predicted yeast proteins,
expressed as Gal4 DNA-binding domain fusion proteins, against the library, and
characterized positives by sequence analysis. These approaches resulted in the
detection of 957 putative interactions involving 1,004 S. cerevisiae proteins.
These data reveal interactions that place functionally unclassified proteins in
a biological context, interactions between proteins involved in the same
biological function, and interactions that link biological functions together
into larger cellular processes. The results of these screens are shown here.
PMID: 10688190 [PubMed - indexed for MEDLINE]
538: Biopolymers 2000 Apr 5;53(4):293-307
Osmolyte-induced changes in protein conformational equilibria.
Saunders AJ, Davis-Searles PR, Allen DL, Pielak GJ, Erie DA.
Department of Biochemistry and Biophysics, University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599, USA.
Examining solute-induced changes in protein conformational equilibria is a
long-standing method for probing the role of water in maintaining protein
stability. Interpreting the molecular details governing the solute-induced
effects, however, remains controversial. We present experimental and theoretical
data for osmolyte-induced changes in the stabilities of the A and N states of
yeast iso-1-ferricytochrome c. Using polyol osmolytes of increasing size, we
observe that osmolytes alone induce A-state formation from acid-denatured
cytochrome c and N state formation from the thermally denatured protein. The
stabilities of the A and N states increase linearly with osmolyte concentration.
Interestingly, osmolytes stabilize the A state to a greater degree than the N
state. To interpret the data, we divide the free energy for the reaction into
contributions from nonspecific steric repulsions (excluded volume effects) and
from binding interactions. We use scaled particle theory (SPT) to estimate the
free energy contributions from steric repulsions, and we estimate the
contributions from water-protein and osmolyte-protein binding interactions by
comparing the SPT calculations to experimental data. We conclude that excluded
volume effects are the primary stabilizing force, with changes in water-protein
and solute-protein binding interactions making favorable contributions to
stability of the A state and unfavorable contributions to the stability of the N
state. The validity of our interpretation is strengthened by analysis of data on
osmolyte-induced protein stabilization from the literature, and by comparison
with other analyses of solute-induced changes in conformational equilibria.
Copyright 2000 John Wiley & Sons, Inc.
PMID: 10685050 [PubMed - indexed for MEDLINE]
539: Nucleic Acids Res 2000 Mar 15;28(6):1407-17
A new double-stranded RNA-binding protein that interacts with PKR.
Coolidge CJ, Patton JG.
Department of Molecular Biology, Box 1820, Station B, Vanderbilt University,
Nashville, TN 37235, USA.
We have identified a 74 kDa double-stranded (ds)RNA-binding protein that shares
extensive homology with the mouse spermatid perinuclear RNA-binding (Spnr)
protein. p74 contains two dsRNA-binding motifs (dsRBMs) that are essential for
preferential binding to dsRNA. Previously, dsRNA-binding proteins were shown to
undergo homo- and heterodimerization, raising the possibility that regulation of
activity could be controlled by interactions between different family members.
Homodimerization is required to activate the dsRNA-dependent protein kinase PKR,
whereas hetero-dimerization between PKR and other dsRNA-binding proteins can
inhibit kinase activity. We have found that p74 also interacts with PKR, both
the wild-type enzyme and a catalytically defective mutant (K296R). While
co-expression of p74 and wild-type PKR in the yeast Saccharomyces cerevisiae did
not alter PKR activity, co-expression of p74 and the catalytically defective
K296R mutant surprisingly resulted in abnormal morphology and cell death in
transformants that maintained a high level of p74 expression. These
transformants could be rescued by overexpression of the alpha-subunit of
wild-type eukaryotic translation initiation factor 2 (eIF2alpha), one of the
known substrates for PKR. We hypothesize that competing heterodimers between
p74-K296R PKR and eIF2alpha-K296R PKR may control cell growth such that
stabilization of the p74-K296R PKR heterodimer induces abnormal morphology and
cell death.
PMID: 10684936 [PubMed - indexed for MEDLINE]
540: Nucleic Acids Res 2000 Mar 15;28(6):1332-9
Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli
3-methyladenine-DNA glycosylases with DNA containing dIMP residues.
Saparbaev M, Mani JC, Laval J.
Groupe 'Reparation des lesions Radio- et Chimio-Induites', UMR 8532 CNRS,
Institut Gustave Roussy, 94805 Villejuif Cedex, France.
In DNA, the deamination of dAMP generates 2'-deoxy-inosine 5'-monophosphate
(dIMP). Hypoxanthine (HX) residues are mutagenic since they give rise to
A.T-->G.C transition. They are excised, although with different efficiencies, by
an activity of the 3-methyl-adenine (3-meAde)-DNA glycosylases from Escherichia
coli (AlkA protein), human cells (ANPG protein), rat cells (APDG protein) and
yeast (MAG protein). Comparison of the kinetic constants for the excision of HX
residues by the four enzymes shows that the E.coli and yeast enzymes are quite
inefficient, whereas for the ANPG and the APDG proteins they repair the HX
residues with an efficiency comparable to that of alkylated bases, which are
believed to be the primary substrates of these DNA glycosylases. Since the use
of various substrates to monitor the activity of HX-DNA glycosylases has
generated conflicting results, the efficacy of the four 3-meAde-DNA glycosylases
of different origin was compared using three different substrates. Moreover,
using oligo-nucleotides containing a single dIMP residue, we investigated a
putative sequence specificity of the enzymes involving the bases next to the HX
residue. We found up to 2-5-fold difference in the rates of HX excision between
the various sequences of the oligonucleotides studied. When the dIMP residue was
placed opposite to each of the four bases, a preferential recognition of dI:T
over dI:dG, dI:dC and dI:dA mismatches was observed for both human (ANPG) and
E.coli (AlkA) proteins. At variance, the yeast MAG protein removed more
efficiently HX from a dI:dG over dI:dC, dI:T and dI:dA mismatches.
PMID: 10684927 [PubMed - indexed for MEDLINE]
541: J Biol Chem 2000 Feb 25;275(8):5767-72
The assembly factor Atp11p binds to the beta-subunit of the mitochondrial
F(1)-ATPase.
Wang ZG, Ackerman SH.
Department of Surgery, Wayne State University School of Medicine, Detroit,
Michigan 48201, USA.
Atp11p is a protein of Saccharomyces cerevisiae required for the assembly of the
F(1) component of the mitochondrial F(1)F(0)-ATP synthase. This study presents
evidence that Atp11p binds selectively to the beta-subunit of F(1). Under
conditions in which avidin-Sepharose beads specifically adsorbed biotinylated
Atp11p from yeast mitochondrial extracts, the F(1) beta-subunit coprecipitated
with the tagged Atp11p protein. Binding interactions between Atp11p and the
entire beta-subunit of F(1) or fragments of the beta-subunit were also revealed
by a yeast two-hybrid screen: Atp11p bound to a region of the nucleotide-binding
domain of the beta-subunit located between Gly(114) and Leu(318). Certain
elements of this sequence that would be accessible to Atp11p in the free
beta-subunit make contact with adjacent alpha-subunits in the assembled enzyme.
This observation suggests that the alpha-subunits may exchange for bound Atp11p
during the process of F(1) assembly.
PMID: 10681564 [PubMed - indexed for MEDLINE]
542: Proc Natl Acad Sci U S A 2000 Feb 29;97(5):2011-6
Anatomy of a proficient enzyme: the structure of orotidine 5'-monophosphate
decarboxylase in the presence and absence of a potential transition state
analog.
Miller BG, Hassell AM, Wolfenden R, Milburn MV, Short SA.
Department of Biochemistry, University of North Carolina, Chapel Hill, NC 27599,
USA. Research Triangle Park, NC 27709, USA.
Orotidine 5'-phosphate decarboxylase produces the largest rate enhancement that
has been reported for any enzyme. The crystal structure of the recombinant
Saccharomyces cerevisiae enzyme has been determined in the absence and presence
of the proposed transition state analog 6-hydroxyuridine 5'-phosphate, at a
resolution of 2.1 A and 2.4 A, respectively. Orotidine 5'-phosphate
decarboxylase folds as a TIM-barrel with the ligand binding site near the open
end of the barrel. The binding of 6-hydroxyuridine 5'-phosphate is accompanied
by protein loop movements that envelop the ligand almost completely, forming
numerous favorable interactions with the phosphoryl group, the ribofuranosyl
group, and the pyrimidine ring. Lysine-93 appears to be anchored in such a way
as to optimize electrostatic interactions with developing negative charge at C-6
of the pyrimidine ring, and to donate the proton that replaces the carboxylate
group at C-6 of the product. In addition, H-bonds from the active site to O-2
and O-4 help to delocalize negative charge in the transition state. Interactions
between the enzyme and the phosphoribosyl group anchor the pyrimidine within the
active site, helping to explain the phosphoribosyl group's remarkably large
contribution to catalysis despite its distance from the site of decarboxylation.
PMID: 10681417 [PubMed - indexed for MEDLINE]
543: EMBO J 2000 Feb 15;19(4):683-90
The novel coactivator C1 (HCF) coordinates multiprotein enhancer formation and
mediates transcription activation by GABP.
Vogel JL, Kristie TM.
Laboratory of Viral Diseases, National Institutes of Health, Building 4, Room
133, 4 Center Drive, Bethesda, MD 20892, USA.
Transcription of the herpes simplex virus 1 (HSV-1) immediate early (IE) genes
is determined by multiprotein enhancer complexes. The core enhancer assembly
requires the interactions of the POU-homeodomain protein Oct-1, the viral
transactivator alphaTIF and the cellular factor C1 (HCF). In this context, the
C1 factor interacts with each protein to assemble the stable enhancer complex.
In addition, the IE enhancer cores contain adjacent binding sites for other
cellular transcription factors such as Sp1 and GA-binding protein (GABP). In
this study, a direct interaction of the C1 factor with GABP is demonstrated,
defining the C1 factor as the critical coordinator of the enhancer complex
assembly. In addition, mutations that reduce the GABP transactivation potential
also impair the C1-GABP interaction, indicating that the C1 factor functions as
a novel coactivator of GABP-mediated transcription. The interaction and
coordinated assembly of the enhancer proteins by the C1 factor may be critical
for the regulation of the HSV lytic-latent cycle.
PMID: 10675337 [PubMed - indexed for MEDLINE]
544: EMBO J 2000 Feb 15;19(4):581-8
Crystal structure of a class I alpha1,2-mannosidase involved in N-glycan
processing and endoplasmic reticulum quality control.
Vallee F, Lipari F, Yip P, Sleno B, Herscovics A, Howell PL.
Structural Biology and Biochemistry, Research Institute, The Hospital for Sick
Children, 555 University Avenue, Toronto, M5G 1X8, Ontario.
Mannose trimming is not only essential for N-glycan maturation in mammalian
cells but also triggers degradation of misfolded glycoproteins. The crystal
structure of the class I alpha1, 2-mannosidase that trims Man(9)GlcNAc(2) to
Man(8)GlcNAc(2 )isomer B in the endoplasmic reticulum of Saccharomyces
cerevisiae reveals a novel (alphaalpha)(7)-barrel in which an N-glycan from one
molecule extends into the barrel of an adjacent molecule, interacting with the
essential acidic residues and calcium ion. The observed protein-carbohydrate
interactions provide the first insight into the catalytic mechanism and
specificity of this eukaryotic enzyme family and may be used to design
inhibitors that prevent degradation of misfolded glycoproteins in genetic
diseases.
PMID: 10675327 [PubMed - indexed for MEDLINE]
545: Gene 2000 Jan 11;241(2):309-15
A highly representative two-hybrid genomic library for the yeast Yarrowia
lipolytica.
Kabani M, Boisrame A, Beckerich JM, Gaillardin C.
Laboratoire de Genetique Moleculaire et Cellulaire, INRA-INA.PG-CNRS BP 01
78850, Thiverval-Grignon, France. kabani@platon.grignon.inra.fr
Since its description by Fields and Song in 1989 (Nature 340, 245-246), the
yeast two-hybrid system has been used extensively to study protein-protein
interactions, becoming increasingly efficient with technological and
methodological improvements. Here, we report the construction of a highly
representative two-hybrid genomic library for the dimorphic yeast Yarrowia
lipolytica based on the system described by James et al. (1996. Genetics 144,
1425-1436). The endoplasmic reticulum protein Slslp was then used as a bait in a
functional test of the library. Indeed, we previously showed that the SLS1 gene
product is involved in protein translocation across the endoplasmic reticulum
membrane and interacts physically in a two-hybrid assay with Kar2p, an essential
luminal member of the HSP70 family (Boisrame et al., 1998. J. Biol. Chem. 273,
30 903-30 908). We developed a mating strategy similar to that used for the
Saccharomyces cerevisiae FRYL library (Fromont-Racine et al., 1997. Nat. Genet.
16, 277-282). No other partner than Kar2p was identified in this screen. As an
interesting result, Kar2p interacts with Slslp through its ATPase domain,
supporting our hypothesis that Slslp is a cofactor of the chaperone protein,
modulating its activity during the HSP70 cycle. Our results indicate that we
have constructed a new and powerful tool for the study of Yarrowia lipolytica,
which we believe is a good alternative model to investigate such complex
biological processes as secretion pathways.
PMID: 10675043 [PubMed - indexed for MEDLINE]
546: Methods 2000 Feb;20(2):219-31
Identification of connexin-interacting proteins: application of the yeast
two-hybrid screen.
Jin C, Lau AF, Martyn KD.
Molecular Carcinogenesis, Cancer Research Center of Hawaii, University of Hawaii
at Manoa, 1236 Lauhala Street, Room 304, Honolulu, Hawaii 96813, USA.
Protein-protein interactions are recognized as one of the fundamental mechanisms
for relaying the intra- and intercellular signals that are required for normal
cellular activities affecting growth, development, and maintenance of
homeostasis in tissues and organs. The yeast two-hybrid screen has become a
valuable tool for identifying protein-protein interactions. The gap junction
protein connexin 43 (Cx43) has been implicated in a number of biological
processes including development and cellular growth control. To further advance
our understanding of the ways in which Cx43 may influence these cellular
activities, and to extend our knowledge of the regulation of Cx43 function
and/or processing, we have employed the yeast two-hybrid screen technique to
identify Cx43-interacting proteins. We present detailed methods for the yeast
two-hybrid screen of a mouse embryonic cDNA library using the C terminus of Cx43
as "bait." We also describe additional methods to confirm the interactions
between Cx43 and the identified proteins. These methods include in vitro binding
assays, coimmunoprecipitation, and subcellular localization using
immunofluorescence microscopy. Copyright 2000 Academic Press.
PMID: 10671315 [PubMed - indexed for MEDLINE]
547: Biol Cell 1999 Dec;91(9):649-63
The Saccharomyces cerevisiae Cdc14 phosphatase is implicated in the structural
organization of the nucleolus.
de Almeida A, Raccurt I, Peyrol S, Charbonneau M.
UMR CNRS/ENS no 5665, Ecole Normale Superieure, Lyon, France.
Cdc14, a dual-specificity protein phosphatase, has been previously implicated in
triggering exit from mitosis in the yeast Saccharomyces cerevisiae. Using
immunofluorescence microscopy and immunogold labeling, we demonstrate that a
functional HA-tagged version of the phosphatase Cdc14 localizes to the
nucleolus. Moreover, Cdc14-HA co-localized with the nucleolar NOP2 and GAR1
proteins. By immunofluorescence, Cdc14-HA was found in the nucleolus during most
of the mitotic cell cycle, except during anaphase-telophase when it
redistributed along the mitotic spindle. While this work was in progress, the
same pattern of Cdc14 localization was described by others (Visintin et al,
Nature 398 (1999) 818). Constitutive overexpression of CDC14 was toxic and led
to cell cycle arrest of cells, mainly in G1. This correlated with the appearance
of abnormal nuclear structures. A genetic search for suppressors of the
lethality associated with CDC14 overexpression identified YJL076W. Because
overproduction of Yj1076w buffered the toxic effect of Cdc14 overproduction,
this suggested that it might be a substrate of Cdc14. This has indeed been found
to be the case by others who recently described Yj1076w/Netl as a nucleolar
protein that physically associates with Cdc14 (Shou et al, Cell 97 (1999) 233).
The present data confirm several recently uncovered aspects of the regulation of
Cdc14 localization and activity and suggest that the level of expression of
CDC14 influences the structural organization of the nucleolus.
PMID: 10668096 [PubMed - indexed for MEDLINE]
548: J Virol 2000 Mar;74(5):2372-82
A chimeric protein containing the N terminus of the adeno-associated virus Rep
protein recognizes its target site in an in vivo assay.
Cathomen T, Collete D, Weitzman MD.
Laboratory of Genetics, The Salk Institute for Biological Studies, San Diego,
California 92186, USA.
The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved
in DNA replication, regulation of gene expression, and targeting site-specific
integration. They bind to a specific Rep recognition sequence (RRS) found in
both the viral inverted terminal repeats and the AAVS1 integration locus on
human chromosome 19. Previous in vitro studies implied that an N-terminal
segment of Rep is involved in DNA recognition, while additional domains might
stabilize binding and mediate multimerization. In order to define the minimal
requirements for Rep to recognize its target site in the human genome, we
developed one-hybrid assays in which DNA-protein interactions are detected in
vivo. Chimeric proteins consisting of the N terminus of Rep fused to different
oligomerization motifs and a transcriptional activation domain were analyzed for
oligomerization, DNA binding, and activation of reporter gene expression.
Expression of reporter genes was driven from RRS motifs cloned upstream of
minimal promoters and examined in mammalian cells from transfected plasmids and
in Saccharomyces cerevisiae from a reporter cassette integrated into the yeast
genome. Our results show for the first time that chimeric proteins containing
the amino-terminal 244 residues of Rep are able to target the RRS in vitro and
in vivo when incorporated into artificial multimers. These studies suggest that
chimeric proteins may be used to harness the unique targeting feature of AAV for
gene therapy applications.
PMID: 10666268 [PubMed - indexed for MEDLINE]
549: Curr Biol 2000 Jan 27;10(2):111-4
Two paralogs involved in transcriptional silencing that antagonistically control
yeast life span.
Roy N, Runge KW.
Department of Molecular Biology, The Lerner Research Institute, Cleveland Clinic
Foundation, Cleveland, OH 44195, USA.
In the yeast Saccharomyces cerevisiae, one determinant of aging or life span is
the accumulation of extrachromosomal copies of rDNA circles in old mother cells
[1]. The production of rDNA circles depends upon intrachromosomal recombination
within the rDNA tandem array, a process regulated by the protein Sir2 (Sir2p).
Together with Sir1p, Sir3p, Sir4p and Orc1p, Sir2p is also involved in
transcriptional silencing of genes at the silent mating-type cassettes, in the
rDNA array, and at telomeres. Using a 'triple silencer' strain that can monitor
an increase or decrease in gene expression at these three loci, we found that
deletion of the ZDS1 gene caused an increase in silencing in the rDNA and at a
silent mating-type cassette at the expense of telomere silencing. The zds1
deletion also resulted in an increase in life span and a decrease in Sir3p
phosphorylation. In contrast, deletion of its paralog ZDS2 caused a decrease in
rDNA silencing, a decrease in life span and an increase in Sir3p
phosphorylation. As Zds2p, but not Zds1p, had strong two-hybrid interactions
with Orc1p and the four Sir proteins, Zds1p might indirectly control Sir3p
through a Sir3p kinase.
PMID: 10662670 [PubMed - indexed for MEDLINE]
550: Virology 2000 Feb 15;267(2):185-98
Multiple interactions among proteins encoded by the mite-transmitted wheat
streak mosaic tritimovirus.
Choi IR, Stenger DC, French R.
School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68583,
USA.
The genome organization of the mite-transmitted wheat streak mosaic virus (WSMV)
appears to parallel that of members of the Potyviridae with monopartite genomes,
but there are substantial amino acid dissimilarities with other potyviral
polyproteins. To initiate studies on the functions of WSMV-encoded proteins, a
protein interaction map was generated using a yeast two-hybrid system. Because
the pathway of proteolytic maturation of the WSMV polyprotein has not been
experimentally determined, random libraries of WSMV cDNA were made both in
DNA-binding domain and activation domain plasmid vectors and introduced into
yeast. Sequence analysis of multiple interacting pairs revealed that
interactions largely occurred between domains within two groups of proteins. The
first involved interactions among nuclear inclusion protein a, nuclear inclusion
protein b, and coat protein (CP), and the second involved helper
component-proteinase (HC-Pro) and cylindrical inclusion protein (CI). Further
immunoblot and deletion mapping analyses of the interactions suggest that
subdomains of CI, HC-Pro, and P1 interact with one another. The two-hybrid assay
was then performed using full-length genes of CI, HC-Pro, P1, P3, and CP, but no
heterologous interactions were detected. In vitro binding assay using
glutathione-S-transferase fusion proteins and in vitro translation products,
however, revealed mutual interactions among CI, HC-Pro, P1, and P3. The failure
to detect interactions between full-length proteins by the two-hybrid assay
might be due to adverse effects of expression of viral proteins in yeast cells.
The capacity to participate in multiple homomeric and heteromeric molecular
interactions is consistent with the pleiotropic nature of many potyviral gene
mutants and suggests mechanisms for regulation of various viral processes via a
network of viral protein complexes. Copyright 2000 Academic Press.
PMID: 10662614 [PubMed - indexed for MEDLINE]
551: J Cell Biol 2000 Feb 7;148(3):441-52
Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in
budding yeast.
Segal M, Clarke DJ, Maddox P, Salmon ED, Bloom K, Reed SI.
Department of Molecular Biology, MB7, The Scripps Research Institute, La Jolla,
California 92037, USA.
The orientation of the mitotic spindle along a polarity axis is critical in
asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss
of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent
kinase activity (cdc28-4 clb5Delta cells) causes a spindle positioning defect
that results in an undivided nucleus entering the bud. Based on time-lapse
digital imaging microscopy of microtubules labeled with green fluorescent
protein fusions to either tubulin or dynein, we observed that the asymmetric
behavior of the spindle pole bodies during spindle assembly was lost in the
cdc28-4 clb5Delta cells. As soon as a spindle formed, both poles were equally
likely to interact with the bud cell cortex. Persistent dynamic interactions
with the bud ultimately led to spindle translocation across the bud neck. Thus,
the mutant failed to assign one spindle pole body the task of organizing astral
microtubules towards the mother cell. Our data suggest that Clb5p-associated
kinase is required to confer mother-bound behavior to one pole in order to
establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and
Clb4p, though partially redundant with Clb5p for an early role in spindle
morphogenesis, preferentially promote spindle assembly.
PMID: 10662771 [PubMed - indexed for MEDLINE]
552: Mol Gen Genet 2000 Jan;262(6):1147-56
SLG1 plays a role during G1 in the decision to enter or exit the cell cycle.
Ivanovska I, Rose MD.
Department of Molecular Biology, Princeton University, NJ 08544-1014, USA.
Saccharomyces cerevisiae cells decide to divide during G1. If nutrients are
abundant, cells pass through START and coordinately undergo DNA replication, bud
emergence, and spindle pole body duplication. Phenotypic analysis of the
slg1delta mutant revealed that this mutation uncouples post-START events. At the
nonpermissive temperature, slg1delta cells that have undergone bud emergence but
not DNA replication or SPB duplication accumulate. Furthermore, while wild-type
cells arrest in GO when starved, the slg1delta mutant fails to arrest at this
point; instead, cells with small buds accumulate. The slg1delta mutation
displayed genetic interactions with cdc34, which encodes a regulator of exit
from G1. This is consistent with a role of SLG1 in G1 regulation. Epitope-tagged
Slg1p cofractionated with the plasma membrane, suggesting that Slglp may
function by integrating external cues and relaying them to the interior of the
cell. We propose that SLG1 plays a regulatory role in bud emergence or
stationary phase.
PMID: 10660075 [PubMed - indexed for MEDLINE]
553: J Mol Biol 2000 Feb 11;296(1):7-17
Domain III of Saccharomyces cerevisiae 25 S ribosomal RNA: its role in binding
of ribosomal protein L25 and 60 S subunit formation.
van Beekvelt CA, Kooi EA, de Graaff-Vincent M, Riet J, Venema J, Raue HA.
Department of Biochemistry and Molecular Biology, IMBW BioCentrum Amsterdam,
Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Domain III of Saccharomyces cerevisiae 25 S rRNA contains the recognition site
for the primary rRNA-binding ribosomal protein L25, which belongs to the
functionally conserved EL23/L25 family of ribosomal proteins. The EL23/L25
binding region is very complex, consisting of several irregular helices held
together by long-distance secondary and tertiary interactions. Moreover, it
contains the eukaryote-specific V9 (D7a) expansion segment. Functional
characterisation of the structural elements of this site by a detailed in vitro
and in vivo mutational analysis indicates the presence of two separate regions
that are directly involved in L25 binding. In particular, mutation of either of
two conserved nucleotides in the loop of helix 49 significantly reduces in vitro
L25 binding, thus strongly supporting their role as attachment sites for the
r-protein. Two other helices appear to be primarily required for the correct
folding of the binding site. Mutations that abolish in vitro binding of L25
block accumulation of 25 S rRNA in vivo because they stall pre-rRNA processing
at the level of its immediate precursor, the 27 S(B) pre-rRNA. Surprisingly,
several mutations that do not significantly affect L25 binding in vitro cause
the same lethal defect in 27 S(B) pre-rRNA processing. Deletion of the V9
expansion segment also leads to under-accumulation of mature 25 S rRNA and a
twofold reduction in growth rate. We conclude that an intact domain III,
including the V9 expansion segment, is essential for normal processing and
assembly of 25 S rRNA. Copyright 2000 Academic Press.
PMID: 10656814 [PubMed - indexed for MEDLINE]
554: J Mol Biol 2000 Jan 28;295(4):927-38
X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity,
and identification of framework interactions determining cation sensitivity.
Albert A, Yenush L, Gil-Mascarell MR, Rodriguez PL, Patel S, Martinez-Ripoll M,
Blundell TL, Serrano R.
Grupo de Cristalografia Macromolecular y Biologia Estructural, Instituto de
Quimica Fisica "Rocasolano", Consejo Superior de Investigaciones Cientificas,
Serrano 119, Madrid, E-28006, Spain. xalbert@iqfr.csic.es
The product of the yeast HAL2 gene (Hal2p) is an in vivo target of sodium and
lithium toxicity and its overexpression improves salt tolerance in yeast and
plants. Hal2p is a metabolic phosphatase which catalyses the hydrolysis of
3'-phosphoadenosine-5'-phosphate (PAP) to AMP. It is, the prototype of an
evolutionarily conserved family of PAP phosphatases and the engineering of
sodium insensitive enzymes of this group may contribute to the generation of
salt-tolerant crops. We have solved the crystal structure of Hal2p in complex
with magnesium, lithium and the two products of PAP hydrolysis, AMP and Pi, at
1.6 A resolution. A functional screening of random mutations of the HAL2 gene in
growing yeast generated forms of the enzyme with reduced cation sensitivity.
Analysis of these mutants defined a salt bridge (Glu238 ellipsis Arg152) and a
hydrophobic bond (Va170 ellipsis Trp293) as important framework interactions
determining cation sensitivity. Hal2p belongs to a larger superfamily of
lithium-sensitive phosphatases which includes inositol monophosphatase. The
hydrophobic interaction mutated in Hal2p is conserved in this superfamily and
its disruption in human inositol monophosphatase also resulted in reduced cation
sensitivity. Copyright 2000 Academic Press.
PMID: 10656801 [PubMed - indexed for MEDLINE]
555: Nat Struct Biol 2000 Feb;7(2):113-7
The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor
into a helix.
Li M, Phylip LH, Lees WE, Winther JR, Dunn BM, Wlodawer A, Kay J, Gustchina A.
Macromolecular Crystallography Laboratory, Program in Structural Biology,
National Cancer Institute-FCRDC, Frederick, Maryland 21702, USA.
Aspartic proteinase A from yeast is specifically and potently inhibited by a
small protein called IA3 from Saccharomyces cerevisiae. Although this inhibitor
consists of 68 residues, we show that the inhibitory activity resides within the
N-terminal half of the molecule. Structures solved at 2.2 and 1.8 A,
respectively, for complexes of proteinase A with full-length IA3 and with a
truncated form consisting only of residues 2-34, reveal an unprecedented mode of
inhibitor-enzyme interactions. Neither form of the free inhibitor has detectable
intrinsic secondary structure in solution. However, upon contact with the
enzyme, residues 2-32 become ordered and adopt a near-perfect alpha-helical
conformation. Thus, the proteinase acts as a folding template, stabilizing the
helical conformation in the inhibitor, which results in the potent and specific
blockage of the proteolytic activity.
PMID: 10655612 [PubMed - indexed for MEDLINE]
556: Proc Natl Acad Sci U S A 2000 Feb 1;97(3):1143-7
Toward a protein-protein interaction map of the budding yeast: A comprehensive
system to examine two-hybrid interactions in all possible combinations between
the yeast proteins.
Ito T, Tashiro K, Muta S, Ozawa R, Chiba T, Nishizawa M, Yamamoto K, Kuhara S,
Sakaki Y.
Human Genome Center, Institute of Medical Science, University of Tokyo, 4-6-1
Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. tito@ims.u-tokyo.ac.jp
Protein-protein interactions play pivotal roles in various aspects of the
structural and functional organization of the cell, and their complete
description is indispensable to thorough understanding of the cell. As an
approach toward this goal, here we report a comprehensive system to examine
two-hybrid interactions in all of the possible combinations between proteins of
Saccharomyces cerevisiae. We cloned all of the yeast ORFs individually as a
DNA-binding domain fusion ("bait") in a MATa strain and as an activation domain
fusion ("prey") in a MATalpha strain, and subsequently divided them into pools,
each containing 96 clones. These bait and prey clone pools were systematically
mated with each other, and the transformants were subjected to strict selection
for the activation of three reporter genes followed by sequence tagging. Our
initial examination of approximately 4 x 10(6) different combinations,
constituting approximately 10% of the total to be tested, has revealed 183
independent two-hybrid interactions, more than half of which are entirely novel.
Notably, the obtained binary data allow us to extract more complex interaction
networks, including the one that may explain a currently unsolved mechanism for
the connection between distinct steps of vesicular transport. The approach
described here thus will provide many leads for integration of various cellular
functions and serve as a major driving force in the completion of the
protein-protein interaction map.
PMID: 10655498 [PubMed - indexed for MEDLINE]
557: Genetics 2000 Feb;154(2):557-71
A yeast heterogeneous nuclear ribonucleoprotein complex associated with RNA
polymerase II.
Conrad NK, Wilson SM, Steinmetz EJ, Patturajan M, Brow DA, Swanson MS, Corden
JL.
Department of Molecular Biology and Genetics, The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205, USA.
Recent evidence suggests a role for the carboxyl-terminal domain (CTD) of the
largest subunit of RNA polymerase II (pol II) in pre-mRNA processing. The yeast
NRD1 gene encodes an essential RNA-binding protein that shares homology with
mammalian CTD-binding proteins and is thought to regulate mRNA abundance by
binding to a specific cis-acting element. The present work demonstrates genetic
and physical interactions among Nrd1p, the pol II CTD, Nab3p, and the CTD kinase
CTDK-I. Previous studies have shown that Nrd1p associates with the CTD of pol II
in yeast two-hybrid assays via its CTD-interaction domain (CID). We show that
nrd1 temperature-sensitive alleles are synthetically lethal with truncation of
the CTD to 9 or 10 repeats. Nab3p, a yeast hnRNP, is a high-copy suppressor of
some nrd1 temperature-sensitive alleles, interacts with Nrd1p in a yeast
two-hybrid assay, and coimmunoprecipitates with Nrd1p. Temperature-sensitive
alleles of NAB3 are suppressed by deletion of CTK1, a kinase that has been shown
to phosphorylate the CTD and increase elongation efficiency in vitro. This set
of genetic and physical interactions suggests a role for yeast RNA-binding
proteins in transcriptional regulation.
PMID: 10655211 [PubMed - indexed for MEDLINE]
558: J Biol Chem 2000 Feb 4;275(5):3128-36
Analysis of the yeast arginine methyltransferase Hmt1p/Rmt1p and its in vivo
function. Cofactor binding and substrate interactions.
McBride AE, Weiss VH, Kim HK, Hogle JM, Silver PA.
Department of Biological Chemistry, Harvard Medical School, Boston,
Massachusetts 02115, USA.
Many eukaryotic RNA-binding proteins are modified by methylation of arginine
residues. The yeast Saccharomyces cerevisiae contains one major arginine
methyltransferase, Hmt1p/Rmt1p, which is not essential for normal cell growth.
However, cells missing HMT1 and also bearing mutations in the mRNA-binding
proteins Npl3p or Cbp80p can no longer survive, providing genetic backgrounds in
which to study Hmt1p function. We now demonstrate that the catalytically active
form of Hmt1p is required for its activity in vivo. Amino acid changes in the
putative Hmt1p S-adenosyl-L-methionine-binding site were generated and shown to
be unable to catalyze methylation of Npl3p in vitro and in vivo or to restore
growth to strains that require HMT1. In addition these mutations affect
nucleocytoplasmic transport of Npl3p. A cold-sensitive mutant of Hmt1p was
generated and showed reduced methylation of Npl3p, but not of other substrates,
at 14 degrees C. These results define new aspects of Hmt1 and reveal the
importance of its activity in vivo.
PMID: 10652296 [PubMed - indexed for MEDLINE]
559: Biochem Biophys Res Commun 2000 Feb 5;268(1):73-7
Investigation of Fanconi anemia protein interactions by yeast two-hybrid
analysis.
Huber PA, Medhurst AL, Youssoufian H, Mathew CG.
Division of Medical Genetics, Guy's, King's and St. Thomas' School of Medicine,
Guy's Hospital, 7th Floor, Guy's Tower, London, SE1 9RT, United Kingdom.
pia.huber@kcl.ac.uk
Fanconi anemia is a chromosomal breakage disorder with eight complementation
groups (A-H), and three genes (FANCA, FANCC, and FANCG) have been identified.
Initial investigations of the interaction between FANCA and FANCC, principally
by co-immunoprecipitation, have proved controversial. We used the yeast
two-hybrid assay to test for interactions of the FANCA, FANCC, and FANCG
proteins. No activation of the reporter gene was observed in yeast co-expressing
FANCA and FANCC as hybrid proteins, suggesting that FANCA does not directly
interact with FANCC. However, a high level of activation was found when FANCA
was co-expressed with FANCG, indicating strong, direct interaction between these
proteins. Both FANCA and FANCG show weak but consistent interaction with
themselves, suggesting that their function may involve dimerisation. The site of
interaction of FANCG with FANCA was investigated by analysis of 12 mutant
fragments of FANCG. Although both N- and C-terminal fragments did interact,
binding to FANCA was drastically reduced, suggesting that more than one region
of the FANCG protein is required for proper interaction with FANCA. Copyright
2000 Academic Press.
PMID: 10652215 [PubMed - indexed for MEDLINE]
560: Eur J Biochem 2000 Feb;267(3):861-8
The structural basis of substrate activation in yeast pyruvate decarboxylase. A
crystallographic and kinetic study.
Lu G, Dobritzsch D, Baumann S, Schneider G, Konig S.
Department of Medical Biochemistry and Biophysics, Karolinska Institutet,
Stockholm, Sweden.
The crystal structure of the complex of the thiamine diphosphate dependent
tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with
the activator pyruvamide has been determined to 2.4 A resolution. The asymmetric
unit of the crystal contains two subunits, and the tetrameric molecule is
generated by crystallographic symmetry. Structure analysis revealed
conformational nonequivalence of the active sites. One of the two active sites
in the asymmetric unit was found in an open conformation, with two active site
loop regions (residues 104-113 and 290-304) disordered. In the other subunit,
these loop regions are well-ordered and shield the active site from the bulk
solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in
the active site channel, and is located in the vicinity of the thiazolium ring
of the cofactor. A second pyruvamide binding site was found at the interface
between the Pyr and the R domains of the subunit in the closed conformation,
about 10 A away from residue C221. This second pyruvamide molecule might
function in stabilizing the unique orientation of the R domain in this subunit
which in turn is important for dimer-dimer interactions in the activated
tetramer. No difference electron density in the close vicinity of the side chain
of residue C221 was found, indicating that this residue does not form a covalent
adduct with an activator molecule. Kinetic experiments showed that substrate
activation was not affected by oxidation of cysteine residues and therefore does
not seem to be dependent on intact thiol groups in the enzyme. The results
suggest that a disorder-order transition of two active-site loop regions is a
key event in the activation process triggered by the activator pyruvamide and
that covalent modification of C221 is not required for this transition to occur.
Based on these findings, a possible mechanism for the activation of PDC by its
substrate, pyruvate, is proposed.
PMID: 10651824 [PubMed - indexed for MEDLINE]
561: Mol Cell Biol 2000 Feb;20(4):1361-9
Yeast meiosis-specific protein Hop1 binds to G4 DNA and promotes its formation.
Muniyappa K, Anuradha S, Byers B.
Department of Biochemistry, Indian Institute of Science, Bangalore 560012,
India.
DNA molecules containing stretches of contiguous guanine residues can assume a
stable configuration in which planar quartets of guanine residues joined by
Hoogsteen pairing appear in a stacked array. This conformation, called G4 DNA,
has been implicated in several aspects of chromosome behavior including
immunoglobulin gene rearrangements, promoter activation, and telomere
maintenance. Moreover, the ability of the yeast SEP1 gene product to cleave DNA
in a G4-DNA-dependent fashion, as well as that of the SGS1 gene product to
unwind G4 DNA, has suggested a crucial role for this structure in meiotic
synapsis and recombination. Here, we demonstrate that the HOP1 gene product,
which plays a crucial role in the formation of synaptonemal complex in
Saccharomyces cerevisiae, binds robustly to G4 DNA. The apparent dissociation
constant for interaction with G4 DNA is 2 x 10(-10), indicative of binding that
is about 1,000-fold stronger than to normal duplex DNA. Oligonucleotides of
appropriate sequence bound Hop1 protein maximally if the DNA was first subjected
to conditions favoring the formation of G4 DNA. Furthermore, incubation of
unfolded oligonucleotides with Hop1 led to their transformation into G4 DNA.
Methylation interference experiments confirmed that modifications blocking G4
DNA formation inhibit Hop1 binding. In contrast, neither bacterial RecA proteins
that preferentially interact with GT-rich DNA nor histone H1 bound strongly to
G4 DNA or induced its formation. These findings implicate specific interactions
of Hop1 protein with G4 DNA in the pathway to chromosomal synapsis and
recombination in meiosis.
PMID: 10648621 [PubMed - indexed for MEDLINE]
562: Mol Cell Biol 2000 Feb;20(4):1321-8
Regulatory interactions between the Reg1-Glc7 protein phosphatase and the Snf1
protein kinase.
Sanz P, Alms GR, Haystead TA, Carlson M.
Departments of Genetics and Development and Microbiology, Columbia University,
New York, New York 10032, USA.
Protein phosphatase 1, comprising the regulatory subunit Reg1 and the catalytic
subunit Glc7, has a role in glucose repression in Saccharomyces cerevisiae.
Previous studies showed that Reg1 regulates the Snf1 protein kinase in response
to glucose. Here, we explore the functional relationships between Reg1, Glc7,
and Snf1. We show that different sequences of Reg1 interact with Glc7 and Snf1.
We use a mutant Reg1 altered in the Glc7-binding motif to demonstrate that Reg1
facilitates the return of the activated Snf1 kinase complex to the autoinhibited
state by targeting Glc7 to the complex. Genetic evidence indicated that the
catalytic activity of Snf1 negatively regulates its interaction with Reg1. We
show that Reg1 is phosphorylated in response to glucose limitation and that this
phosphorylation requires Snf1; moreover, Reg1 is dephosphorylated by Glc7 when
glucose is added. Finally, we show that hexokinase PII (Hxk2) has a role in
regulating the phosphorylation state of Reg1, which may account for the effect
of Hxk2 on Snf1 function. These findings suggest that the phosphorylation of
Reg1 by Snf1 is required for the release of Reg1-Glc7 from the kinase complex
and also stimulates the activity of Glc7 in promoting closure of the complex.
PMID: 10648618 [PubMed - indexed for MEDLINE]
563: J Cell Biol 2000 Jan 24;148(2):353-62
Comment in:
J Cell Biol. 2000 Jan 24;148(2):219-21.
A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p,
and the Arp2/3 complex.
Evangelista M, Klebl BM, Tong AH, Webb BA, Leeuw T, Leberer E, Whiteway M,
Thomas DY, Boone C.
Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada.
Type I myosins are highly conserved actin-based molecular motors that localize
to the actin-rich cortex and participate in motility functions such as
endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail
of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3
(SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both
Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that
link actin assembly and signaling molecules. The myosin-I acidic domain
interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both
sequence similarity and genetic redundancy with the COOH-terminal acidic domain
of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These
findings suggest that myosin-I proteins may participate in a diverse set of
motility functions through a role in actin assembly.
PMID: 10648568 [PubMed - indexed for MEDLINE]
564: Structure Fold Des 1999 Dec 15;7(12):1557-66
The three-dimensional structure of the HRDC domain and implications for the
Werner and Bloom syndrome proteins.
Liu Z, Macias MJ, Bottomley MJ, Stier G, Linge JP, Nilges M, Bork P, Sattler M.
European Molecular Biology Laboratory, Heidelberg, Germany.
BACKGROUND: The HRDC (helicase and RNaseD C-terminal) domain is found at the C
terminus of many RecQ helicases, including the human Werner and Bloom syndrome
proteins. RecQ helicases have been shown to unwind DNA in an ATP-dependent
manner. However, the specific functional roles of these proteins in DNA
recombination and replication are not known. An HRDC domain exists in both of
the human RecQ homologues that are implicated in human disease and may have an
important role in their function. RESULTS: We have determined the
three-dimensional structure of the HRDC domain in the Saccharomyces cerevisiae
RecQ helicase Sgs1p by nuclear magnetic resonance (NMR) spectroscopy. The
structure resembles auxiliary domains in bacterial DNA helicases and other
proteins that interact with nucleic acids. We show that a positively charged
region on the surface of the Sgs1p HRDC domain can interact with DNA. Structural
similarities to bacterial DNA helicases suggest that the HRDC domain functions
as an auxiliary domain in RecQ helicases. Homology models of the Werner and
Bloom HRDC domains show different surface properties when compared with Sgs1p.
CONCLUSIONS: The HRDC domain represents a structural scaffold that resembles
auxiliary domains in proteins that are involved in nucleic acid metabolism. In
Sgs1p, the HRDC domain could modulate the helicase function via auxiliary
contacts to DNA. However, in the Werner and Bloom syndrome helicases the HRDC
domain may have a role in their functional differences by mediating diverse
molecular interactions.
PMID: 10647186 [PubMed - indexed for MEDLINE]
565: J Biol Chem 2000 Jan 28;275(4):2627-35
PAR1 thrombin receptor-G protein interactions. Separation of binding and
coupling determinants in the galpha subunit.
Swift S, Sheridan PJ, Covic L, Kuliopulos A.
Molecular Cardiology Research Institute, Division of Hematology, New England
Medical Center, Boston, Massachusetts 02111, USA.
Signal transfer between the protease-activated PAR1 thrombin receptor and
membrane-associated heterotrimeric G proteins is mediated by protein-protein
interactions. We constructed a yeast signaling system that resolves
domain-specific functions of binding from coupling in the Galpha subunit. The
endogenous yeast Galpha subunit, Gpa1, does not bind to PAR1 and served as a
null structural template. N- and C-terminal portions of mammalian G(i2) and
G(16) were substituted back into the Gpa1 template and gain-of-function
assessed. The C-terminal third of G(16), but not of G(i2), provides sufficient
interactions for coupling to occur with PAR1. The N-terminal two-thirds of G(i2)
also contains sufficient determinants to bind and couple to PAR1 and overcome
the otherwise negative or missing interactions supplied by the C-terminal third
of Gpa1. Replacement of the N-terminal alpha-helix of G(i2), residues 1-34, with
those of Gpa1 abolishes coupling but not binding to PAR1 or to betagamma
subunits. These data support a model that the N-terminal alphaN helix of the
Galpha subunit is physically interposed between PAR1 and the Gbeta subunit and
directly assists in transferring the signal between agonist-activated receptor
and G protein.
PMID: 10644723 [PubMed - indexed for MEDLINE]
566: J Gen Virol 2000 Jan;81(Pt 1):209-18
Interactions in vivo between the proteins of infectious bursal disease virus:
capsid protein VP3 interacts with the RNA-dependent RNA polymerase, VP1.
Tacken MG, Rottier PJ, Gielkens AL, Peeters BP.
Institute for Animal Science and Health (ID-Lelystad), Department of Avian
Virology, PO Box 65, NL-8200 AB Lelystad, The Netherlands.
m.g.j.tacken@id.wag-ur.nl
Little is known about the intermolecular interactions between the viral proteins
of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system,
which allows the detection of protein-protein interactions in vivo, all possible
interactions were tested by fusing the viral proteins to the LexA DNA-binding
domain and the B42 transactivation domain. A heterologous interaction between
VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1,
were found by co-expression of the fusion proteins in Saccharomyces cerevisiae.
The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by
co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1
and VP3 is formed in the cytoplasm and eventually is released into the
cell-culture medium, indicating that VP1-VP3 complexes are present in mature
virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa.
Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it
interacted exclusively with the 95 kDa protein both in infected cells and in the
culture supernatant. These results suggest that the VP1-VP3 complex is involved
in replication and packaging of the IBDV genome.
PMID: 10640560 [PubMed - indexed for MEDLINE]
567: Genes Dev 2000 Jan 1;14(1):97-107
ATP can be dispensable for prespliceosome formation in yeast.
Perriman R, Ares M Jr.
Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of
California, Santa Cruz, Santa Cruz, California 95064, USA.
The first ATP-dependent step in pre-mRNA splicing involves the stable binding of
U2 snRNP to form the prespliceosome. We show that a prespliceosome-like complex
forms in the absence of ATP in yeast extracts lacking the U2 suppressor protein
CUS2. These complexes display the same pre-mRNA and U snRNA requirements as
authentic prespliceosomes and can be chased through the splicing pathway,
indicating that they are a functional intermediate in the spliceosome assembly
pathway. ATP-independent prespliceosome-like complexes are also observed in
extracts containing a mutant U2 snRNA. Loss of CUS2 does not bypass the role of
PRP5, an RNA helicase family member required for ATP-dependent prespliceosome
formation. Genetic interactions between CUS2 and a heat-sensitive prp5 allele
parallel those observed between CUS2 and U2, and suggest that CUS2 mediates
functional interactions between U2 RNA and PRP5. We propose that CUS2 enforces
ATP dependence during formation of the prespliceosome by brokering an
interaction between PRP5 and the U2 snRNP that depends on correct U2 RNA
structure.
PMID: 10640279 [PubMed - indexed for MEDLINE]
568: Nucleic Acids Res 2000 Feb 1;28(3):809-17
Isolation and characterization of human orthologs of yeast CCR4-NOT complex
subunits.
Albert TK, Lemaire M, van Berkum NL, Gentz R, Collart MA, Timmers HT.
Laboratory for Physiological Chemistry and Centre for Biomedical Genetics,
Utrecht University, PO Box 80042, 3508 TA Utrecht, The Netherlands,
The yeast CCR4-NOT protein complex is a global regulator of RNA polymerase II
transcription. It is comprised of yeast NOT1 to NOT5, yeast CCR4 and additional
proteins like yeast CAF1. Here we report the isolation of cDNAs encoding human
NOT2, NOT3, NOT4 and a CAF1-like factor, CALIF. Analysis of their mRNA levels in
different human tissues reveals a common ubiquitous expression pattern. A
multitude of two-hybrid interactions among the human cDNAs suggest that their
encoded proteins also form a complex in mammalian cells. Functional conservation
of these proteins throughout evolution is supported by the observation that the
isolated human NOT3 and NOT4 cDNAs can partially com-plement corresponding not
mutations in yeast. Interestingly, human CALIF is highly homologous to, although
clearly different from, a recently described human CAF1 protein. Conserved
interactions of this factor with both NOT and CCR4 proteins and
co-immunoprecipitation experiments suggest that CALIF is a bona fide component
of the human CCR4-NOT complex.
PMID: 10637334 [PubMed - indexed for MEDLINE]
569: Mol Biol Cell 2000 Jan;11(1):369-91
Kinetic analysis of a molecular model of the budding yeast cell cycle.
Chen KC, Csikasz-Nagy A, Gyorffy B, Val J, Novak B, Tyson JJ.
Department of Biology, Virginia Polytechnic Institute and State University,
Blacksburg Virginia 24061, USA.
The molecular machinery of cell cycle control is known in more detail for
budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism.
In recent years, many elegant experiments on budding yeast have dissected the
roles of cyclin molecules (Cln1-3 and Clb1-6) in coordinating the events of DNA
synthesis, bud emergence, spindle formation, nuclear division, and cell
separation. These experimental clues suggest a mechanism for the principal
molecular interactions controlling cyclin synthesis and degradation. Using
standard techniques of biochemical kinetics, we convert the mechanism into a set
of differential equations, which describe the time courses of three major
classes of cyclin-dependent kinase activities. Model in hand, we examine the
molecular events controlling "Start" (the commitment step to a new round of
chromosome replication, bud formation, and mitosis) and "Finish" (the transition
from metaphase to anaphase, when sister chromatids are pulled apart and the bud
separates from the mother cell) in wild-type cells and 50 mutants. The model
accounts for many details of the physiology, biochemistry, and genetics of cell
cycle control in budding yeast.
PMID: 10637314 [PubMed - indexed for MEDLINE]
570: Mol Biol Cell 2000 Jan;11(1):339-54
Functions and functional domains of the GTPase Cdc42p.
Kozminski KG, Chen AJ, Rodal AA, Drubin DG.
Department of Molecular and Cell Biology, University of California, Berkeley,
California 94720-3202, USA.
Cdc42p, a Rho family GTPase of the Ras superfamily, is a key regulator of cell
polarity and morphogenesis in eukaryotes. Using 37 site-directed cdc42 mutants,
we explored the functions and interactions of Cdc42p in the budding yeast
Saccharomyces cerevisiae. Cytological and genetic analyses of these cdc42
mutants revealed novel and diverse phenotypes, showing that Cdc42p possesses at
least two distinct essential functions and acts as a nodal point of cell
polarity regulation in vivo. In addition, mapping the functional data for each
cdc42 mutation onto a structural model of the protein revealed as functionally
important a surface of Cdc42p that is distinct from the canonical
protein-interacting domains (switch I, switch II, and the C terminus) identified
previously in members of the Ras superfamily. This region overlaps with a region
(alpha5-helix) recently predicted by structural models to be a specificity
determinant for Cdc42p-protein interactions.
PMID: 10637312 [PubMed - indexed for MEDLINE]
571: Mol Biol Cell 2000 Jan;11(1):277-86
The N terminus of the transmembrane protein BP180 interacts with the N-terminal
domain of BP230, thereby mediating keratin cytoskeleton anchorage to the cell
surface at the site of the hemidesmosome.
Hopkinson SB, Jones JC.
Department of Cell and Molecular Biology, Northwestern University Medical
School, Chicago, Illinois 60611, USA.
In epidermal cells, the keratin cytoskeleton interacts with the elements in the
basement membrane via a multimolecular junction called the hemidesmosome. A
major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid
autoantigen (BP230/BPAG1), which connects directly to the keratin-containing
intermediate filaments of the cytoskeleton via its C terminus. A second bullous
pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component
of the hemidesmosome. Using yeast two-hybrid technology and recombinant
proteins, we show that an N-terminal fragment of BP230 can bind directly to an
N-terminal fragment of BP180. We have also explored the consequences of
expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in
vitro. Unexpectedly, this fragment disrupts the distribution of BP180 in
transfected cells but has no apparent impact on the organization of endogenous
BP230 and alpha6beta4 integrin. We propose that the BP230 N terminus competes
with endogenous BP230 protein for BP180 binding and inhibits incorporation of
BP180 into the cell surface at the site of the hemidesmosome. These data provide
new insight into those interactions of the molecules of the hemidesmosome that
are necessary for its function in integrating epithelial and connective tissue
types.
PMID: 10637308 [PubMed - indexed for MEDLINE]
572: J Biol Chem 2000 Jan 21;275(3):2191-8
Lipid-dependent targeting of G proteins into rafts.
Moffett S, Brown DA, Linder ME.
Department of Cell Biology and Physiology, Washington University School of
Medicine, St. Louis, Missouri 63110, USA.
Domains rich in sphingolipids and cholesterol, or rafts, may organize signal
transduction complexes at the plasma membrane. Raft lipids are believed to exist
in a state similar to the liquid-ordered phase. It has been proposed that
proteins with a high affinity for an ordered lipid environment will
preferentially partition into rafts (Melkonian, K. A., Ostermeyer, A. G., Chen,
J. Z., Roth, M. G., and Brown, D. A. (1999) J. Biol. Chem. 274, 3910-3917). We
investigated the possibility that lipid-lipid interactions between
lipid-modified proteins and raft lipids mediate targeting of proteins to these
domains. G protein monomers or trimers were reconstituted in liposomes,
engineered to mimic raft domains. Assay for partitioning of G proteins into
rafts was based on Triton X-100 insolubility. Myristoylation and palmitoylation
of Galpha(i) were necessary and sufficient for association with liposomes and
partitioning into rafts. Strikingly, the amount of fatty-acylated Galpha(i) in
rafts was significantly reduced when myristoylated Galpha(i) was thioacylated
with cis-unsaturated fatty acids instead of saturated fatty acids such as
palmitate. Prenylated betagamma subunits were excluded from rafts, whether
reconstituted alone or with fatty-acylated alpha subunits. These results suggest
that the structural difference between lipids that modify proteins is one basis
for the selectivity of protein targeting to rafts.
PMID: 10636925 [PubMed - indexed for MEDLINE]
573: J Biol Chem 2000 Jan 21;275(3):2130-6
The RNA interacting domain but not the protein interacting domain is highly
conserved in ribosomal protein P0.
Rodriguez-Gabriel MA, Remacha M, Ballesta JP.
Centro de Biologia Molecular "Severo Ochoa," Universidad Autonoma de Madrid and
Consejo Superior de Investigaciones Cientificas, Cantoblanco, 28049 Madrid.
Protein P0 interacts with proteins P1alpha, P1beta, P2alpha, and P2beta, and
forms the Saccharomyces cerevisiae ribosomal stalk. The capacity of RPP0 genes
from Aspergillus fumigatus, Dictyostelium discoideum, Rattus norvegicus, Homo
sapiens, and Leishmania infantum to complement the absence of the homologous
gene has been tested. In S. cerevisiae W303dGP0, a strain containing standard
amounts of the four P1/P2 protein types, all heterologous genes were functional
except the one from L. infantum, some of them inducing an osmosensitive
phenotype at 37 degrees C. The polymerizing activity and the elongation
factor-dependent functions but not the peptide bond formation capacity is
affected in the heterologous P0 containing ribosomes. The heterologous P0
proteins bind to the yeast ribosomes but the composition of the ribosomal stalk
is altered. Only proteins P1alpha and P2beta are found in ribosomes carrying the
A. fumigatus, R. norvegicus, and H. sapiens proteins. When the heterologous
genes are expressed in a conditional null-P0 mutant whose ribosomes are totally
deprived of P1/P2 proteins, none of the heterologous P0 proteins complemented
the conditional phenotype. In contrast, chimeric P0 proteins made of different
amino-terminal fragments from mammalian origin and the complementary
carboxyl-terminal fragments from yeast allow W303dGP0 and D67dGP0 growth at
restrictive conditions. These results indicate that while the P0 protein
RNA-binding domain is functionally conserved in eukaryotes, the regions involved
in protein-protein interactions with either the other stalk proteins or the
elongation factors have notably evolved.
PMID: 10636918 [PubMed - indexed for MEDLINE]
574: Mol Microbiol 2000 Jan;35(1):15-31
Recruitment of the yeast MADS-box proteins, ArgRI and Mcm1 by the pleiotropic
factor ArgRIII is required for their stability.
El Bakkoury M, Dubois E, Messenguy F.
Institut de Recherches Microbiologiques J-M. Wiame, and Laboratoire de
Microbiologie de l'Universit inverted question marke Libre de Bruxelles, Avenue
E. Gryzon, 1, B-1070 Brussels, Belgium.
Regulation of arginine metabolism requires the integrity of four regulatory
proteins, ArgRI, ArgRII, ArgRIII and Mcm1. To characterize further the
interactions between the different proteins, we used the two-hybrid system,
which showed that ArgRI and Mcm1 interact together, and with ArgRII and ArgRIII,
without an arginine requirement. To define the interacting domains of ArgRI and
Mcm1 with ArgRIII, we fused portions of ArgRI and Mcm1 to the DNA-binding domain
of Gal4 (GBD) and created mutations in GBD-ArgRI and GBD-Mcm1. The putative
alpha helix present in the MADS-box domain of ArgRI and Mcm1 is their major
region of interaction with ArgRIII. Interactions between the two MADS-box
proteins and ArgRIII were confirmed using affinity chromatography. The
requirement for ArgRIII in the control of arginine metabolism can be bypassed in
vitro as well as in vivo by overproducing ArgRI or Mcm1, which indicates that
ArgRIII is not present in the protein complex formed with the 'arginine boxes'.
We show that the impairment of arginine regulation in an argRIII deletant strain
is a result of a lack of stability of ArgRI and Mcm1. A mutation in ArgRI,
impairing its interaction with ArgRIII, leads to an unstable ArgRI protein in a
wild-type strain. ArgRIII integrity is crucial for Mcm1 function, as shown by
the marked decreased expression of five genes controlled by Mcm1. However,
ArgRIII is likely to recruit other proteins in the yeast cell, as overexpression
of Mcm1 does not compensate some physiological defects observed in an argRIII
deletant strain.
PMID: 10632874 [PubMed - indexed for MEDLINE]
575: Nucleic Acids Res 2001 Jan 1;29(1):75-9
YPD, PombePD and WormPD: model organism volumes of the BioKnowledge library, an
integrated resource for protein information.
Costanzo MC, Crawford ME, Hirschman JE, Kranz JE, Olsen P, Robertson LS,
Skrzypek MS, Braun BR, Hopkins KL, Kondu P, Lengieza C, Lew-Smith JE, Tillberg
M, Garrels JI.
Proteome, Inc., 100 Cummings Center, Suite 435M, Beverly, MA 01915, USA.
mcc@proteome.com
The BioKnowledge Library is a relational database and web site
(http://www.proteome.com) composed of protein-specific information collected
from the scientific literature. Each Protein Report on the web site summarizes
and displays published information about a single protein, including its
biochemical function, role in the cell and in the whole organism, localization,
mutant phenotype and genetic interactions, regulation, domains and motifs,
interactions with other proteins and other relevant data. This report describes
four species-specific volumes of the BioKnowledge Library, concerned with the
model organisms Saccharomyces cerevisiae (YPD), Schizosaccharomyces pombe
(PombePD) and Caenorhabditis elegans (WormPD), and with the fungal pathogen
Candida albicans (CalPD). Protein Reports of each species are unified in format,
easily searchable and extensively cross-referenced between species. The
relevance of these comprehensively curated resources to analysis of proteins in
other species is discussed, and is illustrated by a survey of model organism
proteins that have similarity to human proteins involved in disease.
PMID: 11125054 [PubMed - indexed for MEDLINE]
576: Genetics 2000 Jan;154(1):83-97
Synthetic genetic interactions with temperature-sensitive clathrin in
Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related
Vps1p in clathrin-dependent protein sorting at the trans-Golgi network.
Bensen ES, Costaguta G, Payne GS.
Department of Biological Chemistry, School of Medicine, University of
California, Los Angeles, California 90095, USA.
Clathrin is involved in selective protein transport at the Golgi apparatus and
the plasma membrane. To further understand the molecular mechanisms underlying
clathrin-mediated protein transport pathways, we initiated a genetic screen for
mutations that display synthetic growth defects when combined with a
temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in
Saccharomyces cerevisiae. Mutations, when present in cells with wild-type
clathrin, were analyzed for effects on mating pheromone alpha-factor precursor
maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of
protein sorting at the yeast trans-Golgi network (TGN) compartment. By these
criteria, two classes of mutants were obtained, those with and those without
defects in protein sorting at the TGN. One mutant with unaltered protein sorting
at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase
with widespread influences. The collection of mutants displaying TGN sorting
defects includes members with mutations in previously identified vacuolar
protein sorting genes (VPS), including the dynamin family member VPS1. Striking
genetic interactions were observed by combining temperature-sensitive alleles of
CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated
vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting
defects are isolates with mutations in the following: RIC1, encoding a product
originally proposed to participate in ribosome biogenesis; LUV1, encoding a
product potentially involved in vacuole and microtubule organization; and INP53,
encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of
INP53, but not the related INP51 and INP52 genes, resulted in alpha-factor
maturation defects and exacerbated alpha-factor maturation defects when combined
with chc1-521. Our findings implicate a wide variety of proteins in
clathrin-dependent processes and provide evidence for the selective involvement
of Inp53p in clathrin-mediated protein sorting at the TGN.
PMID: 10628971 [PubMed - indexed for MEDLINE]
577: Genetics 2000 Jan;154(1):61-71
Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes
involved in pre-mRNA splicing and cell cycle progression.
Ben-Yehuda S, Russell CS, Dix I, Beggs JD, Kupiec M.
Department of Molecular Microbiology and Biotechnology, Tel Aviv University,
Ramat Aviv 69978, Israel.
Biochemical and genetic experiments have shown that the PRP17 gene of the yeast
Saccharomyces cerevisiae encodes a protein that plays a role during the second
catalytic step of the splicing reaction. It was found recently that PRP17 is
identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the
restrictive temperature after the completion of DNA replication. Although the
PRP17/CDC40 gene product is essential only at elevated temperatures, splicing
intermediates accumulate in prp17 mutants even at the permissive temperature. In
this report we describe extensive genetic interactions between PRP17/CDC40 and
the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for
spliceosome assembly and for both catalytic steps of the splicing reaction. We
show that mutations in the PRP8 gene are able to suppress the
temperature-sensitive growth phenotype and the splicing defect conferred by the
absence of the Prp17 protein. In addition, these mutations are capable of
suppressing certain alterations in the conserved PyAG trinucleotide at the 3'
splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover,
other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and
show a reduced ability to splice an intron bearing an altered 3' splice
junction. On the basis of these findings, we propose a model for the mode of
interaction between the Prp8 and Prp17 proteins during the second catalytic step
of the splicing reaction.
PMID: 10628969 [PubMed - indexed for MEDLINE]
578: Anal Biochem 2000 Jan 15;277(2):247-53
Yeast two-hybrid assay for examining human immunodeficiency virus protease
heterodimer formation with dominant-negative inhibitors and multidrug-resistant
variants.
Todd S, Laboissiere MC, Craik CS.
Department of Pharmaceutical Chemistry, University of California, San Francisco,
California 94143-0446, USA.
The yeast two-hybrid assay was used to study the dimerization of engineered and
naturally occurring variants of human immunodeficiency virus (HIV) protease (PR)
monomers. Defective monomers that were previously shown to exhibit a
dominant-negative (D-N) effect in cultured mammalian cells were tested for their
ability to interact in the two-hybrid assay. Similarly, monomers with
dimer-interface substitutions and monomers harboring in vivo selected mutations
that confer multidrug resistance (mdr) in an AIDS patient were tested for
interaction in yeast. Dimer formation between wt monomers with catalytic
aspartates was not detected in yeast, whereas the dimerization of PR monomers
harboring the acid active site substitution D25N was readily demonstrated. The
use of inactive monomers harboring the D25N substitution as a genetic background
for studying additional HIV PR mutations allowed for the probing of interactions
between monomers with mdr-associated mutations with those based on the HIV-1
HXB2R sequence. The HTLVIII/HIV-1 HXB2R clone has been the basis for a large
number of HIV-related plasmids, primers, antibodies, and other specific reagents
throughout the HIV research community. The results of our assay suggest that
HXB2R-based D-N PR inhibitors associate with variant monomers based on the
recently obtained nucleotide sequence from an AIDS patient with a
multidrug-resistant virus. These results further encourage the use of D-N PR
inhibitors as antiviral agents which may complement existing small-molecule
combination therapies. Copyright 2000 Academic Press.
PMID: 10625514 [PubMed - indexed for MEDLINE]
579: J Mol Biol 2000 Jan 21;295(3):393-409
Role of an alpha-helical bulge in the yeast heat shock transcription factor.
Hardy JA, Walsh ST, Nelson HC.
Department of Molecular Biology, University of California, Berkeley, CA,
94720-3206, USA.
The heat shock transcription factor (HSF) is the master transcriptional
regulator of the heat shock response. The identity of a majority of the genes
controlled by HSF and the circumstances under which HSF becomes induced are
known, but the details of the mechanism by which HSF is able to sense and
respond to heat remains an enigma. For example, it is unclear whether HSF senses
the heat shock directly or requires ancillary interactions from a heat-induced
signaling pathway. We present the analysis of a series of mutations in an
alpha-helical bulge in the DNA-binding domain of HSF. Deletion of residues in
this bulged region increases the overall activity of the protein. Yeast
containing the deletion mutant HSF are able to survive growth temperatures that
are lethal to yeast containing wild-type HSF, and they are also constitutively
thermotolerant. The increase in activity can be measured as an increase in both
constitutive and induced transcriptional activity. The mutant proteins bind DNA
more tightly than the wild-type protein does, but this is unlikely to account
fully for the increase in transcriptional activity as yeast HSF is
constitutively bound to its binding site in vivo. The stability of the mutant
proteins to thermal denaturation is lower than wild-type, though their
native-state structures are still well-folded. Therefore, the mutants may be
structurally analogous to the heat-induced state of HSF, and suggest that the
DNA-binding domain of HSF may be capable of sensing heat shock directly.
Copyright 2000 Academic Press.
PMID: 10623534 [PubMed - indexed for MEDLINE]
580: Biotechnol Bioeng 2000 Feb 5;67(3):300-11
Performance modeling and simulation of biochemical process sequences with
interacting unit operations.
Groep ME, Gregory ME, Kershenbaum LS, Bogle ID.
Centre for Process Systems Engineering, Imperial College, London SW7 2BY.
Many biochemical processes consist of a sequence of operations for which optimal
operating conditions (setpoints) have to be determined. If such optimization is
performed for each operation separately with respect to objectives defined for
each operation individually, overall process performance is likely to be
suboptimal. Interactions between unit operations have to be considered, and a
unique objective has to be defined for the whole process. This paper shows how a
suitable optimization problem can be formulated and solved to obtain the best
overall set of operating conditions for a process. A typical enzyme production
process has been chosen as an example. In order to arrive at a demonstrative
model for the entire sequence of unit operations, it is shown how interaction
effects may be accommodated in the models. Optimal operating conditions are then
determined subject to a global process objective and are shown to be different
from those resulting from optimization of each separate operation. As this
strategy may result in an economic benefit, it merits further research into
interaction modeling and performance optimization. Copyright 2000 John Wiley &
Sons, Inc.
PMID: 10620260 [PubMed - indexed for MEDLINE]
581: EMBO J 2000 Jan 4;19(1):37-47
The WD-repeat protein pfs2p bridges two essential factors within the yeast
pre-mRNA 3'-end-processing complex.
Ohnacker M, Barabino SM, Preker PJ, Keller W.
Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse
70, CH-4056 Basel, Switzerland.
In the yeast Saccharomyces cerevisiae, pre-mRNA 3'-end processing requires six
factors: cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor
II (CF II), polyadenylation factor I (PF I), poly(A) polymerase (Pap1p) and
poly(A)-binding protein I (Pab1p). We report the characterization of Pfs2p, a
WD-repeat protein previously identified in a multiprotein complex carrying PF
I-Pap1p activity. The 3'-end-processing defects of pfs2 mutant strains and the
results of immunodepletion and immunoinactivation experiments indicate an
essential function for Pfs2p in cleavage and polyadenylation. With a one-step
affinity purification method that exploits protein A-tagged Pfs2p, we showed
that this protein is part of a CF II-PF I complex. Pull-down experiments with
GST fusion proteins revealed direct interactions of Pfs2p with subunits of CF
II-PF I and CF IA. These results show that Pfs2p plays an essential role in
3'-end formation by bridging different processing factors and thereby promoting
the assembly of the processing complex.
PMID: 10619842 [PubMed - indexed for MEDLINE]
582: Carbohydr Res 1999 Oct 15;321(3-4):143-56
Synthesis and glycosidase inhibitory activity of 5-thioglucopyranosylamines.
Molecular modeling of complexes with glucoamylase.
Randell KD, Frandsen TP, Stoffer B, Johnson MA, Svensson B, Pinto BM.
Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
The synthesis of a series of 5-thio-D-glucopyranosylarylamines by reaction of
5-thio-D-glucopyranose pentaacetate with the corresponding arylamine and
mercuric chloride catalyst is reported. The products were obtained as anomeric
mixtures of the tetraacetates which can be separated and crystallized. The
tetraacetates were deprotected to give alpha/beta mixtures of the parent
compounds which were evaluated as inhibitors of the hydrolysis of maltose by
glucoamylase G2 (GA). A transferred NOE NMR experiment with an alpha/beta
mixture of 7 in the presence of GA showed that only the alpha isomer is bound by
the enzyme. The Ki values, calculated on the basis of specific binding of the
alpha isomers, are 0.47 mM for p-methoxy-N-phenyl-5-thio-D-glucopyranosylamine
(7), 0.78 mM for N-phenyl-5-thio-D-glucopyranosylamine (8), 0.27 mM for
p-nitro-N-phenyl-5-thio-D-glucopyranosylamine (9) and 0.87 mM for
p-trifluoromethyl-N-phenyl-5-thio-D-glucopyranosylamine (10), and the K(m)
values for the substrates maltose and p-nitrophenyl alpha-D-glucopyranoside are
1.2 and 3.7 mM, respectively. Methyl
4-amino-4-deoxy-4-N-(5'-thio-alpha-D-glucopyranosyl)-alpha-D-glucopyrano side
(11) is a competitive inhibitor of GA wild-type (Ki 4 microM) and the active
site mutant Trp120-->Phe GA (Ki 0.12 mM). Compounds 7, 8, and 11 are also
competitive inhibitors of alpha-glucosidase from brewer's yeast, with Ki values
of 1.05 mM, > 10 mM, and 0.5 mM, respectively. Molecular modeling of the
inhibitors in the catalytic site of GA was used to probe the ligand-enzyme
complementary interactions and to offer insight into the differences in
inhibitory potencies of the ligands.
PMID: 10614065 [PubMed - indexed for MEDLINE]
583: J Cell Biol 1999 Dec 27;147(7):1493-502
Adenine nucleotide translocase-1, a component of the permeability transition
pore, can dominantly induce apoptosis.
Bauer MK, Schubert A, Rocks O, Grimm S.
Max-Planck-Institute for Biochemistry, 82152 Martinsried, Germany.
Here, we describe the isolation of adenine nucleotide translocase-1 (ANT-1) in a
screen for dominant, apoptosis-inducing genes. ANT-1 is a component of the
mitochondrial permeability transition complex, a protein aggregate connecting
the inner with the outer mitochondrial membrane that has recently been
implicated in apoptosis. ANT-1 expression led to all features of apoptosis, such
as phenotypic alterations, collapse of the mitochondrial membrane potential,
cytochrome c release, caspase activation, and DNA degradation. Both point
mutations that impair ANT-1 in its known activity to transport ADP and ATP as
well as the NH(2)-terminal half of the protein could still induce apoptosis.
Interestingly, ANT-2, a highly homologous protein could not lead to cell death,
demonstrating the specificity of the signal for apoptosis induction. In contrast
to Bax, a proapoptotic Bcl-2 gene, ANT-1 was unable to elicit a form of cell
death in yeast. This and the observed repression of apoptosis by the
ANT-1-interacting protein cyclophilin D suggest that the suicidal effect of
ANT-1 is mediated by specific protein-protein interactions within the
permeability transition pore.
PMID: 10613907 [PubMed - indexed for MEDLINE]
584: Annu Rev Cell Dev Biol 1999;15:63-80
Cooperation between microtubule- and actin-based motor proteins.
Brown SS.
Department of Anatomy and Cell Biology, University of Michigan Medical School,
Ann Arbor 48109, USA. susanbb@umich.edu
Organelle transport has been proposed to proceed in two steps: long-range
transport along microtubules and local delivery via actin filaments. This model
is supported by recent studies of pigment transport in several cell types and
transport in neurons, and in several cases, class V myosin has been implicated
as the actin-based motor. Mutations in mice (dilute) and yeast (myo2) have also
implicated this class of myosin in organelle transport, and genetic interactions
in yeast have indicated that a kinesin-related protein (Smy1p) plays a
supporting role. This link between members of two different motor superfamilies
has now taken a surprising turn: There is evidence for a physical interaction
between class V myosins and kinesin or Smy1p in both mice and yeast.
Publication Types:
Review
Review, Tutorial
PMID: 10611957 [PubMed - indexed for MEDLINE]
585: Biochimie 1999 Dec;81(12):1079-87
Display of Ras on filamentous phage through cysteine replacement.
Wind T, Kjaer S, Clark BF.
University of Aarhus, Department of Molecular and Structural Biology, Denmark.
Phage display technology has been used in a variety of contexts to understand
and manipulate biomolecular interactions between proteins and other
biomolecules. In this paper we describe the establishment of a phage display
system for elucidation of the interactions between the GTPase Ras and its panel
of effectors. It is shown how technical problems associated with phage display
of a protein with unpaired cysteines, likely to be caused by the oxidizing
environment of the bacterial periplasm into which the protein is directed, can
be overcome by cysteine replacement based on functional and structural studies.
First, the catalytic domain (residues 1-166) of mammalian H-Ras (Ras) was
observed to be displayed on phage in an incorrect conformation not detectable by
antibodies recognizing conformational epitopes on Ras. Although truncation of
the phage coat protein used as fusion partner (g3p) resulted in minor
improvements in the display, Ras was tailored for phage display by cysteine
replacement. By replacing the three cysteines at positions 51, 80 and 118 of Ras
with the corresponding residues in Saccharomyces cerevisiae RAS1, the resulting
fusion-phage is recognized by the conformation-dependent anti-Ras antibodies.
Furthermore, display of cysteine-free Ras is demonstrated by GTP-analogue
dependent binding to the Ras-binding domain of the Ras-effector Raf1. These data
pave the way for analysis of Ras-effector interactions using phage display
technology yet demonstrate that phage display of proteins with normally reduced
cysteines should be approached with caution.
PMID: 10607402 [PubMed - indexed for MEDLINE]
586: RNA 1999 Dec;5(12):1615-31
Splicing factor SF1 from Drosophila and Caenorhabditis: presence of an
N-terminal RS domain and requirement for viability.
Mazroui R, Puoti A, Kramer A.
Departement de Biologie Cellulaire, Universite de Geneve, Switzerland.
Splicing factor SF1 contributes to the recognition of the 3' splice site by
interacting with U2AF65 and binding to the intron branch site during the
formation of the early splicing complex E. These interactions and the essential
functional domains of SF1 are highly conserved in Saccharomyces cerevisiae. We
have isolated cDNAs encoding SF1 from Drosophila (Dm) and Caenorhabditis (Ce).
The encoded proteins share the U2AF65 interaction domain, a hnRNP K homology
domain, and one or two zinc knuckles required for RNA binding as well as
Pro-rich C-terminal sequences with their yeast and mammalian counterparts. In
contrast to SF1 in other species, DmSF1 and CeSF1 are characterized by an
N-terminal region enriched in Ser, Arg, Lys, and Asp residues with homology to
the RS domains of several splicing proteins. These domains mediate
protein-protein or protein-RNA interactions, suggesting an additional role for
DmSF1 and CeSF1 in pre-mRNA splicing. Human (Hs), fly, and worm SF1 interact
equally well with HsU2AF65 or the Drosophila homolog DmU2AF50. Moreover, DmSF1
lacking its N terminus is functional in prespliceosome formation in a HeLa
splicing system, emphasizing the conserved nature of interactions at an early
step in spliceosome assembly. The Ce-SF1 gene is located in a polycistronic
transcription unit downstream of the genes encoding U2AF35 (uaf-2) and a
cyclophilin (cyp-13), implying the coordinate transcriptional regulation of
these genes. Injection of double-stranded RNA into C. elegans results in
embryonic lethality; thus, the SF1 gene is essential not only in yeast but also
in at least one metazoan.
PMID: 10606272 [PubMed - indexed for MEDLINE]
587: RNA 1999 Dec;5(12):1526-34
Crystallographic structure of the amino terminal domain of yeast initiation
factor 4A, a representative DEAD-box RNA helicase.
Johnson ER, McKay DB.
Department of Structural Biology, Stanford University School of Medicine,
California 94305-5400, USA.
The eukaryotic translation initiation factor 4A (elF4A) is a representative of
the DEAD-box RNA helicase protein family. We have solved the crystallographic
structure of the amino-terminal domain (residues 1-223) of yeast elF4A. The
domain is built around a core scaffold, a parallel alpha-beta motif with five
beta strands, that is found in other RNA and DNA helicases, as well as in the
RecA protein. The amino acid sequence motifs that are conserved within the
helicase family are localized to the beta strand-->alpha helix junctions within
the core. The core of the amino terminal domain of elF4A is amplified with
additional structural elements that differ from those of other helicases. The
phosphate binding loop (the Walker A motif) is in an unusual closed
conformation. The crystallographic structure reveals specific interactions
between amino acid residues of the phosphate binding loop, the DEAD motif, and
the SAT motif, whose alteration is known to impair coupling between the ATPase
cycle and the RNA unwinding activity of elF4A.
PMID: 10606264 [PubMed - indexed for MEDLINE]
588: EMBO J 1999 Dec 15;18(24):7041-55
SIR repression of a yeast heat shock gene: UAS and TATA footprints persist
within heterochromatin.
Sekinger EA, Gross DS.
Department of Biochemistry and Molecular Biology, Louisiana State University
Medical Center, Shreveport, LA 71130-3932, USA.
Previous work has suggested that products of the Saccharomyces cerevisiae Silent
Information Regulator (SIR) genes form a complex with histones, nucleated by
cis-acting silencers or telomeres, which represses transcription in a
position-dependent but sequence-independent fashion. While it is generally
thought that this Sir complex works through the establishment of
heterochromatin, it is unclear how this structure blocks transcription while
remaining fully permissive to other genetic processes such as recombination or
integration. Here we examine the molecular determinants underlying the silencing
of HSP82, a transcriptionally potent, stress-inducible gene. We find that HSP82
is efficiently silenced in a SIR-dependent fashion, but only when HMRE
mating-type silencers are configured both 5' and 3' of the gene. Accompanying
dominant repression are novel wrapped chromatin structures within both core and
upstream promoter regions. Strikingly, DNase I footprints mapping to the binding
sites for heat shock factor (HSF) and TATA-binding protein (TBP) are
strengthened and broadened, while groove-specific interactions, as detected by
dimethyl sulfate, are diminished. Our data are consistent with a model for SIR
repression whereby transcriptional activators gain access to their cognate sites
but are rendered unproductive by a co-existing heterochromatic complex.
PMID: 10601026 [PubMed - indexed for MEDLINE]
589: J Mol Biol 1999 Dec 17;294(5):1311-25
Crystal structure of the histone acetyltransferase Hpa2: A tetrameric member of
the Gcn5-related N-acetyltransferase superfamily.
Angus-Hill ML, Dutnall RN, Tafrov ST, Sternglanz R, Ramakrishnan V.
Department of Biochemistry, University of Utah School of Medicine, Salt Lake
City, UT 84132, USA.
We report the crystal structure of the yeast protein Hpa2 in complex with acetyl
coenzyme A (AcCoA) at 2.4 A resolution and without cofactor at 2.9 A resolution.
Hpa2 is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, a
family of enzymes with diverse substrates including histones, other proteins,
arylalkylamines and aminoglycosides. In vitro, Hpa2 is able to acetylate
specific lysine residues of histones H3 and H4 with a preference for Lys14 of
histone H3. Hpa2 forms a stable dimer in solution and forms a tetramer upon
binding AcCoA. The crystal structure reveals that the Hpa2 tetramer is
stabilized by base-pair interactions between the adenine moieties of the bound
AcCoA molecules. These base-pairs represent a novel method of stabilizing an
oligomeric protein structure. Comparison of the structure of Hpa2 with those of
other GNAT superfamily members illustrates a remarkably conserved fold of the
catalytic domain of the GNAT family even though members of this family share low
levels of sequence homology. This comparison has allowed us to better define the
borders of the four sequence motifs that characterize the GNAT family, including
a motif that is not discernable in histone acetyltransferases by sequence
comparison alone. We discuss implications of the Hpa2 structure for the
catalytic mechanism of the GNAT enzymes and the opportunity for multiple histone
tail modification created by the tetrameric Hpa2 structure. Copyright 1999
Academic Press.
PMID: 10600387 [PubMed - indexed for MEDLINE]
590: Protein Sci 1999 Nov;8(11):2465-73
Structures of yeast vesicle trafficking proteins.
Tishgarten T, Yin FF, Faucher KM, Dluhy RA, Grant TR, Fischer von Mollard G,
Stevens TH, Lipscomb LA.
Department of Biochemistry & Molecular Biology, University of Georgia, Athens
30602, USA.
In protein transport between organelles, interactions of v- and t-SNARE proteins
are required for fusion of protein-containing vesicles with appropriate target
compartments. Mammalian SNARE proteins have been observed to interact with NSF
and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This
observation led to the hypothesis that, despite low sequence homology, SNARE
proteins are structurally similar among eukaryotes. SNARE proteins can be
classified into two groups depending on whether they interact with SNARE binding
partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the
published structural data available is for SNAREs involved in exocytosis (either
in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier
transform infrared spectroscopy, and dynamic light scattering data for a set of
yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in
intracellular trafficking. Our results suggest that the secondary structure of
Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of
solution conditions. In these respects, Vti1p appears to be distinct from
R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is
similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE,
is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms
dimers under many of the solution conditions tested in our experiments. The
results presented in this paper are among the first to suggest heterogeneity in
the functioning of SNARE complexes.
PMID: 10595551 [PubMed - indexed for MEDLINE]
591: Mol Cell Biol 2000 Jan;20(1):104-12
Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the
recruitment of mRNA processing machinery to RNA polymerase II.
Rodriguez CR, Cho EJ, Keogh MC, Moore CL, Greenleaf AL, Buratowski S.
Department of Biological Chemistry, Harvard Medical School, Boston,
Massachusetts 02115, USA.
The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is
mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal
domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping
enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with
the CTD and that serine 5, the major site of phosphorylation within the CTD
heptamer consensus YSPTSPS, is particularly important. We sought to identify the
CTD kinase responsible for capping enzyme targeting. The candidate kinases
Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione
S-transferase-CTD fusion protein such that capping enzyme can bind in vitro.
However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit
genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do
not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for
proper capping enzyme targeting in vivo. Interestingly, levels of the
polyadenylation factor Pta1 are also reduced in kin28 mutants, while several
other polyadenylation factors remain stable. Pta1 in yeast extracts binds
specifically to the phosphorylated CTD, suggesting that this interaction may
mediate coupling of polyadenylation and transcription.
PMID: 10594013 [PubMed - indexed for MEDLINE]
592: Mol Cell Biol 2000 Jan;20(1):26-33
Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms
a second Ras-binding site which mediates its Ras-dependent activation.
Shima F, Okada T, Kido M, Sen H, Tanaka Y, Tamada M, Hu CD, Yamawaki-Kataoka Y,
Kariya K, Kataoka T.
Department of Physiology II, Kobe University School of Medicine, Chuo-ku, Kobe
650-0017, Japan.
Posttranslational modification, in particular farnesylation, of Ras is crucial
for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1). Based on the
previous observation that association of CYR1 with cyclase-associated protein
(CAP) is essential for its activation by posttranslationally modified Ras, we
postulated that the associated CAP might contribute to the formation of a
Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary
Ras-binding site, the leucine-rich repeat domain. Here, we observed a
posttranslational modification-dependent association of Ras with a complex
between CAP and CYR1 C-terminal region. When CAP mutants defective in Ras
signaling but retaining the CYR1-binding activity were isolated by screening of
a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained
three mutants failed to be activated efficiently by modified Ras and exhibited a
severely impaired ability to bind Ras, providing a genetic evidence for the
importance of the physical association with Ras at the second Ras-binding site.
On the other hand, CYR1, complexed with the other CAP mutant, failed to be
activated by Ras but exhibited a greatly enhanced binding to Ras. Conversely, a
Ras mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP
complex, failed to activate CYR1 efficiently. Thus, the strength of interaction
at the second Ras-binding site appears to be a critical determinant of CYR1
regulation by Ras: too-weak and too-strong interactions are both detrimental to
CYR1 activation. These results, taken together with those obtained with
mammalian Raf, suggest the importance of the second Ras-binding site in effector
regulation.
PMID: 10594005 [PubMed - indexed for MEDLINE]
593: Mol Cell Biol 2000 Jan;20(1):12-25
Pan1p, End3p, and S1a1p, three yeast proteins required for normal cortical actin
cytoskeleton organization, associate with each other and play essential roles in
cell wall morphogenesis.
Tang HY, Xu J, Cai M.
Institute of Molecular and Cell Biology, National University of Singapore,
Singapore 117609, Singapore.
The EH domain proteins Pan1p and End3p of budding yeast have been known to form
a complex in vivo and play important roles in organization of the actin
cytoskeleton and endocytosis. In this report, we describe new findings
concerning the function of the Pan1p-End3p complex. First, we found that the
Pan1p-End3p complex associates with Sla1p, another protein known to be required
for the assembly of cortical actin structures. Sla1p interacts with the first
long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving
the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and
the C-terminal repeat region of End3p, undisturbed. Second, Pan1p, End3p, and
Sla1p are also required for normal cell wall morphogenesis. Each of the Pan1-4,
sla1Delta, and end3Delta mutants displays the abnormal cell wall morphology
previously reported for the act1-1 mutant. These cell wall defects are also
exhibited by wild-type cells overproducing the C-terminal region of Sla1p that
is responsible for interactions with Pan1p and End3p. These results indicate
that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may
depend on the formation of a heterotrimeric complex. Interestingly, the cell
wall abnormalities exhibited by these cells are independent of the actin
cytoskeleton organization on the cell cortex, as they manifest despite the
presence of apparently normal cortical actin cytoskeleton. Examination of
several act1 mutants also supports this conclusion. These observations suggest
that the Pan1p-End3p-Sla1p complex is required not only for normal actin
cytoskeleton organization but also for normal cell wall morphogenesis in yeast.
PMID: 10594004 [PubMed - indexed for MEDLINE]
594: Mol Cell Biol 2000 Jan;20(1):1-11
Scanning mutagenesis of Mcm1: residues required for DNA binding, DNA bending,
and transcriptional activation by a MADS-box protein.
Acton TB, Mead J, Steiner AM, Vershon AK.
Waksman Institute of Microbiology, Department of Molecular Biology, Rutgers
University, Piscataway, New Jersey 08854-8020, USA.
MCM1 is an essential gene in the yeast Saccharomyces cerevisiae and is a member
of the MADS-box family of transcriptional regulatory factors. To understand the
nature of the protein-DNA interactions of this class of proteins, we have made a
series of alanine substitutions in the DNA-binding domain of Mcm1 and examined
the effects of these mutations in vivo and in vitro. Our results indicate which
residues of Mcm1 are important for viability, transcriptional activation, and
DNA binding and bending. Substitution of residues in Mcm1 which are highly
conserved among the MADS-box proteins are lethal to the cell and abolish DNA
binding in vitro. These positions have almost identical interactions with DNA in
both the serum response factor-DNA and alpha2-Mcm1-DNA crystal structures,
suggesting that these residues make up a conserved core of protein-DNA
interactions responsible for docking MADS-box proteins to DNA. Substitution of
residues which are not as well conserved among members of the MADS-box family
play important roles in contributing to the specificity of DNA binding. These
results suggest a general model of how MADS-box proteins recognize and bind DNA.
We also provide evidence that the N-terminal extension of Mcm1 may have
considerable conformational freedom, possibly to allow binding to different DNA
sites. Finally, we have identified two mutants at positions which are critical
for Mcm1-mediated DNA bending that have a slow-growth phenotype. This finding is
consistent with our earlier results, indicating that DNA bending may have a role
in Mcm1 function in the cell.
PMID: 10594003 [PubMed - indexed for MEDLINE]
595: Yeast 1999 Dec;15(16):1761-8
New tools for protein linkage mapping and general two-hybrid screening.
Durfee T, Draper O, Zupan J, Conklin DS, Zambryski PC.
Department of Plant and Microbial Biology, University of California, Berkeley,
CA 94720, USA. durf@nature.berkeley.edu
The two-hybrid system has proved to be a facile method for detecting and
analyzing protein-protein interactions. An expanded application of this system,
protein linkage mapping, provides a means of identifying interactions on a
global scale and should prove a powerful tool in analyzing whole genomes as
their sequences become available. To overcome some of the inherent difficulties
in such a large-scale approach, we have constructed a set of new strains and
vectors that will allow for more efficient screening. The strains contain a
GAL1-URA3 reporter for positive and negative selection, as well as a UAS(G)-lacZ
reporter. The strains are of opposite mating types, permitting libraries present
in one strain to be easily screened against a second library in the companion
strain. We also constructed a family of CEN-based vectors for expression of both
Gal4 DNA-binding and activation domain fusions. These plasmids include a
hemagglutinin epitope tag and different polylinkers to increase the ease of
subcloning. CEN-based vectors are maintained at 1-2 copies per cell, limiting
the number of individual cells containing multiple plasmids that can confuse
further analyses, and ensuring that fusions are not expressed at toxic levels.
Using these vectors, both homo- and heterodimeric interactions resulted in up to
10-fold higher reporter gene transcription than obtained with 2micro;-based
plasmids, despite significantly lower protein levels. In addition to protein
linkage mapping, these reagents should be generally useful in standard
two-hybrid applications. Copyright 1999 John Wiley & Sons, Ltd.
PMID: 10590464 [PubMed - indexed for MEDLINE]
596: Yeast 1999 Dec;15(16):1719-31
RGD1 genetically interacts with MID2 and SLG1, encoding two putative sensors for
cell integrity signalling in Saccharomyces cerevisiae.
de Bettignies G, Barthe C, Morel C, Peypouquet MF, Doignon F, Crouzet M.
Laboratoire de Biologie Moleculaire et de Sequencage, UPR CNRS 9026, BP 64, 146
rue Leo Saignat, 33076 Bordeaux cedex, France.
The RGD1 gene was identified during systematic genome sequencing of
Saccharomyces cerevisiae. To further understand Rgd1p function, we set up a
synthetic lethal screen for genes interacting with RGD1. Study of one lethal
mutant made it possible to identify the SLG1 and MID2 genes. The gene
SLG1/HCS77/WSC1 was mutated in the original synthetic lethal strain, whereas
MID2/SMS1 acted as a monocopy suppressor. The SLG1 gene has been described to be
an upstream component in the yeast PKC pathway and encodes a putative cell
surface sensor for the activation of cell integrity signalling. First identified
by viability loss of shmoos after pheromone exposure, and since found in
different genetic screens, MID2 was recently reported as also encoding an
upstream activator of the PKC pathway. The RGD1 gene showed genetic interactions
with both sensors of cell integrity pathway. The rgd1 slg1 synthetic lethality
was rescued by osmotic stabilization, as expected for mutants altered in cell
wall integrity. The slight viability defect of rgd1 in minimal medium, which was
exacerbated by mid2, was not osmoremediated. As for mutants altered in PKC
pathway, the accumulation of small-budded dead cells in slg1, rgd1 and mid2
after heat shock was prevented by 1 M sorbitol. In addition, the rgd1 strain
also displayed dead shmoos after pheromone treatment, like mid2. Taken together,
the present results indicate close functional links between RGD1, MID2 and SLG1
and suggest that RGD1 and MID2 interact in a cell integrity signalling
functionally linked to the PKC pathway. Copyright 1999 John Wiley & Sons, Ltd.
PMID: 10590461 [PubMed - indexed for MEDLINE]
597: Mol Biol Cell 1999 Dec;10(12):4121-33
The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its
role in actin polarity.
Adamo JE, Rossi G, Brennwald P.
Department of Cell Biology, Cell Biology, and Genetics, Weill Medical College of
Cornell University, New York, New York 10021, USA.
Budding yeast grow asymmetrically by the polarized delivery of proteins and
lipids to specific sites on the plasma membrane. This requires the coordinated
polarization of the actin cytoskeleton and the secretory apparatus. We
identified Rho3 on the basis of its genetic interactions with several
late-acting secretory genes. Mutational analysis of the Rho3 effector domain
reveals three distinct functions in cell polarity: regulation of actin polarity,
transport of exocytic vesicles from the mother cell to the bud, and docking and
fusion of vesicles with the plasma membrane. We provide evidence that the
vesicle delivery function of Rho3 is mediated by the unconventional myosin Myo2
and that the docking and fusion function is mediated by the exocyst component
Exo70. These data suggest that Rho3 acts as a key regulator of cell polarity and
exocytosis, coordinating several distinct events for delivery of proteins to
specific sites on the cell surface.
PMID: 10588647 [PubMed - indexed for MEDLINE]
598: Biochem J 1999 Dec 15;344 Pt 3:633-42
Polyamine transport in bacteria and yeast.
Igarashi K, Kashiwagi K.
Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku,
Chiba 263-8522, Japan. iga16077@p.chiba-u.ac.jp
The polyamine content of cells is regulated by biosynthesis, degradation and
transport. In Escherichia coli, the genes for three different polyamine
transport systems have been cloned and characterized. Two uptake systems
(putrescine-specific and spermidine-preferential) were ABC transporters, each
consisting of a periplasmic substrate-binding protein, two transmembrane
proteins and a membrane-associated ATPase. The crystal structures of the
substrate-binding proteins (PotD and PotF) have been solved. They consist of two
domains with an alternating beta-alpha-beta topology, similar to other
periplasmic binding proteins. The polyamine-binding site is in a cleft between
the two domains, as determined by crystallography and site-directed mutagenesis.
Polyamines are mainly recognized by aspartic acid and glutamic acid residues,
which interact with the NH(2)- (or NH-) groups, and by tryptophan and tyrosine
residues that have hydrophobic interactions with the methylene groups of
polyamines. The precursor of one of the substrate binding proteins, PotD,
negatively regulates transcription of the operon for the spermidine-preferential
uptake system, thus providing another level of regulation of cellular
polyamines. The third transport system, catalysed by PotE, mediates both uptake
and excretion of putrescine. Uptake of putrescine is dependent on membrane
potential, whereas excretion involves an exchange reaction between putrescine
and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport
protein (TPO1) was identified. The properties of this protein are similar to
those of PotE, and TPO1 is located on the vacuolar membrane.
Publication Types:
Review
Review, Tutorial
PMID: 10585849 [PubMed - indexed for MEDLINE]
599: Anal Biochem 1999 Dec 1;276(1):18-26
Usefulness of statistic experimental designs in enzymology: example with
recombinant hCYP3A4 and 1A2.
Bournique B, Petry M, Gousset G.
Rhone-Poulenc Rorer, Drug Metabolism and Pharmacokinetics, and Pharmaceutical
Sciences, 13 Quai Jules Guesdes, Vitry s/Seine Cedex, 94403, France.
bruno.bournique@rp-rorer.fr
First, the effects of 10 incubation factors were screened altogether on
nifedipine dehydrogenase (NIF) and methoxyresorufin O-deethylase (MROD)
activities catalyzed by recombinant human CYP3A4 and 1A2, respectively. Using a
statistic experimental design, only 36 assays were needed to be exhaustive.
Eight factors influenced CYP3A4-mediated NIF activity: buffer type, pH,
temperature, Mg/EDTA, cytochrome b5, NADPH-P450 reductase, NADH, and solvent.
Two factors had no significant effect: total ionic concentration and
catalase/reduced glutathione. Six factors influenced CYP1A2-mediated MROD rates:
buffer type, pH, temperature, Mg/EDTA, NADH, and glycerol. Four factors had no
significant effect: total ionic concentration, cytochrome b5, reductase, and
NAD+. Secondly, the combined effects of ionic strength and Mg concentration on
NIF/CYP3A4 were studied and easily modeled using another statistic experimental
design. The effect of Mg was strong at a constant ionic strength of 100 mM and
negligible at a constant ionic strength of 500 mM. Thirdly, the effects of
influencing factors and their interactions on MROD/CYP1A2 were modeled after 40
assays using a third statistic experimental design. Later experiments confirmed
the predictivity of the models and the efficiency of optimized conditions. This
approach can be applied to other biochemistry areas. Copyright 1999 Academic
Press.
PMID: 10585740 [PubMed - indexed for MEDLINE]
600: Mol Gen Genet 1999 Oct;262(3):508-14
Mechanism of transcription termination: PTRF interacts with the largest subunit
of RNA polymerase I and dissociates paused transcription complexes from yeast
and mouse.
Jansa P, Grummt I.
Division of Molecular Biology of the Cell II, German Cancer Research Center,
Heidelberg.
Transcription termination by RNA polymerase I (Pol I) is a stepwise process.
First the elongating RNA polymerase is forced to pause by DNA-bound
transcription termination factor (TTF-I). Then the ternary transcription complex
is dissociated by PTRF, a novel factor that promotes release of both nascent
transcripts and Pol I from the template. In this study we have investigated the
ability of PTRF to liberate transcripts from ternary transcription complexes
isolated from yeast and mouse. Using immobilized, tailed templates that contain
terminator sequences from Saccharomyces cerevisiae and mouse, respectively, we
demonstrate that PTRF promotes release of terminated transcripts, irrespective
of whether mouse Pol I has interacted with the murine termination factor TTF-I
or its yeast homolog Reb1p. In contrast, mouse Pol I paused by the lac repressor
remains bound to the template both in the presence and absence of PTRF. We
demonstrate that PTRF interacts with the largest subunit of murine Pol I, with
TTF-I and Reb1p, but not the lac repressor. The results imply that Pol I
transcription termination in yeast and mouse is mediated by conserved
interactions between Pol I, Reb1p/TTF-I and PTRF.
PMID: 10589839 [PubMed - indexed for MEDLINE]
601: Mol Gen Genet 1999 Oct;262(3):473-80
Budding yeast Cdc6p induces re-replication in fission yeast by inhibition of
SCF(Pop)-mediated proteolysis.
Wolf DA, McKeon F, Jackson PK.
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, MA
02115, USA. dwolf@hsph.harvard.edu
In fission yeast, overexpression of the replication initiator protein Cdc18p
induces re-replication, a phenotype characterized by continuous DNA synthesis in
the absence of cell division. In contrast, overexpression of Cdc6p, the budding
yeast homolog of Cdc18p, does not cause re-replication in S. cerevisiae.
However, we have found that Cdc6p has the ability to induce rereplication in
fission yeast. Cdc6p cannot functionally replace Cdc18p, but instead interferes
with the proteolysis of both Cdc18p and Rum1p, the inhibitor of the protein
kinase Cdc2p. This activity of Cdc6p is entirely contained within a short
N-terminal peptide, which forms a tight complex with Cdc2p and the
F-box/WD-repeat protein Sud1p/Pop2p, a component of the SCF(Pop) ubiquitin
ligase in fission yeast. These interactions are mediated by two distinct regions
within the N-terminal region of Cdc6p and depend on the integrity of its Cdc2p
phosphorylation sites. The data suggest that disruption of re-replication
control by overexpression of Cdc6p in fission yeast is a consequence of
sequestration of Cdc2p and Pop2p, two factors involved in the negative
regulation of Rum1p, Cdc18p and potentially other replication proteins.
PMID: 10589835 [PubMed - indexed for MEDLINE]
602: Cell 1999 Nov 24;99(5):533-43
Structure and mechanism of yeast RNA triphosphatase: an essential component of
the mRNA capping apparatus.
Lima CD, Wang LK, Shuman S.
Biochemistry Department, Weill Medical College of Cornell University, New York,
New York 10021, USA.
RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the
first step in cap formation. The 2.05 A crystal structure of yeast RNA
triphosphatase Cet1p reveals a novel active site fold whereby an eight-stranded
beta barrel forms a topologically closed triphosphate tunnel. Interactions of a
sulfate in the center of the tunnel with a divalent cation and basic amino acids
projecting into the tunnel suggest a catalytic mechanism that is supported by
mutational data. Discrete surface domains mediate Cet1p homodimerization and
Cet1p binding to the guanylyltransferase component of the capping apparatus. The
structure and mechanism of fungal RNA triphosphatases are completely different
from those of mammalian mRNA capping enzymes. Hence, RNA triphosphatase presents
an ideal target for structure-based antifungal drug discovery.
PMID: 10589681 [PubMed - indexed for MEDLINE]
603: J Biol Chem 1999 Dec 10;274(50):35583-90
Self-association of the alpha subunit of phosphorylase kinase as determined by
two-hybrid screening.
Ayers NA, Wilkinson DA, Fitzgerald TJ, Carlson GM.
Division of Molecular Biology, School of Biological Sciences, University of
Missouri, Kansas City, Missouri 64110-2499, USA.
The structural organization of the (alphabetagammadelta)(4) phosphorylase kinase
complex has been studied using the yeast two-hybrid screen for the purpose of
elucidating regions of alpha subunit interactions. By screening a rabbit
skeletal muscle cDNA library with residues 1-1059 of the alpha subunit of
phosphorylase kinase, we have isolated 16 interacting, independent, yet
overlapping transcripts of the alpha subunit containing its C-terminal region.
Domain mapping of binary interactions between alpha constructs revealed two
regions involved in the self-association of the alpha subunit: residues 833-854,
a previously unrecognized leucine zipper, and an unspecified region within
residues 1015-1237. The cognate binding partner for the latter domain has been
inferred to lie within the stretch from residues 864-1059. Indirect evidence
from the literature suggests that the interacting domains contained within the
latter two, overlapping regions may be further narrowed to the stretches from
1057 to 1237 and from 864 to 971. Cross-linking of the nonactivated holoenzyme
with N-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced
intramolecularly cross-linked alpha-alpha dimers, consistent with portions of
two alpha subunits in the holoenyzme being in sufficient proximity to associate.
This is the first report to identify potential areas of contact between the
alpha subunits of phosphorylase kinase. Additionally, issues regarding the
general utility of two-hybrid screening as a method for studying homodimeric
interactions are discussed.
PMID: 10585434 [PubMed - indexed for MEDLINE]
604: Nat Struct Biol 1999 Dec;6(12):1139-47
Comment in:
Nat Struct Biol. 1999 Dec;6(12):1081-3.
A novel loop-loop recognition motif in the yeast ribosomal protein L30
autoregulatory RNA complex.
Mao H, White SA, Williamson JR.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, USA.
The yeast Saccharomyces cerevisiae ribosomal protein L30 negatively
autoregulates its production by binding to a helix-loop-helix structure formed
in its pre-mRNA and its mRNA. A three-dimensional solution structure of the L30
protein in complex with its regulatory RNA has been solved using NMR
spectroscopy. In the complex, the helix-loop-helix RNA adopts a sharply bent
conformation at the internal loop region. Unusual RNA features include a purine
stack, a reverse Hoogsteen base pair (G11anti-G56syn) and highly distorted
backbones. The L30 protein is folded in a three-layer alpha/beta/alpha sandwich
topology, and three loops at one end of the sandwich make base-specific contacts
with the RNA internal loop. The protein-RNA binding interface is divided into
two clusters, including hydrophobic and aromatic stacking interactions centering
around G56, and base-specific hydrogen-bonding contacts to A57, G58 and G10-U60
wobble base pair. Both the protein and the RNA exhibit a partially induced fit
for binding, where loops in the protein and the internal loop in the RNA become
more ordered upon complex formation. The specific interactions formed between
loops on L30 and the internal loop on the mRNA constitute a novel loop-loop
recognition motif where an intimate RNA-protein interface is formed between
regions on both molecules that lack regular secondary structure.
PMID: 10581556 [PubMed - indexed for MEDLINE]
605: Nat Struct Biol 1999 Dec;6(12):1081-3
Comment on:
Nat Struct Biol. 1999 Dec;6(12):1139-47.
If the loop fits...
Frankel AD.
The structure of the yeast L30 ribosomal protein bound to its autoregulatory RNA
site has been determined by NMR spectroscopy. The intricate architecture of the
RNA internal loop and the structure of the binding region of the protein both
are stabilized in the complex, highlighting the importance of mutually-induced
fit in RNA-protein interactions.
Publication Types:
Comment
News
PMID: 10581539 [PubMed - indexed for MEDLINE]
606: Appl Environ Microbiol 1999 Dec;65(12):5451-8
Permeabilization of fungal membranes by plant defensins inhibits fungal growth.
Thevissen K, Terras FR, Broekaert WF.
F. A. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, B-3001
Heverlee-Leuven, Belgium.
We used an assay based on the uptake of SYTOX Green, an organic compound that
fluoresces upon interaction with nucleic acids and penetrates cells with
compromised plasma membranes, to investigate membrane permeabilization in fungi.
Membrane permeabilization induced by plant defensins in Neurospora crassa was
biphasic, depending on the plant defensin dose. At high defensin levels (10 to
40 microM), strong permeabilization was detected that could be strongly
suppressed by cations in the medium. This permeabilization appears to rely on
direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1
microM), a weaker, but more cation-resistant, permeabilization occurred at
concentrations that correlated with the inhibition of fungal growth.
Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus
sativus, failed to induce cation-resistant permeabilization in N. crassa.
Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane
permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its
antifungal activity. However, Dm-AMP1 could not induce cation-resistant
permeabilization in the Dm-AMP1-resistant S. cerevisiae mutant DM1, which has a
drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant
permeabilization is binding site mediated and linked to the primary cause of
fungal growth inhibition induced by plant defensins.
PMID: 10584003 [PubMed - indexed for MEDLINE]
607: EMBO J 1999 Dec 1;18(23):6832-44
The E2-E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is
required for the synthesis of multiubiquitin chain.
Xie Y, Varshavsky A.
Division of Biology, 147-75, California Institute of Technology, 1200 East
California Boulevard, Pasadena, CA 91125, USA.
We dissected physical and functional interactions between the
ubiquitin-conjugating (E2) enzyme Ubc2p and Ubr1p, the E3 component of the N-end
rule pathway in Saccharomyces cerevisiae. The binding of the 20 kDa Ubc2p by the
225 kDa Ubr1p is shown to be mediated largely by the basic residue-rich (BRR)
region of Ubr1p. However, mutations of the BRR domain that strongly decrease the
interaction between Ubr1p and Ubc2p do not prevent the degradation of N-end rule
substrates. In contrast, this degradation is completely dependent on the RING-H2
finger of Ubr1p adjacent to the BRR domain. Specifically, the first cysteine of
RING-H2 is required for the ubiquitylation activity of the Ubr1p-Ubc2p complex,
although this cysteine plays no detectable role in either the binding of N-end
rule substrates by Ubr1p or the physical affinity between Ubr1p and Ubc2p. These
results defined the topography of the Ubc2p-Ubr1p interaction and revealed the
essential function of the RING-H2 finger, a domain that is present in many
otherwise dissimilar E3 proteins of the ubiquitin system.
PMID: 10581257 [PubMed - indexed for MEDLINE]
608: EMBO J 1999 Dec 1;18(23):6672-81
Gal83 mediates the interaction of the Snf1 kinase complex with the transcription
activator Sip4.
Vincent O, Carlson M.
Departments of Genetics and Development, Columbia University, 701 West 168th
Street, New York, NY 10032, USA.
The Snf1/AMPK protein kinase family is widely conserved in eukaryotes. In
Saccharomyces cerevisiae, the Snf1 kinase is an essential element of the glucose
response pathway and has diverse regulatory roles. The Snf1 complex contains one
of the related proteins Sip1, Sip2 and Gal83, which are also conserved in higher
eukaryotes. Previous studies showed that the Sip1/Sip2/Gal83 component plays a
structural role in the complex. We present evidence that this component also
mediates the interaction of the Snf1 kinase complex with specific targets. We
show that Gal83 mediates the association of the kinase with Sip4, a
Snf1-regulated transcription activator of gluconeogenic genes. Gal83 interacts
with Sip4 in two-hybrid assays in vivo, and bacterially expressed proteins bind
in vitro. Moreover, Gal83 is required for the two-hybrid interaction of Sip4
with the Snf1 kinase. Gal83 also facilitates the rapid Snf1-dependent
phosphorylation and activation of Sip4 in response to glucose limitation,
indicating that Gal83 mediates the functional interaction of Snf1 with Sip4.
Evidence indicates that Sip1 and Sip2 do not interact with Sip4. We propose that
members of the Sip1/Sip2/Gal83 family confer specificity to the kinase complex
in its interactions with target proteins.
PMID: 10581241 [PubMed - indexed for MEDLINE]
609: EMBO J 1999 Dec 1;18(23):6662-71
A new regulatory domain on the TATA-binding protein.
Cang Y, Auble DT, Prelich G.
Department of Molecular Genetics, Albert Einstein College of Medicine, 1300
Morris Park Avenue, Bronx, NY 10461, USA.
Recognition of the TATA box by the TATA-binding protein (TBP) is a highly
regulated step in RNA polymerase II-dependent transcription. Several proteins
have been proposed to regulate TBP activity, yet the TBP domains responsive to
all these regulators have not been defined. Here we describe a new class of TBP
mutants that increase transcription from core promoters in vivo. The majority of
these mutations alter amino acids that cluster tightly on the TBP surface,
defining a new TBP regulatory domain. The mutant TBP proteins are defective for
binding the transcriptional regulator yNC2, are resistant to inhibition by yNC2
in vitro and exhibit allele-specific genetic interactions with yNC2. These
results provide strong biochemical and genetic evidence that TBP is directly
repressed in vivo, and define a new TBP domain important for transcriptional
regulation.
PMID: 10581240 [PubMed - indexed for MEDLINE]
610: Biochem Biophys Res Commun 1999 Dec 9;266(1):135-40
Functional genomic analysis reveals the utility of the I/LWEQ module as a
predictor of protein:actin interaction.
McCann RO, Craig SW.
Department of Biological Chemistry, Johns Hopkins University School of Medicine,
725 N. Wolfe Street, Baltimore, Maryland, 21205-2185, USA.
The I/LWEQ module is a conserved sequence that we have identified as an
actin-binding motif in the metazoan focal adhesion protein talin and the yeast
protein Sla2p. Both of these proteins are associated with the actin cytoskeleton
in cells. To better establish the value of the I/LWEQ module for prediction of
actin-binding function, we have applied a functional genomics approach. Analysis
of the 23 available I/LWEQ module sequences supports the division of I/LWEQ
protein superfamily into four groups: (1) metazoan talin, (2) Dictyostelium
discoideum talin homologs TalA/B, (3) metazoan Hip1p, and (4) yeast Sla2p. We
show here that I/LWEQ modules from each major group bind to F-actin in vitro and
that GFP-fusion proteins of the I/LWEQ modules of talin and Sla2p bind to
F-actin in vivo. Therefore, the presence of an I/LWEQ module is strongly
predictive of protein-actin interactions. The structural and functional
conservation of the I/LWEQ module across the phylogenetic distance between
cellular slime molds and mammals implies that the role of the I/LWEQ module is
to connect diverse proteins involved in distinct cellular processes, including
cell adhesion, cytoskeletal organization, and cell differentiation, to the actin
cytoskeleton. Copyright 1999 Academic Press.
PMID: 10581178 [PubMed - indexed for MEDLINE]
611: RNA 1999 Nov;5(11):1509-16
StreptoTag: a novel method for the isolation of RNA-binding proteins.
Bachler M, Schroeder R, von Ahsen U.
Institute of Microbiology & Genetics, University of Vienna, Austria.
We describe a fast and simple one-step affinity-purification method for the
isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA
consisting of an aptamer sequence with high binding specificity to the
antibiotic streptomycin and a putative protein-binding RNA sequence is incubated
with crude extract. After complex formation, the sample is applied to an
affinity column containing streptomycin immobilized to Sepharose. The binding of
the in vitro-assembled RNA-protein complex to streptomycin-Sepharose is mediated
by the aptamer RNA and the specifically bound proteins are recovered from the
affinity matrix by elution with the antibiotic. Employing two well-characterized
RNA-protein interactions, we tested the performance of this new method. The
spliceosomal U1A protein and the bacteriophage MS2 coat protein could be
isolated via their appropriate RNA motif containing hybrid RNA from crude yeast
extracts in high yield and purity after only one round of affinity purification.
As the purification principle is independent of the extract source, this new
affinity chromatography strategy that makes use of an in vitro-selected
antibiotic-binding RNA as a tag, "StreptoTag," should be applicable to extracts
from other organisms as well. Therefore, we propose StreptoTag to be a versatile
tool for the isolation of unknown RNA-binding proteins.
PMID: 10580480 [PubMed - indexed for MEDLINE]
612: RNA 1999 Nov;5(11):1470-81
Characterization of U6 snRNA-protein interactions.
Vidal VP, Verdone L, Mayes AE, Beggs JD.
Institute of Cell and Molecular Biology, University of Edinburgh, United
Kingdom.
Through a combination of in vitro snRNP reconstitution, photocross-linking and
immunoprecipitation techniques, we have investigated the interaction of proteins
with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5
tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with
this spliceosomal snRNA, three were shown to contact the RNA directly, and to
maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs,
the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved
region that contributes to the formation of the catalytic core of the
spliceosome. Other tri-snRNP-specific contacts were also detected, indicating
the dynamic nature of protein interactions with this important snRNA. The
uridine-rich extreme 3' end of U6 RNA was shown to be essential but not
sufficient for the association of the Lsm proteins. Interestingly, the Lsm
proteins associate efficiently with the 3' half of U6, which contains the 3'
stem-loop and uridine-rich 3' end, suggesting that the Lsm and Sm proteins may
recognize similar features in RNAs.
PMID: 10580475 [PubMed - indexed for MEDLINE]
613: Front Biosci 1999 Dec 1;4:D824-33
Transcription factors in DNA replication.
Murakami Y, Ito Y.
Department of Viral Oncology, Institute for Virus Research, Kyoto University,
Sakyo-ku, Kyoto 606-8507, Japan. yota@virus.kyoto-u.ac.jp
Accumulating evidence suggests the involvement of transcription factors in the
regulation of DNA replication in eukaryotic cells. Almost all eukaryotic DNA
viruses contain binding sites for transcription factors which function as
auxiliary elements for DNA replication initiation at replication origins, and,
indeed, the binding of transcription factors to these elements has been shown to
stimulate DNA replication. Transcription factors also regulate some of the
chromosome DNA replication origins of budding yeast, indicating that
transcription factor involvement in DNA replication is not restricted to
viruses. Consistent with this notion, recently determined replication origins of
higher eukaryotes have been found occasionally to associate with transcription
factor binding sites, although there is no direct evidence for the involvement
of the factors that bind to these sequences in DNA replication. Analyses using
viral and yeast systems have suggested that transcription factors stimulate the
formation of the replication initiation complex by engaging in specific
interactions with proteins of the initiation complex and/or by modulating the
repressive chromatin structure around origins of replication. These mechanisms
are analogous to those advanced to explain stimulation of transcription by
transcription factors. The accumulated data suggests that transcription factors
play a general role in the formation of functional complexes on chromosomes.
Publication Types:
Review
Review, Tutorial
PMID: 10577391 [PubMed - indexed for MEDLINE]
614: Biochimie 1999 Nov;81(11):1015-23
In vitro assembly of yeast 5S rRNA and a fusion protein containing ribosomal
protein L5 and maltose binding protein.
Pakhomova ON, Yeh LC, Monette J, Lee JC.
Department of Biochemistry, The University of Texas Health Science Center at San
Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7760 USA.
Binding of yeast ribosomal protein L5 with 5S rRNA has long been considered a
promising model for studying molecular mechanisms of protein-RNA interactions.
However, in vitro assembly of a ribonucleoprotein (RNP) complex from purified
yeast ribosomal protein L5 (also known as L1, L1a, or YL3) and 5S rRNA proved to
be difficult, thus limiting the utility of this model. In the present report, we
present data on the successful in vitro assembly of a RNP complex using a fusion
(MBP-L5) protein consisting of the yeast ribosomal protein L5 fused to the
carboxyl terminus of the E. coli maltose-binding protein (MBP). We demonstrated
that: 1) the MBP-L5 protein binds yeast 5S rRNA but not 5.8S rRNA in vitro; 2)
the MBP protein itself does not bind yeast 5S rRNA; 3) formation of the RNP
complex is proportional to the concentration of MBP-L5 protein and 5S rRNA; and
4) the MBP moiety of the fusion protein in the RNP complex can be removed with
factor Xa. The electrophoretic mobility of the resultant RNP complex is
indistinguishable from that of L5-5S rRNA complex isolated from the ribosome.
Using this new experimental approach, we further showed that the RNA binding
capability of a mutant L5 protein is decreased by 60% compared to the wild-type
protein. Additionally, the mutant RNP complex migrates slower than the wild-type
RNP complex suggesting that the mutant RNP complex has a less compact
conformation. The finding provides a probable explanation for an earlier
observation that the 60S ribosomal subunit containing the mutant protein is
unstable.
PMID: 10575356 [PubMed - indexed for MEDLINE]
615: Nature 1999 Nov 4;402(6757):83-6
Comment in:
Nature. 1999 Nov 4;402(6757):23, 25-6.
A combined algorithm for genome-wide prediction of protein function.
Marcotte EM, Pellegrini M, Thompson MJ, Yeates TO, Eisenberg D.
Molecular Biology Institute, UCLA-DOE Laboratory of Structural Biology and
Molecular Medicine, University of California, Los Angeles 90095, USA.
The availability of over 20 fully sequenced genomes has driven the development
of new methods to find protein function and interactions. Here we group proteins
by correlated evolution, correlated messenger RNA expression patterns and
patterns of domain fusion to determine functional relationships among the 6,217
proteins of the yeast Saccharomyces cerevisiae. Using these methods, we discover
over 93,000 pairwise links between functionally related yeast proteins. Links
between characterized and uncharacterized proteins allow a general function to
be assigned to more than half of the 2,557 previously uncharacterized yeast
proteins. Examples of functional links are given for a protein family of
previously unknown function, a protein whose human homologues are implicated in
colon cancer and the yeast prion Sup35.
PMID: 10573421 [PubMed - indexed for MEDLINE]
616: Nucleic Acids Res 1999 Dec 15;27(24):4695-702
Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmc1
homolog, in Pyrococcus furiosus.
Hayashi I, Morikawa K, Ishino Y.
Department of Molecular Biology, Biomolecular Engineering Research Institute
(BERI), 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan.
Pyrococcus furiosus has an operon containing the DNA polymerase II (PolD) gene
and three other genes. Using a two-hybrid screening to examine the interactions
of the proteins encoded by the operon, we identified a specific interaction
between the second subunit of PolD (DP1) and a Rad51/Dmc1 homologous protein
(RadB). To ensure the specific interaction between these two proteins, each gene
in the operon was expressed in Escherichia coli or insect cells separately and
the products were purified. The in vitro analyses using the purified proteins
also showed the interaction between DP1 and RadB. The deletion mutant analysis
of DP1 revealed that a region important for binding with RadB is located in the
central part of the sequence (amino acid residues 206-498). This region has an
overlap to the C-terminal half (amino acids 334-613), which is highly conserved
among euryarchaeal DP1s and is essential for the activity of PolD. Our results
suggest that, although RadB does not noticeably affect the primer extension
ability of PolD in vitro, PolD may utilize the RadB protein in DNA synthesis
under certain conditions.
PMID: 10572168 [PubMed - indexed for MEDLINE]
617: Proc Natl Acad Sci U S A 1999 Nov 23;96(24):13650-5
RNA-controlled polymorphism in the in vivo assembly of 180-subunit and
120-subunit virions from a single capsid protein.
Krol MA, Olson NH, Tate J, Johnson JE, Baker TS, Ahlquist P.
Institute for Molecular Virology, University of Wisconsin, Madison, WI 53706,
USA.
Repeated, specific interactions between capsid protein (CP) subunits direct
virus capsid assembly and exemplify regulated protein-protein interactions. The
results presented here reveal a striking in vivo switch in CP assembly. Using
cryoelectron microscopy, three-dimensional image reconstruction, and molecular
modeling, we show that brome mosaic virus (BMV) CP can assemble in vivo two
remarkably distinct capsids that selectively package BMV-derived RNAs in the
absence of BMV RNA replication: a 180-subunit capsid indistinguishable from
virions produced in natural infections and a previously unobserved BMV capsid
type with 120 subunits arranged as 60 CP dimers. Each such dimer contains two
CPs in distinct, nonequivalent environments, in contrast to the quasi-equivalent
CP environments throughout the 180-subunit capsid. This 120-subunit capsid
utilizes most of the CP interactions of the 180-subunit capsid plus
nonequivalent CP-CP interactions. Thus, the CP of BMV, and perhaps other
viruses, can encode CP-CP interactions that are not apparent from mature virions
and may function in assembly or disassembly. Shared structural features suggest
that the 120- and 180-subunit capsids share assembly steps and that a common
pentamer of CP dimers may be an important assembly intermediate. The ability of
a single CP to switch between distinct capsids by means of alternate
interactions also implies reduced evolutionary barriers between different capsid
structures. The in vivo switch between alternate BMV capsids is controlled by
the RNA packaged: a natural BMV genomic RNA was packaged in 180-subunit capsids,
whereas an engineered mRNA containing only the BMV CP gene was packaged in
120-subunit capsids. RNA features can thus direct the assembly of a
ribonucleoprotein complex between alternate structural pathways.
PMID: 10570127 [PubMed - indexed for MEDLINE]
618: Mol Pharmacol 1999 Dec;56(6):1105-15
Domain interactions affecting human DNA topoisomerase I catalysis and
camptothecin sensitivity.
Fiorani P, Amatruda JF, Silvestri A, Butler RH, Bjornsti MA, Benedetti P.
Istituto di Biologia Cellulare, "Campus Adriano Buzzati-Traverso" Consiglio
Nazionale delle Ricerche, Rome, Italy.
DNA topoisomerase I (Top1p) relaxes supercoiled DNA by the formation of a
covalent intermediate in which the active site tyrosine is transiently bound to
the severed DNA strand. The antineoplastic agent camptothecin (Cpt) specifically
targets Top1p and several mutations have been isolated that render the enzyme
Cpt resistant. The mutated residues, although located in different regions of
the enzyme, may constitute part of the Cpt binding site. To begin identifying
the structural features of DNA Top1p important for Cpt-induced cytotoxicity, we
developed a novel yeast genetic screen to isolate catalytically active, yet
Cpt-resistant enzymes from a pool of human top1 mutants. Among the mutations
isolated were substitutions of Ser or Val for Gly363, which like the Gly363 to
Cys mutation previously reported by us, suppressed the Cpt sensitivity of Top1p.
In contrast, each amino-acid substitution differed in its ability to suppress
the lethal phenotype and catalytic activity of a human top1 mutant top1T718A
that resembles Cpt by stabilizing the covalent intermediate. Biochemical
analyses and molecular modeling support a model where interactions between two
conserved domains, a central "lip" region containing residue Gly363 and the
residues around the active site tyrosine (Tyr723), directly affect the formation
of the Cpt-binding site and enzyme catalysis.
PMID: 10570037 [PubMed - indexed for MEDLINE]
619: Biochemistry 1999 Nov 23;38(47):15580-6
DNA topoisomerases as targets for the anticancer drug TAS-103: DNA interactions
and topoisomerase catalytic inhibition.
Fortune JM, Velea L, Graves DE, Utsugi T, Yamada Y, Osheroff N.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232-0146, USA.
TAS-103 is a novel anticancer drug that kills cells by increasing levels of DNA
cleavage mediated by topoisomerase II. While most drugs that stimulate
topoisomerase II-mediated DNA scission (i.e., topoisomerase II poisons) also
inhibit the catalytic activity of the enzyme, they typically do so only at
concentrations above the clinical range. TAS-103 is unusual in that it
reportedly inhibits the catalytic activity of both topoisomerase I and II and
does so at physiologically relevant concentrations [Utsugi, T., et al. (1997)
Jpn. J. Cancer Res. 88, 992-1002]. Without a topoisomerase activity to relieve
accumulating torsional stress, the DNA tracking systems that promote the action
of TAS-103 as a topoisomerase II poison would be undermined. Therefore, the
effects of TAS-103 on the catalytic activity of topoisomerase I and II were
characterized. DNA binding and unwinding assays indicate that the drug
intercalates into DNA with an apparent dissociation constant of approximately
2.2 microM. Furthermore, DNA strand passage assays with mammalian topoisomerase
I indicate that TAS-103 does not inhibit the catalytic activity of the type I
enzyme. Rather, the previously reported inhibition of topoisomerase I-catalyzed
DNA relaxation results from a drug-induced alteration in the apparent topology
of the nucleic acid substrate. TAS-103 does inhibit the catalytic activity of
human topoisomerase IIalpha, apparently by blocking the DNA religation reaction
of the enzyme. The lack of inhibition of topoisomerase I catalytic activity by
TAS-103 explains how the drug is able to function as a topoisomerase II poison
in treated cells.
PMID: 10569942 [PubMed - indexed for MEDLINE]
620: Biochemistry 1999 Nov 23;38(47):15573-9
DNA topoisomerases as targets for the anticancer drug TAS-103: primary cellular
target and DNA cleavage enhancement.
Byl JA, Fortune JM, Burden DA, Nitiss JL, Utsugi T, Yamada Y, Osheroff N.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232-0146, USA.
TAS-103 is a novel antineoplastic agent that is active against in vivo tumor
models [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. This drug
is believed to be a dual topoisomerase I/II-targeted agent, because it enhances
both topoisomerase I- and topoisomerase II-mediated DNA cleavage in treated
cells. However, the relative importance of these two enzymes for the cytotoxic
actions of TAS-103 is not known. Therefore, the primary cellular target of the
drug and its mode of action were determined. TAS-103 stimulated DNA cleavage
mediated by mammalian topoisomerase I and human topoisomerase IIalpha and beta
in vitro. The drug was less active than camptothecin against the type I enzyme
but was equipotent to etoposide against topoisomerase IIalpha. A yeast genetic
system that allowed manipulation of topoisomerase activity and drug sensitivity
was used to determine the contributions of topoisomerase I and II to drug
cytotoxicity. Results indicate that topoisomerase II is the primary cellular
target of TAS-103. In addition, TAS-103 binds to human topoisomerase IIalpha in
the absence of DNA, suggesting that enzyme-drug interactions play a role in
formation of the ternary topoisomerase II.drug.DNA complex. TAS-103 induced
topoisomerase II-mediated DNA cleavage at sites similar to those observed in the
presence of etoposide. Like etoposide, it enhanced cleavage primarily by
inhibiting the religation reaction of the enzyme. Based on these findings, it is
suggested that TAS-103 be classified as a topoisomerase II-targeted drug.
PMID: 10569941 [PubMed - indexed for MEDLINE]
621: FEBS Lett 1999 Nov 19;461(3):253-7
Interactions between the full complement of human RNA polymerase II subunits.
Schaller S, Grandemange S, Shpakovski GV, Golemis EA, Kedinger C, Vigneron M.
Institut de Genetique et de Biologie Moleculaire et Cellulaire
(CNRS/INSERM/ULP), BP 163, 67404, Illkirch, France.
As an approach to elucidating the rules governing the assembly of human RNA
polymerase II (hRPB), interactions between its subunits have been systematically
analyzed. Eleven of the 12 expected hRPB subunits have previously been tested
for reciprocal interactions (J. Biol. Chem. 272 (1997) 16815-16821). We now
report the results obtained for the last subunit (hRPB4; Mol. Cell. Biol. 18
(1998) 1935-1945) and propose an essentially complete picture of the potential
interactions occurring within hRPB. Finally, complementation experiments in
yeast indicated that hRPB4 expression efficiently cured both heat and
cold-sensitivity of RPB4-lacking strains, supporting the existence of conserved
functional subunit interactions.
PMID: 10567706 [PubMed - indexed for MEDLINE]
622: Mol Cell Biol 1999 Dec;19(12):8673-85
Analysis of TFIIA function In vivo: evidence for a role in TATA-binding protein
recruitment and gene-specific activation.
Liu Q, Gabriel SE, Roinick KL, Ward RD, Arndt KM.
Department of Biological Sciences, University of Pittsburgh, Pittsburgh,
Pennsylvania 15260, USA.
Activation of transcription can occur by the facilitated recruitment of TFIID to
promoters by gene-specific activators. To investigate the role of TFIIA in TFIID
recruitment in vivo, we exploited a class of yeast TATA-binding protein (TBP)
mutants that is activation and DNA binding defective. We found that
co-overexpression of TOA1 and TOA2, the genes that encode yeast TFIIA, overcomes
the activation defects caused by the TBP mutants. Using a genetic screen, we
isolated a new class of TFIIA mutants and identified three regions on TFIIA that
are likely to be involved in TBP recruitment or stabilization of the TBP-TATA
complex in vivo. Amino acid replacements in only one of these regions enhance
TFIIA-TBP-DNA complex formation in vitro, suggesting that the other regions are
involved in regulatory interactions. To determine the relative importance of
TFIIA in the regulation of different genes, we constructed yeast strains to
conditionally deplete TFIIA levels prior to gene activation. While the
activation of certain genes, such as INO1, was dramatically impaired by TFIIA
depletion, activation of other genes, such as CUP1, was unaffected. These data
suggest that TFIIA facilitates DNA binding by TBP in vivo, that TFIIA may be
regulated by factors that target distinct regions of the protein, and that
promoters vary significantly in the degree to which they require TFIIA for
activation.
PMID: 10567590 [PubMed - indexed for MEDLINE]
623: Mol Microbiol 1999 Nov;34(4):780-91
Interaction between the F plasmid TraA (F-pilin) and TraQ proteins.
Harris RL, Sholl KA, Conrad MN, Dresser ME, Silverman PM.
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation,
Oklahoma City, OK 73104, USA.
Elaboration of conjugative (F) pili by F+ strains of Escherichia coli requires
the activities of over a dozen F-encoded DNA transfer (Tra) proteins. The
organization and functions of these proteins are largely unknown. Using the
yeast two-hybrid assay, we have begun to analyse binary interactions among the
Tra proteins required for F-pilus formation. We focus here on interactions
involving F-pilin, the only known F-pilus subunit. Using a library of F tra DNA
fragments that contained all the F genes required for F pilus formation in a
yeast GAL4 activation domain vector (pACTII), we transformed yeast containing a
plasmid (pAS1CYH2traA) encoding a GAL4 DNA-binding domain-F-pilin fusion. Doubly
transformed cells were screened for GAL4-dependent gene expression. This screen
repeatedly identified only a single Tra protein, TraQ, previously identified as
a likely F-pilin chaperone. The F-pilin-TraQ interaction appeared to be
specific, as no transcriptional activation was detected in yeast transformants
containing pACTIItraQ plasmids and the Salmonella typhi pED208 traA gene cloned
in pAS1CYH2. Two traQ segments isolated in the screen against F-pilin were
tested for complementation of a traQ null allele in E. coli. One, lacking the
first 11 (of 94) TraQ amino acids, restored DNA donor activity, donor-specific
bacteriophage sensitivity and membrane F-pilin accumulation to wild-type levels.
The second, lacking the first 21 amino acids, was much less effective in these
assays. Both TraQ polypeptides accumulated in E. coli as transmembrane proteins.
The longer, biologically active segment was fused to the GAL4 DNA-binding domain
gene of pAS1CYH2 and used to screen the tra fragment library. The only positives
from this screen identified traA segments. The fusion sites between the traA and
GAL4 segments identified the hydrophobic, C-terminal domain IV of F-pilin as
sufficient for the interaction. As TraQ is the only Tra protein required for the
accumulation of inner membrane F-pilin, the interaction probably reflects a
specific, chaperone-like function for TraQ in E. coli. Attempts to isolate an
F-pilin-TraQ complex from E. coli were unsuccessful, suggesting that the
interaction between the two is normally transient, as expected from previous
studies of the kinetics of TraA membrane insertion and processing to F-pilin.
PMID: 10564517 [PubMed - indexed for MEDLINE]
624: Biochim Biophys Acta 1999 Nov 16;1435(1-2):147-52
A novel mammalian nuclear protein similar to Schizosaccharomyces pombe
Prp1p/Zer1p and Saccharomyces cerevisiae Prp6p pre-mRNA splicing factors.
Nishikimi A, Mukai J, Kioka N, Yamada M.
Laboratory of Reproductive Physiology, Graduate School of Agriculture, Kyoto
University, Kyoto, Japan.
We have cloned a novel 100-kDa mammalian protein, which was recognized by an
anti-peptide antibody against an epitope-containing nuclear localization signal
of NF-kappaB p65 subunit. Predicted amino acid sequence of the protein is
similar to those of yeast splicing factors, Prp1p/Zer1p of Schizosaccharomyces
pombe and Prp6p of Saccharomyces cerevisiae. Among these proteins, tetratrico
peptide repeat (TPR) motif, which mediates protein-protein interactions, is
conserved, whereas leucine zipper motif is found only in the 100-kDa protein.
Indirect immunofluorescent staining showed that the 100-kDa protein localized in
the nucleus in HeLa cells.
PMID: 10561546 [PubMed - indexed for MEDLINE]
625: Mol Biol Cell 1999 Nov;10(11):3549-65
Shr3p mediates specific COPII coatomer-cargo interactions required for the
packaging of amino acid permeases into ER-derived transport vesicles.
Gilstring CF, Melin-Larsson M, Ljungdahl PO.
Ludwig Institute for Cancer Research, S-171 77 Stockholm, Sweden.
The SHR3 gene of Saccharomyces cerevisiae encodes an integral membrane component
of the endoplasmic reticulum (ER) with four membrane-spanning segments and a
hydrophilic, cytoplasmically oriented carboxyl-terminal domain. Mutations in
SHR3 specifically impede the transport of all 18 members of the amino acid
permease (aap) gene family away from the ER. Shr3p does not itself exit the ER.
Aaps fully integrate into the ER membrane and fold properly independently of
Shr3p. Shr3p physically associates with the general aap Gap1p but not Sec61p,
Gal2p, or Pma1p in a complex that can be purified from
N-dodecylmaltoside-solubilized membranes. Pulse-chase experiments indicate that
the Shr3p-Gap1p association is transient, a reflection of the exit of Gap1p from
the ER. The ER-derived vesicle COPII coatomer components Sec13p, Sec23p, Sec24p,
and Sec31p but not Sar1p bind Shr3p via interactions with its carboxyl-terminal
domain. The mutant shr3-23p, a nonfunctional membrane-associated protein, is
unable to associate with aaps but retains the capacity to bind COPII components.
The overexpression of either Shr3p or shr3-23p partially suppresses the
temperature-sensitive sec12-1 allele. These results are consistent with a model
in which Shr3p acts as a packaging chaperone that initiates ER-derived transport
vesicle formation in the proximity of aaps by facilitating the membrane
association and assembly of COPII coatomer components.
PMID: 10564255 [PubMed - indexed for MEDLINE]
626: Genes Dev 1999 Nov 1;13(21):2811-27
A specific protein-protein interaction accounts for the in vivo substrate
selectivity of Ptp3 towards the Fus3 MAP kinase.
Zhan XL, Guan KL.
Department of Biological Chemistry, University of Michigan Medical School, Ann
Arbor, Michigan 48109-0606, USA.
The mitogen-activated protein kinases (MAPKs) play critical roles in many signal
transduction processes. Several MAPKs have been found in Saccharomyces
cerevisiae, including Fus3 in the mating pathway and Hog1 in the osmotic-stress
response pathway. Cells lacking Fus3 or Hog1 activity are deficient in mating or
adaptation to osmotic shock, respectively. However, constitutive activation of
either Fus3 or Hog1 is lethal. Therefore, yeast cells have to tightly regulate
both the activation and inactivation of Fus3 and Hog1 MAPKs, which are
controlled mainly by phosphorylation and dephosphorylation. Previous studies
have shown that Fus3 activity is negatively regulated by protein tyrosine
phosphatase Ptp3. In contrast, the Hog1 MAPK is mainly dephosphorylated by Ptp2
even though the two phosphatases share a high degree of sequence similarity. To
understand the mechanisms of MAPK regulation, we examined the molecular basis
underlying the in vivo substrate specificity between phosphatases and MAPKs. We
observed that the amino-terminal noncatalytic domain of Ptp3 directly interacts
with Fus3 via CH2 (Cdc25 homology) domain conserved among yeast PTPases and
mammalian MAP kinase phosphatases and is responsible for the in vivo substrate
selectivity of the phosphatase. Interaction between Ptp3 and Fus3 is required
for dephosphorylation and inactivation of Fus3 under physiological conditions.
Mutations in either Ptp3 or Fus3 that abolish this interaction cause a
dysregulation of the Fus3 MAPK. Our data demonstrate that the specificity of MAP
kinase inactivation in vivo by phosphatases is determined by specific
protein-protein interactions outside of the phosphatase catalytic domain.
PMID: 10557209 [PubMed - indexed for MEDLINE]
627: J Mol Biol 1999 Nov 19;294(1):121-37
The dimerization/repression domain of RFX1 is related to a conserved region of
its yeast homologues Crt1 and Sak1: a new function for an ancient motif.
Katan-Khaykovich Y, Spiegel I, Shaul Y.
Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot,
76100, Israel.
The RFX protein family includes members from yeast to humans, which function in
various biological systems, and share a DNA-binding domain and a conserved
C-terminal region. In the human transcription regulator RFX1, the conserved C
terminus is an independent functional domain, which mediates dimerization and
transcriptional repression. This dimerization domain has a unique ability to
mediate the formation of two alternative homodimeric DNA-protein complexes, the
upper of which has been linked to repression. Here, we localize the complex
formation capacity to several different RFX1 C-terminal subregions, each of
which can function independently to generate the upper complex and repress
transcription, thus correlating complex formation with repression. To gain an
evolutionary perspective, we have examined whether the different properties of
the RFX1 C terminus exist in the two yeast RFX proteins, which are involved in
signaling pathways. Replacement of the RFX1 C terminus with those of Sak1 and
Crt1, its orthologues from Schizosaccharomyces pombe and Saccharomyces
cerevisiae, respectively, and analysis of fusions with the Gal4 DNA-binding
domain, revealed that the ability to generate the two alternative complexes is
conserved in the RFX family, from S. cerevisiae to man. While sharing this
unique biochemical property, the three C termini differed from each other in
their ability to mediate dimerization and transcriptional repression. In both
functions, RFX1, Sak1, and Crt1 showed high capacity, moderate capacity, and no
capacity, respectively. This comparative analysis of the RFX proteins,
representing different evolutionary stages, suggests a gradual development of
the conserved C terminus, from the appearance of the ancestral motif (Crt1), to
the later acquisition of the dimerization/repression functions (Sak1), and
finally to the enhancement of these functions to generate a domain mediating
highly stable protein-protein interactions and potent transcriptional repression
(RFX1). Copyright 1999 Academic Press.
PMID: 10556033 [PubMed - indexed for MEDLINE]
628: Biochim Biophys Acta 1999 Nov 16;1422(3):235-54
Mechanisms of protein translocation into mitochondria.
Voos W, Martin H, Krimmer T, Pfanner N.
Institut fur Biochemie und Molekularbiologie, Universitat Freiburg,
Hermann-Herder-Str. 7, D-79104, Freiburg, Germany. voos@ruf.uni-freiburg.de
Mitochondrial biogenesis utilizes a complex proteinaceous machinery for the
import of cytosolically synthesized preproteins. At least three large
multisubunit protein complexes, one in the outer membrane and two in the inner
membrane, have been identified. These translocase complexes cooperate with
soluble proteins from the cytosol, the intermembrane space and the matrix. The
translocation of presequence-containing preproteins through the outer membrane
channel includes successive electrostatic interactions of the charged
mitochondrial targeting sequence with a chain of import components.
Translocation across the inner mitochondrial membrane utilizes the energy of the
proton motive force of the inner membrane and the hydrolysis of ATP. The matrix
chaperone system of the mitochondrial heat shock protein 70 forms an
ATP-dependent import motor by interaction with the polypeptide chain in transit
and components of the inner membrane translocase. The precursors of integral
inner membrane proteins of the metabolite carrier family interact with newly
identified import components of the intermembrane space and are inserted into
the inner membrane by a second translocase complex. A comparison of the full set
of import components between the yeast Sacccharomyces cerevisiae and the
nematode Caenorhabditis elegans demonstrates an evolutionary conservation of
most components of the mitochondrial import machinery with a possible greater
divergence for the import pathway of the inner membrane carrier proteins.
Publication Types:
Review
Review, Tutorial
PMID: 10548718 [PubMed - indexed for MEDLINE]
629: J Cell Sci 1999 Nov;112 ( Pt 22):4123-34
Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a
retro-translocation complex mediating protein transport for ER degradation.
Plemper RK, Bordallo J, Deak PM, Taxis C, Hitt R, Wolf DH.
Institut fur Biochemie, Universitat Stuttgart, D-70569 Stuttgart, Germany.
The endoplasmic reticulum contains a quality control system that subjects
misfolded or unassembled secretory proteins to rapid degradation via the
cytosolic ubiquitin proteasome system. This requires retrograde protein
transport from the endoplasmic reticulum back to the cytosol. The Sec61 pore,
the central component of the protein import channel into the endoplasmic
reticulum, was identified as the core subunit of the retro-translocon as well.
As import of mutated proteins into the endoplasmic reticulum lumen is
successfully terminated, a new targeting mechanism must exist that mediates
re-entering of misfolded proteins into the Sec61 pore from the lumenal side de
novo. The previously identified proteins Der3p/Hrd1p and, as we show here, Hrd3p
of the yeast Saccharomyces cerevisiae, are localised in the endoplasmic
reticulum membrane and are essential for the degradation of several substrates
of the endoplasmic reticulum degradation machinery. Based on genetic studies we
demonstrate that they functionally interact with each other and with Sec61p,
probably establishing the central part of the retro-translocon. In the absence
of Hrd3p, the otherwise stable protein Der3p/Hrd1p becomes rapidly degraded.
This depends on a functional ubiquitin proteasome system and the presence of
substrate molecules of the endoplasmic reticulum degradation system. When
overexpressed, Der3p/Hrd1p accelerates CPY* degradation in Delta(hrd3) cells.
Our data suggest a recycling process of Der3p/Hrd1p through Hrd3p. The
retro-translocon seems to be build up at least by the Sec61 pore, Der3p/Hrd1p
and Hrd3p and mediates both retrograde transport and ubiquitination of substrate
molecules.
PMID: 10547371 [PubMed - indexed for MEDLINE]
630: Hum Mol Genet 1999 Nov;8(12):2263-73
Functional analysis of human FEN1 in Saccharomyces cerevisiae and its role in
genome stability.
Greene AL, Snipe JR, Gordenin DA, Resnick MA.
Laboratory of Molecular Genetics, National Institute of Environmental Health
Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA.
The flap endonuclease, FEN1, is an evolutionarily conserved component of DNA
replication from archaebacteria to humans. Based on in vitro results, it
processes Okazaki fragments during replication and is involved in base excision
repair. FEN1 removes the last primer ribonucleotide on the lagging strand and it
cleaves a 5' flap that may result from strand displacement during replication or
during base excision repair. Its biological importance has been revealed largely
through studies in the yeast Saccharomyces cerevisiae where deletion of the
homologous gene RAD27 results in genome instability and mutagen sensitivity.
While the in vivo function of Rad27 has been well characterized through genetic
and biochemical approaches, little is understood about the in vivo functions of
human FEN1. Guided by our recent results with yeast RAD27, we explored the
function of human FEN1 in yeast. We found that the human FEN1 protein
complements a yeast rad27 null mutant for a variety of defects including mutagen
sensitivity, genetic instability and the synthetic lethal interactions of a
rad27 rad51 and a rad27 pol3-01 mutant. Furthermore, a mutant form of FEN1
lacking nuclease function exhibits dominant-negative effects on cell growth and
genome instability similar to those seen with the homologous yeast rad27
mutation. This genetic impact is stronger when the human and yeast PCNA-binding
domains are exchanged. These data indicate that the human FEN1 and yeast Rad27
proteins act on the same substrate in vivo. Our study defines a sensitive yeast
system for the identification and characterization of mutations in FEN1.
PMID: 10545607 [PubMed - indexed for MEDLINE]
631: EMBO J 1999 Nov 1;18(21):6095-105
Erratum in:
EMBO J 2000 Jan 17;19(2):315
Cellular transcription factors recruit viral replication proteins to activate
the Epstein-Barr virus origin of lytic DNA replication, oriLyt.
Baumann M, Feederle R, Kremmer E, Hammerschmidt W.
GSF-National Research Center for Environment and Health, Institute of Clinical
Molecular Biology and Tumor Genetics, Department of Gene Vectors, Munchen,
Germany.
DNA replication of Epstein-Barr virus (EBV) during the productive phase of the
life cycle of this herpesvirus depends on the cis-acting element oriLyt. It
consists of two essential domains, the upstream and the downstream component.
Whereas the upstream component contains several DNA-binding motifs for the viral
activator protein BZLF1, the downstream component is known to be the binding
site of several cellular proteins. We identified cellular transcription factors
that bind synergistically to a functionally relevant subsequence of the
downstream component, the TD element. Two of these transcription factors, ZBP-89
and Sp1, stimulate replication as shown by protein fusions with the GAL4
DNA-binding domain and a single GAL4 DNA-binding motif inserted into the TD
element. In protein binding assays, we observed an interaction of Sp1 and ZBP-89
with the viral DNA polymerase and its processivity factor. Our data indicate
that cellular transcriptional activators tether viral replication proteins to
the lytic origin via direct protein-protein interactions to assemble the viral
replication complex at oriLyt.
PMID: 10545120 [PubMed - indexed for MEDLINE]
632: Toxicol Appl Pharmacol 1999 Nov 1;160(3):297-303
A human aryl hydrocarbon receptor signaling pathway constructed in yeast
displays additive responses to ligand mixtures.
Miller CA 3rd.
Environmental Health Sciences Department, Tulane University School of Public
Health and Tropical Medicine, Tulane-Xavier Center for Bioenvironmental
Research, 1430 Tulane Avenue, New Orleans, Louisiana, 70112, USA.
An optimized signal transduction pathway that reproduces the response of human
aryl hydrocarbon (Ah) receptor to ligands has been established in Saccharomyces
cerevisiae. Ligand treatment induced a 50-fold increase in beta-galactosidase
activity from a reporter plasmid in yeast engineered to express human Ah
receptor and Ah nuclear translocator (Arnt) proteins. The archetypal Ah receptor
ligand, 2,3,7,8-tetrachlorodibenzo(p)dioxin, activated Ah receptor and induced
lacZ reporter activity at concentrations of >/=0.3 nM. Mixtures of halogenated
and nonhalogenated Ah receptor ligands produced additive signaling responses in
this yeast bioassay. These results were consistent with the existence of a
common binding site and mechanism of ligand-mediated Ah receptor activation.
Although yeast have no natural counterpart to the Ah receptor pathway,
expression of human Ah receptor and Arnt under the appropriate conditions
provides a functional model system for studying Ah receptor activation and
signal transduction. Copyright 1999 Academic Press.
PMID: 10544064 [PubMed - indexed for MEDLINE]
633: Curr Biol 1999 Oct 7;9(19):1111-4
A retention mechanism for distribution of mitochondria during cell division in
budding yeast.
Yang HC, Palazzo A, Swayne TC, Pon LA.
Department of Anatomy and Cell Biology Columbia University College of Physicians
and Surgeons New York, New York, 10032, USA.
Mitochondria are indispensable for normal eukaryotic cell function. As they
cannot be synthesized de novo and are self-replicating, mitochondria must be
transferred from mother to daughter cells. Studies in the budding yeast
Saccharomyces cerevisiae indicate that mitochondria enter the bud immediately
after bud emergence, interact with the actin cytoskeleton for linear, polarized
movement of mitochondria from mother to bud, but are equally distributed among
mother and daughter cells [1] [2] [3]. It is not clear how the mother cell
maintains its own supply of mitochondria. Here, we found that mother cells
retain mitochondria by immobilization of some mitochondria in the 'retention
zone', the base of the mother cell distal to the bud. Retention requires the
actin cytoskeleton as mitochondria colocalized with actin cables in the
retention zone, and mutations that perturb actin dynamics or actin-mitochondrial
interactions produced retention defects. Our results support the model that
equal distribution of mitochondria during cell division is a consequence of two
actin-dependent processes: movement of some mitochondria into the daughter bud
and immobilization of others in the mother cell.
PMID: 10531006 [PubMed - indexed for MEDLINE]
634: Appl Microbiol Biotechnol 1999 Sep;52(3):311-20
Yeast cells as tools for target-oriented screening.
Munder T, Hinnen A.
Hans-Knoll-Institut fur Naturstoff-Forschung e V. Jena, Germany.
Information about biomolecular interaction networks is crucial for understanding
cellular functions and the development of disease processes. Many diseases are
known to be based on aberrations of DNA sequences encoding proteins with key
functions in the cellular metabolism. Alterations in the respective proteins
often lead to disturbances in biomolecular interactions caused by unbalanced
stoichiometries, and thus result in alterations of molecule fluxes, cell
architecture and signalling pathways. Drug discovery programmes have been
designed to find promising chemical lead structures with the help of
target-oriented bioassay systems. These are, in most cases, based upon the
interaction of small molecules to specific macromolecular targets in vivo or in
vitro, as exemplified by enzyme assays or small-ligand-based receptor systems.
In addition, interactions between large biomolecules, such as proteins or
nucleic acids, offer a huge arsenal of potential drug targets that can be
addressed by small chemical compounds. This latter approach is gaining
considerable attention because many potential target structures are becoming
available through genomic research. Funnelling these new targets into
high-throughput screening programs represents a major challenge for today's
pharmaceutical research. An important outcome of the ongoing genome projects is
the fact that the basic cellular structures, pathways and signalling principles
show a high degree of conservation. Model organisms that are easily approachable
by genetic, biochemical and physiological means can thus play an important role
in the design of target-oriented screening systems. They offer the possibility
to express individual proteins, nucleic acids or even more complex aggregates of
biomolecules such as protein-interaction networks or transcription-initiation
complexes, which can be addressed by small effector molecules in vivo. Combining
these targets with biological signalling systems is an attractive way of
creating robust cellular assay systems.
Publication Types:
Review
Review, Tutorial
PMID: 10531642 [PubMed - indexed for MEDLINE]
635: Infect Immun 1999 Nov;67(11):6040-7
Overexpression of the Candida albicans ALA1 gene in Saccharomyces cerevisiae
results in aggregation following attachment of yeast cells to extracellular
matrix proteins, adherence properties similar to those of Candida albicans.
Gaur NK, Klotz SA, Henderson RL.
Department of Research, Veterans Affairs Medical Center, Kansas City, Missouri
64128, USA. nkgaur@kuhub.cc.ukans.edu
Candida albicans maintains a commensal relationship with human hosts, probably
by adhering to mucosal tissue in a variety of physiological conditions. We show
that adherence due to the C. albicans gene ALA1 when transformed into
Saccharomyces cerevisiae, is comprised of two sequential steps. Initially, C.
albicans rapidly attaches to extracellular matrix (ECM) protein-coated magnetic
beads in small numbers (the attachment phase). This is followed by a relatively
slower step in which cell-to-cell interactions predominate (the aggregation
phase). Neither of these phases is observed in S. cerevisiae. However,
expression of the C. albicans ALA1 gene from a low-copy vector causes S.
cerevisiae transformants to attach to ECM-coated magnetic beads without
appreciable aggregation. Expression of ALA1 from a high-copy vector results in
both attachment and aggregation. Moreover, transcriptional fusion of ALA1 with
the galactose-inducible promoters GALS, GALL, and GAL1, allowing for low,
moderate, and high levels of inducible transcription, respectively, causes
attachment and aggregation that correlates with the strength of the GAL
promoter. The adherence of C. albicans and S. cerevisiae overexpressing ALA1 to
a number of protein ligands occurs over a broad pH range, is resistant to shear
forces generated by vortexing, and is unaffected by the presence of sugars, high
salt levels, free ligands, or detergents. Adherence is, however, inhibited by
agents that disrupt hydrogen bonds. The similarities in the adherence and
aggregation properties of C. albicans and S. cerevisiae overexpressing ALA1
suggest a role in adherence and aggregation for ALA1 and ALA1-like genes in C.
albicans.
PMID: 10531265 [PubMed - indexed for MEDLINE]
636: Methods 1999 Oct;19(2):338-49
Genetic approaches to the study of protein-protein interactions.
Appling DR.
Department of Chemistry and Biochemistry, The Biochemical Institute, Austin,
Texas 78712, USA. dappling@mail.utexas.edu
This article describes genetic approaches to the study of heterologous
protein-protein interactions, focusing on the yeast Saccharomyces cerevisiae as
a useful eukaryotic model system. Several methods are described that can be used
to search for new interactions, including extragenic suppression, multicopy
suppression, synthetic lethality, and transdominant inhibition. Strategies for
screening, genetic characterization, and clone identification are described,
along with recent examples from the literature. In addition, genetic methods are
discussed that can be used to further characterize a newly discovered
protein-protein interaction. These include the creation of mutant libraries of a
given protein by chemical mutagenesis or polymerase chain reaction, the
production of dominant-negative mutants, and strategies for introducing these
mutant alleles back into yeast for analysis. Although these genetic methods are
quite powerful, they are often just a starting point for further biochemical or
cell biological experiments. Copyright 1999 Academic Press.
PMID: 10527736 [PubMed - indexed for MEDLINE]
637: Methods 1999 Oct;19(2):330-7
Applications of the yeast two-hybrid system.
McAlister-Henn L, Gibson N, Panisko E.
Department of Biochemistry, University of Texas Health Science Center, San
Antonio, Texas 78284-7760, USA. henn@uthscsa.edu
In recent years, the yeast two-hybrid system has become the method of choice for
detection and analysis of protein-protein interactions in an in vivo context.
This system, which capitalizes on the significant genetic history and ease of
protocols for manipulation of Saccharomyces cerevisiae, is accessible to most
laboratories and is applicable to the pursuit of a large variety of experimental
goals. To date, the two-hybrid system has seen widespread application for
identification of interaction partners by screening methods using a particular
protein of interest as a "bait." Large-scale ventures are also in progress, for
example, a cataloging of interactions among the cellular proteins in yeast.
However, this method also has tremendous potential for more focused analyses of
specific proteins and should become more routine as an alternative or adjunct
approach for many structure-function investigations. Copyright 1999 Academic
Press.
PMID: 10527735 [PubMed - indexed for MEDLINE]
638: Mutat Res 1999 Sep 13;435(1):23-33
Erratum in:
Mutat Res 2000 Mar 20;459(2):171-2
Protein complexes in nucleotide excision repair.
Araujo SJ, Wood RD.
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms,
Hertfordshire, UK.
The main pathway by which mammalian cells remove DNA damage caused by UV light
and some other mutagens is nucleotide excision repair (NER). The best
characterised components of the human NER process are those proteins defective
in the inherited disorder xeroderma pigmentosum (XP). The proteins known to be
involved in the first steps of the NER reaction (damage recognition and
incision-excision) are heterotrimeric RPA, XPA, the 6 to 9 subunit TFIIH,
XPC-hHR23B, XPG, and ERCC1-XPF. Many interactions between these proteins have
been found in recent years using different methods both in mammalian cells and
for the homologous proteins in yeast. There are virtually no quantitative
measurements of the relative strengths of these interactions. Higher order
associations between these proteins in solution and even the existence of a
complete "repairosome" complex have been reported, which would have implications
both for the mechanism of repair and for the interplay between NER and other
cellular processes. Nevertheless, evidence for a completely pre-assembled
functional repairosome in solution is inconclusive and the order of action of
repair factors on damaged DNA is uncertain.
Publication Types:
Review
Review, Tutorial
PMID: 10526214 [PubMed - indexed for MEDLINE]
639: Mutat Res 1999 Sep 13;435(1):1-11
Genetic interactions between error-prone and error-free postreplication repair
pathways in Saccharomyces cerevisiae.
Xiao W, Chow BL, Fontanie T, Ma L, Bacchetti S, Hryciw T, Broomfield S.
Department of Microbiology and Immunology, University of Saskatchewan,
Saskatoon, Canada. xiaow@sask.usask.ca
Evidence obtained from recent studies supports the existence of an error-free
postreplication repair (PRR) and a mutagenesis pathway within the Saccharomyces
cerevisiae RAD6 DNA repair group. The MMS2 gene is the only known yeast gene
involved in error-free PRR that, when mutated, significantly increases the
spontaneous mutation rate. In this study, the mutational spectrum of the mms2
mutator was determined and compared to the wild type strain. In addition,
mutagenenic effects and genetic interactions of the mms2 mutator and rev3
anti-mutator were examined with respect to forward mutations, frameshift
reversions as well as amber and ochre suppressions. It was concluded from these
results that the mms2 mutator phenotype is largely dependent on the functional
REV3 gene. The synergistic effects of mms2 and rev3 mutations towards killing by
a variety of DNA-damaging agents ruled out the possibility that MMS2 simply acts
to suppress REV3 activity and favored the hypothesis that MMS2 and REV3 form two
alternative subpathways within the RAD6 DNA repair pathway. Taken together, we
propose that two pathways represented by MMS2 and REV3 deal with a similar range
of endogenous and environmental DNA damage but with different biological
consequences, namely, error-free repair and mutagenesis, respectively.
PMID: 10526212 [PubMed - indexed for MEDLINE]
640: FEBS Lett 1999 Oct 15;459(3):458-62
The yeast Rgd1p is a GTPase activating protein of the Rho3 and rho4 proteins.
Doignon F, Weinachter C, Roumanie O, Crouzet M.
Laboratoire de Biologie Moleculaire et de Sequencage, UPR CNRS 9026, BP Box 64,
146 rue Leo Saignat, 33076, Bordeaux, France.
The RGD1 gene, identified during sequencing of the Saccharomyces cerevisiae
genome, encodes a protein with a Rho-GTPase activating protein (GAP) domain at
the carboxy-terminal end. The Rgd1 protein showed two-hybrid interactions with
the activated forms of Rho2p, Rho3p and Rho4p. Using in vitro assays, we
demonstrated that Rgd1p stimulated the GTPase activity of both Rho3p and Rho4p;
no stimulation was observed on Rho2p. In addition, the rho3Deltargd1Delta double
mutant exhibited a dramatic growth defect compared to the single mutants,
suggesting that Rgd1p has a GAP activity in vivo. The present study allowed the
identification of the first GAP of Rho3p and Rho4p.
PMID: 10526184 [PubMed - indexed for MEDLINE]
641: FEBS Lett 1999 Oct 15;459(3):427-32
Analysis by atomic force microscopy of Med8 binding to cis-acting regulatory
elements of the SUC2 and HXK2 genes of saccharomyces cerevisiae.
Moreno-Herrero F, Herrero P, Colchero J, Baro AM, Moreno F.
Departamento de Bioquimica y Biologia Molecular, Instituto Universitario de
Biotecnologia de Asturias, Universidad de Oviedo, 33006, Oviedo, Spain.
Med8 protein is a regulator that specifically binds to upstream activating
sequences (UASs) of SUC2 promoter, to downstream repressing sequences (DRSs) of
the HXK2 gene and to the carboxy-terminal domain of the RNA polymerase II.
Atomic force microscopy has allowed for direct visualization of Med8
interactions with a 305 bp fragment of SUC2 promoter and with a 676 bp fragment
of HXK2 gene, containing respectively the UASs and DRSs regulatory regions. This
approach has provided complementary information about the position and the
structure of the DNA-protein complexes. Med8 binding to DNA results in total
covering of one of the two existing 7 bp motives (consensus, (A/C)(A/G)GAAAT) in
the studied DNA fragments. No preference for binding either of the two UASs of
SUC2 promoter as well as for the two DRSs of HXK2 gene has been found. We also
discuss whether this protein works as dimer or as a monomer.
PMID: 10526178 [PubMed - indexed for MEDLINE]
642: Chromosoma 1999 Sep;108(5):278-90
Tel2p, a regulator of yeast telomeric length in vivo, binds to single-stranded
telomeric DNA in vitro.
Kota RS, Runge KW.
The Lerner Research Institute, Cleveland Clinic Foundation, Department of
Molecular Biology, NC 20, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
The telomeres of the yeast Saccharomyces cerevisiae consist of a duplex region
of TG(1-3) repeats that acquire a single-stranded 3' extension of the TG(1-3)
strand at the end of S-phase. The length of these repeats is kept within a
defined range by regulators such as the TEL2-encoded protein (Tel2p). Here we
show that Tel2p can specifically bind to single-stranded TG(1-3). Tel2p binding
produced several shifted bands; however, only the slowest migrating band
contained Tel2p. Methylation protection and interference experiments as well as
gel shift experiments using inosine-containing probes indicated that the faster
migrating bands resulted from Tel2p-mediated formation of DNA secondary
structures held together by G-G interactions. Tel2p bound to single-stranded
substrates that were at least 19 bases in length and contained 14 bases of
TG(1-3), and also to double-stranded/single-stranded hybrid substrates with a 3'
TG(1-3) overhang. Tel2p binding to a hybrid substrate with a 24 base
single-stranded TG(1-3) extension also produced a band characteristic of
G-G-mediated secondary structures. These data suggest that Tel2p could regulate
telomeric length by binding to the 3' single-stranded TG(1-3) extension present
at yeast telomeres.
PMID: 10525964 [PubMed - indexed for MEDLINE]
643: Biotechniques 1999 Oct;27(4):790-6
Identification of ribonucleoprotein (RNP)-specific protein interactions using a
yeast RNP interaction trap assay (RITA).
Bouffard P, Briere F, Wellinger RJ, Boire G.
Rheumatology Division, Faculty of Medicine, Universite de Sherbrooke, QC,
Canada.
We describe an adaptation of the yeast three-hybrid system that allows the
reconstitution in vivo of tripartite (protein-RNA-protein) ribonucleoproteins
(RNPs). To build and try this system that we called RNP interaction trap assay
(RITA), we used as a model the autoantigenic Ro RNPs. hY RNAs bear distinct
binding sites for Ro60 and La proteins, and Ro RNPs are thus physiologically
tripartite (Ro60/hY RNA/La). Using recombinant La (rLa) and Ro60 (rRo60)
proteins and recombinant hY RNAs (rhY) co-expressed in yeast, we found that RNPs
made of rRo60/rhY/rLa were readily reassembled. Reconstitution of tripartite
RNPs was critically dependent on the presence of an appropriate Ro60 binding
site on the recombinant RNA. The RITA assay was further used to detect
(rRo60/rhY RNP)-binding proteins from a HeLa cell cDNA library, allowing
specific identification of La and of a novel Ro RNP-binding protein (RoBPI) in
more than 70% of positive clones. RITA assay may complement already available
two- and three-hybrid systems to characterize RNP-binding proteins by allowing
the in vivo identification of interactions strictly dependent upon the
simultaneous presence of a protein and of its cognate RNA.
PMID: 10524322 [PubMed - indexed for MEDLINE]
644: Mol Cell Biol 1999 Nov;19(11):7661-71
Control of meiotic recombination and gene expression in yeast by a simple
repetitive DNA sequence that excludes nucleosomes.
Kirkpatrick DT, Wang YH, Dominska M, Griffith JD, Petes TD.
Department of Biology University of North Carolina, Chapel Hill, North Carolina
27599-3280, USA.
Tandem repeats of the pentanucleotide 5'-CCGNN (where N indicates any base) were
previously shown to exclude nucleosomes in vitro (Y. -H. Wang and J. D.
Griffith, Proc. Natl. Acad. Sci. USA 93:8863-8867, 1996). To determine the in
vivo effects of these sequences, we replaced the upstream regulatory sequences
of the HIS4 gene of Saccharomyces cerevisiae with either 12 or 48 tandem copies
of CCGNN. Both tracts activated HIS4 transcription. We found that (CCGNN)(12)
tracts elevated meiotic recombination (hot spot activity), whereas the
(CCGNN)(48) tract repressed recombination (cold spot activity). In addition, a
"pure" tract of (CCGAT)(12) activated both transcription and meiotic
recombination. We suggest that the cold spot activity of the (CCGNN)(48) tract
is related to the phenomenon of the suppressive interactions of adjacent hot
spots previously described in yeast (Q.-Q. Fan, F. Xu, and T. D. Petes, Mol.
Cell. Biol. 15:1679-1688, 1995; Q.-Q. Fan, F. Xu, M. A. White, and T. D. Petes,
Genetics 145:661-670, 1997; T.-C. Wu and M. Lichten, Genetics 140:55-66, 1995;
L. Xu and N. Kleckner, EMBO J. 16:5115-5128, 1995).
PMID: 10523654 [PubMed - indexed for MEDLINE]
645: J Biol Chem 1999 Oct 22;274(43):30393-401
An RNA binding motif in the Cbp2 protein required for protein-stimulated RNA
catalysis.
Tirupati HK, Shaw LC, Lewin AS.
Department of Molecular Genetics, University of Florida College of Medicine,
Gainesville, Florida 32605, USA.
The fifth and terminal intron of yeast cytochrome b pre-mRNA (a group I intron)
requires a protein encoded by the nuclear gene CBP2 for splicing. Because
catalysis is intrinsic to the RNA, the protein is believed to promote formation
of secondary and tertiary structure of the RNA, resulting in a catalytically
competent intron. In vitro, this mitochondrial intron can be made to self-splice
or undergo protein-facilitated splicing by varying the Mg(2+) and monovalent
salt concentrations. This two-component system, therefore, provides a good model
for understanding the role of proteins in RNA folding. A UV cross-linking
experiment was initiated to identify RNA binding sites on Cbp2 and gain insights
into Cbp2-intron interactions. A 12-amino acid region containing a presumptive
contact site near the amino terminus was targeted for mutagenesis, and mutant
proteins were characterized for RNA binding and stimulation of splicing.
Mutations in this region resulted in partial or complete loss of function,
demonstrating the importance of this determinant for stimulation of RNA
splicing. Several of the mutations that severely reduced splicing did not
significantly shift the overall binding isotherm of Cbp2 for the precursor RNA,
suggesting that contacts critical for activity are not necessarily reflected in
the dissociation constant. This analysis has identified a unique RNA binding
motif of alternating basic and aromatic residues that is essential for protein
facilitated splicing.
PMID: 10521416 [PubMed - indexed for MEDLINE]
646: FEBS Lett 1999 Sep 10;458(1):11-6
Functional phage display of leech-derived tryptase inhibitor (LDTI):
construction of a library and selection of thrombin inhibitors.
Tanaka AS, Silva MM, Torquato RJ, Noguti MA, Sampaio CA, Fritz H, Auerswald EA.
Departamento de Bioquimica, UNIFESP-EPM, Sao Paulo, Brazil. tanaka.bioq@epm.br
The recombinant phage antibody system pCANTAB 5E has been used to display
functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the
filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants
was created with a synthetic LDTI gene, using a degenerated oligonucleotide and
the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions
of the reactive site. Fusion phages and appropriate host strains containing the
phagemids were selected after binding to thrombin and DNA sequencing. The
variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S,
P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a
Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T,
prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and
trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or
neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit
it. The relevant reactive site sequences of the thrombin inhibiting variants
showed a strong preference for arginine in position P1 (K8R) and for valine in
P1' (I9V). The data indicate further that LDTI-5T might be a model candidate for
generation of active-site directed thrombin inhibitors and that LDTI in general
may be useful to generate specific inhibitors suitable for a better
understanding of enzyme-inhibitor interactions.
PMID: 10518924 [PubMed - indexed for MEDLINE]
647: Proc Natl Acad Sci U S A 1999 Oct 12;96(21):11752-7
An internal targeting signal directing proteins into the mitochondrial
intermembrane space.
Diekert K, Kispal G, Guiard B, Lill R.
Institut fur Zytobiologie und Zytopathologie der Philipps-Universitat Marburg,
Robert-Koch-Strasse 5, 35033 Marburg, Germany.
Import of most nucleus-encoded preproteins into mitochondria is mediated by
N-terminal presequences and requires a membrane potential and ATP hydrolysis.
Little is known about the chemical nature and localization of other
mitochondrial targeting signals or of the mechanisms by which they facilitate
membrane passage. Mitochondrial heme lyases lack N-terminal targeting
information. These proteins are localized in the intermembrane space and are
essential for the covalent attachment of heme to c type cytochromes. For import
of heme lyases, the translocase of the mitochondrial outer membrane complex is
both necessary and sufficient. Here, we report the identification of the
targeting signal of mitochondrial heme lyases in the third quarter of these
proteins. The targeting sequence is highly conserved among all known heme
lyases. Its chemical character is hydrophilic because of a large fraction of
both positively and negatively charged amino acid residues. These features
clearly distinguish this signal from classical presequences. When inserted into
a cytosolic protein, the targeting sequence directs the fusion protein into the
intermembrane space, even in the absence of a membrane potential or ATP
hydrolysis. The heme lyase targeting sequence represents the first topogenic
signal for energy-independent transport into the intermembrane space and harbors
two types of information. It assures accurate recognition and translocation by
the translocase of the mitochondrial outer membrane complex, and it is
responsible for driving the import reaction by undergoing high-affinity
interactions with components of the intermembrane space.
PMID: 10518522 [PubMed - indexed for MEDLINE]
648: Mol Cell 1999 Sep;4(3):387-94
The FHA domain is a modular phosphopeptide recognition motif.
Durocher D, Henckel J, Fersht AR, Jackson SP.
Wellcome Trust and Cancer Research Campaign, Institute of Cancer and
Developmental Biology, Cambridge, United Kingdom.
FHA domains are conserved sequences of 65-100 amino acid residues found
principally within eukaryotic nuclear proteins, but which also exist in certain
prokaryotes. The FHA domain is thought to mediate protein-protein interactions,
but its mode of action has yet to be elucidated. Here, we show that the two
highly divergent FHA domains of Saccharomyces cerevisiae Rad53p, a protein
kinase involved in cell cycle checkpoint control, possess phosphopeptide-binding
specificity. We also demonstrate that other FHA domains bind peptides in a
phospho-dependent manner. These findings indicate that the FHA domain is a
phospho-specific protein-protein interaction motif and have important
implications for mechanisms of intracellular signaling in both eukaryotes and
prokaryotes.
PMID: 10518219 [PubMed - indexed for MEDLINE]
649: Biotechnol Bioeng 1999 Dec 5;65(5):550-7
Visualizing integrated bioprocess designs through "windows of operation".
Zhou YH, Titchener-Hooker NJ.
The Advanced Centre for Biochemical Engineering, Department of Biochemical
Engineering, University College London, Torrington Place, London WC1E 7JE, UK.
y.zhou@ucl.ac.uk
This paper demonstrates a simple graphical approach for the design and analysis
of a bioprocess flowsheet in which process interactions are significant. Results
are presented showing how the feasible space for operation can be simulated and
used both to address key design and operating decisions and to identify suitable
trade-offs between operating variables, such as fermentation growth rate and
disruption conditions, in order to achieve prespecified levels of process
performance. Using verified models to describe the production and isolation of
an intracellular protein alcohol dehydrogenase (ADH) in yeast as a test bed, a
series of so-called "windows of operation" are developed at growth rates in the
range of 0.06-0.28 h(-1) and for a range of overall process specifications. The
effects of altering the process design performance specification as defined by
the level of cell debris removal and the overall process productivity on the
size and position of the feasible space were investigated to demonstrate the
sensitivity of the flowsheet to changes in process objectives. Using the
approach it has been possible to visualise the processing trade-offs required to
increase performance in terms of the level of cell debris removal by 50% and the
overall process productivity by 400% from a defined base level. The approach
provides a convenient tool when designing integrated bioprocesses by enabling
process options to be compared visually and can help in achieving better process
designs and accelerating process development for the biological process
industry. Copyright 1999 John Wiley & Sons, Inc.
PMID: 10516581 [PubMed - indexed for MEDLINE]
650: J Biol Chem 1999 Oct 15;274(42):30052-8
Phospholipase C binds to the receptor-like GPR1 protein and controls
pseudohyphal differentiation in Saccharomyces cerevisiae.
Ansari K, Martin S, Farkasovsky M, Ehbrecht IM, Kuntzel H.
Max-Planck-Institut fur Experimentelle Medizin, Hermann-Rein-Strasse 3, D-37075
Gottingen, Germany.
The hormone receptor-like protein Gpr1p physically interacts with
phosphatidylinositol-specific phospholipase C (Plc1p) and with the Galpha
protein Gpa2p, as shown by two-hybrid assays and co-immune precipitation of
epitope-tagged proteins. Plc1p binds to Gpr1p in either the presence or absence
of Gpa2, whereas the Gpr1p/Gpa2p association depends on the presence of Plc1p.
Genetic interactions between the null mutations plc1Delta, gpr1Delta, gpa2Delta,
and ras2Delta suggest that Plc1p acts together with Gpr1p and Gpa2p in a growth
control pathway operating in parallel to the Ras2p function. Diploid cells
lacking Gpr1p, Plc1p, or Gpa2p fail to form pseudohyphae upon nitrogen
depletion, and the filamentation defect of gpr1Delta and plc1Delta strains is
rescued by activating a mitogen-activated protein kinase pathway via STE11-4 or
by activating a cAMP pathway via overexpressed Tpk2p. Plc1p is also required for
efficient expression of the FG(TyA)::lacZ reporter gene under nitrogen
depletion. In conclusion, we have identified two physically interacting
proteins, Gpr1p and Plc1p, as novel components of a nitrogen signaling pathway
controlling the developmental switch from yeast-like to pseudohyphal growth. Our
data suggest that phospholipase C modulates the interaction of the putative
nutrient sensor Gpr1p with the Galpha protein Gpa2p as a downstream effector of
filamentation control.
PMID: 10514491 [PubMed - indexed for MEDLINE]
651: Mol Biol Cell 1999 Oct;10(10):3389-400
Association of the cell cycle transcription factor Mbp1 with the Skn7 response
regulator in budding yeast.
Bouquin N, Johnson AL, Morgan BA, Johnston LH.
Division of Yeast Genetics, National Institute for Medical Research, The
Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
We previously isolated the SKN7 gene in a screen designed to isolate new
components of the G1-S cell cycle transcription machinery in budding yeast. We
have now found that Skn7 associates with Mbp1, the DNA-binding component of the
G1-S transcription factor DSC1/MBF. SKN7 and MBP1 show several genetic
interactions. Skn7 overexpression is lethal and is suppressed by a mutation in
MBP1. Similarly, high overexpression of Mbp1 is lethal and can be suppressed by
skn7 mutations. SKN7 is also required for MBP1 function in a mutant compromised
for G1-specific transcription. Gel-retardation assays indicate that Skn7 is not
an integral part of MBF. However, a physical interaction between Skn7 and Mbp1
was detected using two-hybrid assays and GST pulldowns. Thus, Skn7 and Mbp1 seem
to form a transcription factor independent of MBF. Genetic data suggest that
this new transcription factor could be involved in the bud-emergence process.
PMID: 10512874 [PubMed - indexed for MEDLINE]
652: Biochemistry 1999 Sep 7;38(36):11634-42
An NMR analysis of ubiquitin recognition by yeast ubiquitin hydrolase: evidence
for novel substrate recognition by a cysteine protease.
Sakamoto T, Tanaka T, Ito Y, Rajesh S, Iwamoto-Sugai M, Kodera Y, Tsuchida N,
Shibata T, Kohno T.
Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194-8511, Japan.
Yeast ubiquitin hydrolase 1 (YUH1), a cysteine protease that catalyzes the
removal of ubiquitin C-terminal adducts, is important for the generation of
monomeric ubiquitin. Heteronuclear NMR spectroscopy has been utilized to map the
YUH1 binding surface on ubiquitin. When YUH1 was titrated into a sample of
ubiquitin, approximately 50% of the (1)H-(15)N correlation peaks of ubiquitin
were affected to some degree, as a result of binding to YUH1. It is noteworthy
that the amide resonances of the basic residues (Arg42, Lys48, Arg72, and Lys74)
were highly perturbed. These positively charged basic residues may be involved
in direct interactions with the negatively charged acidic residues on YUH1. In
addition to the electrostatic surface, the hydrophobic surfaces on ubiquitin
(Leu8, Ile44, Phe45, Val70, Leu71, and Leu73) and YUH1 are also likely to
contribute to the binding interaction. Furthermore, the amide resonances of
Ile13, Leu43, Leu50, and Leu69, the side chains of which are not on the surface,
were also highly perturbed, indicating substrate-induced changes in the
environments of these residues as well. These large changes, observed from
residues located throughout the five-stranded beta-sheet surface and the
C-terminus, suggest that substrate recognition by YUH1 involves a wider area on
ubiquitin.
PMID: 10512618 [PubMed - indexed for MEDLINE]
653: Biospectroscopy 1999;5(5 Suppl):S42-52
Influence of protein environment on magnetic circular dichroism spectral
properties of ferric and ferrous ligand complexes of yeast cytochrome c
peroxidase.
Pond AE, Sono M, Elenkova EA, Goodin DB, English AM, Dawson JH.
Department of Chemistry and Biochemistry, University of South Carolina, Columbia
29208, USA.
The addition of exogenous ligands to the ferric and ferrous states of yeast
cytochrome c peroxidase (CCP) is investigated with magnetic circular dichroism
(MCD) at 4 degrees C to determine the effect the protein environment may
exercise on spectral properties. The MCD spectrum of each derivative is directly
compared to those of analogous forms of horseradish peroxidase (HRP) and
myoglobin (Mb), two well-characterized histidine-ligated heme proteins. The
ferric azide adduct of CCP is a hexacoordinate, largely low-spin species with an
MCD spectrum very similar to that of ferric azide HRP. This complex displays an
MCD spectrum dissimilar from that of the Mb derivative, possibly because of the
stabilizing interaction between the azide ligand and the distal arginine of CCP
(Arg 48). For the ferric fluoride derivative all three proteins display varied
MCD data, indicating that the differences in the distal pocket of each protein
influences their respective MCD characteristics. The MCD data for the
cyanoferric complexes are similar for all three proteins, demonstrating that a
strong field ligand bound in the sixth axial position dominates the MCD
characteristics of the derivative. Similarly, the ferric NO complexes of the
three proteins show MCD spectra similar in feature position and shape, but vary
somewhat in intensity. Reduction of CCP at neutral pH yields a typical
pentacoordinate high-spin complex with an MCD spectrum similar to that of
deoxyferrous HRP. Formation of the NO and cyanide complexes of ferrous CCP gives
derivatives with MCD spectra similar to the analogous forms of HRP and Mb in
both feature position and shape. Addition of CO to deoxyferrous CCP results in a
ferrous-CO complex with MCD spectral similarity to that of ferrous-CO HRP but
not Mb, indicating that interactions between the ligand and the distal residues
affects the MCD characteristics. Examination of alkaline (pH 9.7) deoxyferrous
CCP indicates that a pH dependent conformational change has occurred, leading to
a coordination structure similar to that of ferrous cytochrome b5, a known
bis-histidine complex. Exposure of this complex to CO further confirms that a
conformational change has taken place in that the MCD spectral characteristics
of the resulting complex are similar to those of ferrous-CO Mb but not
ferrous-CO HRP.
PMID: 10512537 [PubMed - indexed for MEDLINE]
654: Genetics 1999 Oct;153(2):643-52
Mutational analysis of yeast TFIIB. A functional relationship between Ssu72 and
Sub1/Tsp1 defined by allele-specific interactions with TFIIB.
Wu WH, Pinto I, Chen BS, Hampsey M.
Department of Biochemistry, Louisiana State University Medical Center,
Shreveport, Louisiana 71130, USA.
TFIIB is an essential component of the RNA polymerase II core transcriptional
machinery. Previous studies have defined TFIIB domains required for interaction
with other transcription factors and for basal transcription in vitro. In the
study reported here we investigated the TFIIB structural requirements for
transcription initiation in vivo. A library of sua7 mutations encoding altered
forms of yeast TFIIB was generated by error-prone polymerase chain reaction and
screened for conditional growth defects. Twenty-two single amino acid
replacements in TFIIB were defined and characterized. These replacements are
distributed throughout the protein and occur primarily at phylogenetically
conserved positions. Most replacements have little or no effect on the
steady-state protein levels, implying that each affects TFIIB function rather
than synthesis or stability. In contrast to the initial sua7 mutants, all
replacements, with one exception, have no effect on start site selection,
indicating that specific TFIIB structural defects affect transcriptional
accuracy. This collection of sua7 alleles, including the initial sua7 alleles,
was used to investigate the allele specificity of interactions between ssu72 and
sub1, both of which were initially identified as either suppressors (SUB1 2mu)
or enhancers (sub1Delta, ssu72-1) of sua7 mutations. We show that the
interactions of ssu72-1 and sub1Delta with sua7 are allele specific; that the
allele specificities of ssu72 and sub1 overlap; and that each of the sua7
alleles that interacts with ssu72 and sub1 affects the accuracy of transcription
start site selection. These results demonstrate functional interactions among
TFIIB, Ssu72, and Sub1 and suggest that these interactions play a role in the
mechanism of start site selection by RNA polymerase II.
PMID: 10511545 [PubMed - indexed for MEDLINE]
655: EMBO J 1999 Oct 1;18(19):5370-9
Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and
GLOBOSA is involved in the control of floral architecture in Antirrhinum majus.
Egea-Cortines M, Saedler H, Sommer H.
Max-Planck-Institut fur Zuchtungsforschung, Carl-von-Linne Weg 10, 50829 Koln,
Germany. Marcos.Egea@etsia.upct.es
In Antirrhinum, floral meristems are established by meristem identity genes.
Floral meristems give rise to floral organs in whorls, with their identity
established by combinatorial activities of organ identity genes. Double mutants
of the floral meristem identity gene SQUAMOSA and organ identity genes DEFICIENS
or GLOBOSA produce flowers in which whorled patterning is partially lost. In
yeast, SQUA, DEF and GLO proteins form ternary complexes via their C-termini,
which in gel-shift assays show increased DNA binding to CArG motifs compared
with DEF/GLO heterodimers or SQUA/SQUA homodimers. Formation of ternary
complexes by plant MADS-box factors increases the complexity of their regulatory
functions and might be the molecular basis for establishment of whorled
phyllotaxis and combinatorial interactions of floral organ identity genes.
PMID: 10508169 [PubMed - indexed for MEDLINE]
656: J Biol Chem 1999 Oct 8;274(41):29211-9
Structure-function analysis of the protein-binding domains of Mac1p, a
copper-dependent transcriptional activator of copper uptake in Saccharomyces
cerevisiae.
Serpe M, Joshi A, Kosman DJ.
Department of Biochemistry, School of Medicine, State University of New York at
Buffalo, Buffalo, New York 14214, USA.
The Mac1 protein in Saccharomyces cerevisiae is essential for the expression of
yeast high affinity copper uptake. A positive transcription factor, Mac1p binds
via its N-terminal domain to GCTC elements in the promoters of CTR1 and FRE1,
encoding a copper permease and metal reductase, respectively. Mac1p-dependent
transcriptional activation is negatively regulated by copper. We have mapped the
domains in Mac1p responsible for its nuclear localization and for the
protein-protein interactions that underlie its transcriptional activity.
Immunofluorescence studies indicate that Mac1p contains two nuclear localization
signals, one each in the N- and C-terminal halves of the protein. Yeast
one-hybrid analysis demonstrates that the copper-dependent transcriptional
activity in Mac1p resides primarily in a cysteine-rich element encompassing
residues 264-279. Two-hybrid analysis indicates that a copper-independent
Mac1p-Mac1p interaction linked to DNA binding is due primarily to a predicted
helix in the C-terminal region of the protein encompassing residues 388-406.
Point mutations within this putative helix abrogate the Mac1-Mac1 interaction in
vivo and formation of a ternary (Mac1p)(2).DNA complex in vitro. When produced
in normal abundance, Mac1pI396D and Mac1pF400D helix mutants do not support
transcriptional activation in vivo consistent with an essential Mac1p
dimerization in transcriptional activation. Lastly, the one- and two-hybrid data
indicate that an intramolecular interaction between the DNA-binding and
transactivation domains negatively modulates Mac1p activity.
PMID: 10506178 [PubMed - indexed for MEDLINE]
657: Mol Biotechnol 1999 Jun;11(3):213-20
Reconstitution of fibroblast growth factor receptor interactions in the yeast
two hybrid system.
Aloni-Grinstein R, Seddon A, Yayon A.
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot,
Israel.
Fibroblast growth factors (FGF) activate their receptors through the formation
of trimolecular complexes, composed of a ligand, a receptor, and a heparan
sulfate oligosaccharide, all of which are members of particularly large families
capable of multiple interactions in a combinatorial fashion. Understanding this
large network of interactions not only presents a great challenge, but is
practically beyond the capacity of most classical techniques routinely used to
study ligand receptor interactions. We have used the yeast two hybrid system to
study protein-protein interactions in the FGF family. Both ligand and receptor
ectodomains are properly folded and functional in the yeast. Basic FGF (bFGF)
expressed in the yeast dimerizes spontaneously. This self-assembly occurs at low
affinity, which can be greatly enhanced by the introduction of heparin,
supporting a defined role for heparin in bFGF dimerization. Screening a rat
embryo cDNA library with bFGF in the yeast two hybrid system identified a short
variant of FGF receptor 1, found most frequently in embryonal and tumor cells
and which possesses affinity toward bFGF that is significantly greater than that
of the more abundant, full-length receptor. We find the yeast two hybrid system,
a most suitable alternative method for the analysis of growth factor-receptor
interactions as well as for screening for novel interacting proteins and
modulators of FGF and its receptors.
PMID: 10503237 [PubMed - indexed for MEDLINE]
658: Proc Natl Acad Sci U S A 1999 Sep 28;96(20):11206-10
Chitin synthase III: synthetic lethal mutants and "stress related" chitin
synthesis that bypasses the CSD3/CHS6 localization pathway.
Osmond BC, Specht CA, Robbins PW.
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA
02139, USA.
We screened Saccharomyces strains for mutants that are synthetically lethal with
deletion of the major chitin synthase gene CHS3. In addition to finding, not
surprisingly, that mutations in major cell wall-related genes such as FKS1
(glucan synthase) and mutations in any of the Golgi glycosylation complex genes
(MNN9 family) are lethal in combination with chs3Delta, we found that a mutation
in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3
deletion. In extending studies of fks1-chitin synthase 3 interactions, we made
the surprising discovery that deletion of CSD3/CHS6, a gene normally required
for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact,
that lack of Csd3p/Chs6p did not decrease the high level of stress-related
chitin made in the fks1 mutant. This finding suggests that "stress response"
chitin synthesis proceeds through an alternate Chs3p targeting pathway.
PMID: 10500155 [PubMed - indexed for MEDLINE]
659: J Biol Inorg Chem 1999 Apr;4(2):209-19
X-ray crystal structures of active site mutants of the vanadium-containing
chloroperoxidase from the fungus Curvularia inaequalis.
Macedo-Ribeiro S, Hemrika W, Renirie R, Wever R, Messerschmidt A.
Max-Planck Institut fur Biochemie, Abteilung Strukturforschung, Martinsried,
Germany.
The X-ray structures of the chloroperoxidase from Curvularia inaequalis,
heterologously expressed in Saccharomyces cerevisiae, have been determined both
in its apo and in its holo forms at 1.66 and 2.11 A resolution, respectively.
The crystal structures reveal that the overall structure of this enzyme remains
nearly unaltered, particularly at the metal binding site. At the active site of
the apo-chloroperoxidase structure a clearly defined sulfate ion was found,
partially stabilised through electrostatic interactions and hydrogen bonds with
positively charged residues involved in the interactions with the vanadate in
the native protein. The vanadate binding pocket seems to form a very rigid frame
stabilising oxyanion binding. The rigidity of this active site matrix is the
result of a large number of hydrogen bonding interactions involving side chains
and the main chain of residues lining the active site. The structures of single
site mutants to alanine of the catalytic residue His404 and the vanadium protein
ligand His496 have also been analysed. Additionally we determined the structural
effects of mutations to alanine of residue Arg360, directly involved in the
compensation of the negative charge of the vanadate group, and of residue Asp292
involved in forming a salt bridge with Arg490 which also interacts with the
vanadate. The enzymatic chlorinating activity is drastically reduced to
approximately 1% in mutants D292A, H404A and H496A. The structures of the
mutants confirm the view of the active site of this chloroperoxidase as a rigid
matrix providing an oxyanion binding site. No large changes are observed at the
active site for any of the analysed mutants. The empty space left by replacement
of large side chains by alanines is usually occupied by a new solvent molecule
which partially replaces the hydrogen bonding interactions to the vanadate. The
new solvent molecules additionally replace part of the interactions the mutated
side chains were making to other residues lining the active site frame. When
this is not possible, another side chain in the proximity of the mutated residue
moves in order to satisfy the hydrogen bonding potential of the residues located
at the active site frame.
PMID: 10499093 [PubMed - indexed for MEDLINE]
660: J Biol Chem 1999 Oct 1;274(40):28803-7
Isolation of the protein kinase TAO2 and identification of its mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase binding domain.
Chen Z, Hutchison M, Cobb MH.
Department of Pharmacology, University of Texas Southwestern Medical Center,
Dallas, Texas 75235-9041, USA.
We previously reported the cloning of the thousand and one-amino acid protein
kinase 1 (TAO1), a rat homolog of the Saccharomyces cerevisiae protein kinase
sterile 20 protein. Here we report the complete sequence and properties of a
related rat protein kinase TAO2. Like TAO1, recombinant TAO2 selectively
activated mitogen-activated protein/extracellular signal-regulated kinase
kinases (MEKs) 3, 4, and 6 of the stress-responsive mitogen-activated protein
kinase pathways in vitro and copurified with MEK3 endogenous to Sf9 cells. To
examine TAO2 interactions with MEKs, the MEK binding domain of TAO2 was
localized to an approximately 135-residue sequence just C-terminal to the TAO2
catalytic domain. In vitro this MEK binding domain associated with MEKs 3 and 6
but not MEKs 1, 2, or 4. Using chimeric MEK proteins, we found that the MEK N
terminus was sufficient for binding to TAO2. Catalytic activity of full-length
TAO2 enhanced its binding to MEKs. However, neither the autophosphorylation of
the MEK binding domain of TAO2 nor the activity of MEK itself was required for
MEK binding. These results suggest that TAO proteins lie in stress-sensitive
kinase cascades and define a mechanism by which these kinases may organize
downstream targets.
PMID: 10497253 [PubMed - indexed for MEDLINE]
661: J Biol Chem 1999 Oct 1;274(40):28246-55
Molecular analysis of yeast and human type II topoisomerases. Enzyme-DNA and
drug interactions.
Strumberg D, Nitiss JL, Dong J, Kohn KW, Pommier Y.
Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National
Institutes of Health, Bethesda, Maryland 20892-4255, USA.
The DNA sequence selectivity of topoisomerase II (top2)-DNA cleavage complexes
was examined for the human (top2alpha), yeast, and Escherichia coli (i.e.
gyrase) enzymes in the absence or presence of anticancer or antibacterial drugs.
Species-specific differences were observed for calcium-promoted DNA cleavage.
Similarities and differences in DNA cleavage patterns and nucleic acid sequence
preferences were also observed between the human, yeast, and E. coli top2
enzymes in the presence of the non-intercalators fluoroquinolone CP-115,953,
etoposide, and azatoxin and the intercalators amsacrine and mitoxantrone.
Additional base preferences were generally observed for the yeast when compared
with the human top2alpha enzyme. Preferences in the immediate flanks of the
top2-mediated DNA cleavage sites are, however, consistent with the drug stacking
model for both enzymes. We also analyzed and compared homologous mutations in
yeast and human top2, i.e. Ser(740) --> Trp and Ser(763) --> Trp, respectively.
Both mutations decreased the reversibility of the etoposide-stabilized cleavage
sites and produced consistent base sequence preference changes. These data
demonstrate similarities and differences between human and yeast top2 enzymes.
They also indicate that the structure of the enzyme/DNA interface plays a key
role in determining the specificity of top2 poisons and cleavage sites for both
the intercalating and non-intercalating drugs.
PMID: 10497180 [PubMed - indexed for MEDLINE]
662: Mol Cell Biol 1999 Oct;19(10):6729-41
Regulation of cell cycle transcription factor Swi4 through auto-inhibition of
DNA binding.
Baetz K, Andrews B.
Department of Molecular and Medical Genetics, University of Toronto, Toronto,
Ontario, Canada M5S 1A8.
In Saccharomyces cerevisiae, two transcription factors, SBF (SCB binding factor)
and MBF (MCB binding factor), promote the induction of gene expression at the
G(1)/S-phase transition of the mitotic cell cycle. Swi4 and Mbp1 are the DNA
binding components of SBF and MBF, respectively. The Swi6 protein is a common
subunit of both transcription factors and is presumed to play a regulatory role.
SBF binding to its target sequences, the SCBs, is a highly regulated event and
requires the association of Swi4 with Swi6 through their C-terminal domains.
Swi4 binding to SCBs is restricted to the late M and G(1) phases, when Swi6 is
localized to the nucleus. We show that in contrast to Swi6, Swi4 remains nuclear
throughout the cell cycle. This finding suggests that the DNA binding domain of
Swi4 is inaccessible in the full-length protein when not complexed with Swi6. To
explore this hypothesis, we expressed Swi4 and Swi6 in insect cells by using the
baculovirus system. We determined that partially purified Swi4 cannot bind SCBs
in the absence of Swi6. However, Swi4 derivatives carrying point mutations or
alterations in the extreme C terminus were able to bind DNA or activate
transcription in the absence of Swi6, and the C terminus of Swi4 inhibited Swi4
derivatives from binding DNA in trans. Full-length Swi4 was determined to be
monomeric in solution, suggesting an intramolecular mechanism for
auto-inhibition of binding to DNA by Swi4. We detected a direct in vitro
interaction between a C-terminal fragment of Swi4 and the N-terminal 197 amino
acids of Swi4, which contain the DNA binding domain. Together, our data suggest
that intramolecular interactions involving the C-terminal region of Swi4
physically prevent the DNA binding domain from binding SCBs. The interaction of
the carboxy-terminal region of Swi4 with Swi6 alleviates this inhibition,
allowing Swi4 to bind DNA.
PMID: 10490612 [PubMed - indexed for MEDLINE]
663: Mol Cell Biol 1999 Oct;19(10):6642-51
The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and
functionally separated from NOT2, NOT4, and NOT5.
Bai Y, Salvadore C, Chiang YC, Collart MA, Liu HY, Denis CL.
Department of Biochemistry and Molecular Biology, University of New Hampshire,
Durham, New Hampshire 03824, USA.
The CCR4-NOT complex (1 mDa in size), consisting of the proteins CCR4, CAF1, and
NOT1 to NOT5, regulates gene expression both positively and negatively and is
distinct from other large transcriptional complexes in Saccharomyces cerevisiae
such as SNF/SWI, TFIID, SAGA, and RNA polymerase II holoenzyme. The physical and
genetic interactions between the components of the CCR4-NOT complex were
investigated in order to gain insight into how this complex affects the
expression of diverse genes and processes. The CAF1 protein was found to be
absolutely required for CCR4 association with the NOT proteins, and CCR4 and
CAF1, in turn, physically interacted with NOT1 through its central amino acid
region from positions 667 to 1152. The NOT3, NOT4, and NOT5 proteins had no
significant effect on the association of CCR4, CAF1, and NOT1 with each other.
In contrast, the NOT2, NOT4, and NOT5 interacted with the C-terminal region
(residues 1490 to 2108) of NOT1 in which NOT2 and NOT5 physically associated in
the absence of CAF1, NOT3, and NOT4. These and other data indicate that the
physical ordering of these proteins in the complex is CCR4-CAF1-NOT1-(NOT2,
NOT5), with NOT4 and NOT3 more peripheral to NOT2 and NOT5. The physical
separation of CCR4 and CAF1 from other components of the CCR4-NOT complex
correlated with genetic analysis indicating partially separate functions for
these two groups of proteins. ccr4 or caf1 deletion suppressed the increased
3-aminotriazole resistance phenotype conferred by not mutations, resulted in
opposite effects on gene expression as compared to several not mutations, and
resulted in a number of synthetic phenotypes in combination with not mutations.
These results define the CCR4-NOT complex as consisting of at least two
physically and functionally separated groups of proteins.
PMID: 10490603 [PubMed - indexed for MEDLINE]
664: Mol Cell Biol 1999 Oct;19(10):6543-53
Genetic and physical interactions involving the yeast nuclear cap-binding
complex.
Fortes P, Kufel J, Fornerod M, Polycarpou-Schwarz M, Lafontaine D, Tollervey D,
Mattaj IW.
European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.
Yeast strains lacking the yeast nuclear cap-binding complex (yCBC) are viable,
although impaired in growth. We have taken advantage of this observation to
carry out a genetic screen for components that show synthetic lethality (SL)
with a cbp20-Delta cbp80-Delta double mutation. One set of SL interactions was
due to mutations that were complemented by components of U1 small nuclear RNP
(snRNP) and the yeast splicing commitment complex. These interactions confirm
the role of yCBC in commitment complex formation. Physical interaction of yCBC
with the commitment complex components Mud10p and Mud2p, which may directly
mediate yCBC function, was demonstrated. Unexpectedly, we identified multiple SL
mutations that were complemented by Cbf5p and Nop58p. These are components of
the two major classes of yeast small nucleolar RNPs, which function in the
maturation of rRNA precursors. Mutants lacking yCBC were found to be defective
in rRNA processing. Analysis of the yCBC deletion phenotype suggests that this
is likely to be due to a defect in the splicing of a subset of ribosomal protein
mRNA precursors.
PMID: 10490594 [PubMed - indexed for MEDLINE]
665: Mol Cell 1999 Aug;4(2):153-66
Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role
for the nuclear export signal of 14-3-3 in ligand binding.
Rittinger K, Budman J, Xu J, Volinia S, Cantley LC, Smerdon SJ, Gamblin SJ,
Yaffe MB.
Divison of Protein Structure, National Institute for Medical Research, London,
United Kingdom.
We have solved the high-resolution X-ray structure of 14-3-3 bound to two
different phosphoserine peptides, representing alternative substrate-binding
motifs. These structures reveal an evolutionarily conserved network of
peptide-protein interactions within all 14-3-3 isotypes, explain both binding
motifs, and identify a novel intrachain phosphorylation-mediated loop structure
in one of the peptides. A 14-3-3 mutation disrupting Raf signaling alters the
ligand-binding cleft, selecting a different phosphopeptide-binding motif and
different substrates than the wild-type protein. Many 14-3-3: peptide contacts
involve a C-terminal amphipathic alpha helix containing a putative nuclear
export signal, implicating this segment in both ligand and Crm1 binding.
Structural homology between the 14-3-3 NES structure and those within I kappa B
alpha and p53 reveals a conserved topology recognized by the Crm1 nuclear export
machinery.
PMID: 10488331 [PubMed - indexed for MEDLINE]
666: Mol Gen Genet 1999 Jul;261(6):967-76
Genetic interactions between a null allele of the RIT1 gene encoding an
initiator tRNA-specific modification enzyme and genes encoding translation
factors in Saccharomyces cerevisiae.
Astrom SU, Nordlund ME, Erickson FL, Hannig EM, Bystrom AS.
Department of Microbiology, Umea University, Sweden.
The Saccharomyces cerevisiae gene RIT1 encodes a phospho-ribosyl transferase
that exclusively modifies the initiator tRNA (tRNAMet(i)) by the addition of a
2'-O-ribosyl phosphate group to Adenosine 64. As a result, tRNAMet(i) is
prevented from participating in the elongation steps of protein synthesis. We
previously showed that the modification is not essential for the function of
tRNAMet(i) in the initiation of translation, since rit1 null strains are viable
and show no obvious growth defects. Here, we demonstrate that yeast strains in
which a rit1 null allele is combined with mutations in any of the genes for the
three subunits of eukaryotic initiation factor-2 (eIF-2), or with disruption
alleles of two of the four initiator methionine tRNA (IMT) genes, show
synergistic growth defects. A multicopy plasmid carrying an IMT gene can
alleviate these defects. On the other hand, introduction of a high-copy-number
plasmid carrying the TEF2 gene, which encodes the eukaryotic elongation factor
1alpha (eEF-1alpha), into rit1 null strains with two intact IMT genes had the
opposite effect, indicating that increased levels of eEF-1alpha are deleterious
to these strains, presumably due to sequestration of the unmodified
met-tRNAMet(i) for elongation. Thus, under conditions in which the components of
the ternary met-tRNAMet(i):GTP:eIF-2 complex become limiting or are functionally
impaired, the presence of the 2'-O-ribosyl phosphate modification in tRNAMet(i)
is important for the provision of adequate amounts of tRNAMet(i) for formation
of this ternary complex.
PMID: 10485288 [PubMed - indexed for MEDLINE]
667: Cell 1999 Aug 20;98(4):453-63
The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is
negatively regulated by HID.
Wang SL, Hawkins CJ, Yoo SJ, Muller HA, Hay BA.
Division of Biology MC 156-29, California Institute of Technology, Pasadena
91125, USA.
Drosophila Reaper (RPR), Head Involution Defective (HID), and GRIM induce
caspase-dependent cell death and physically interact with the cell death
inhibitor DIAP1. Here we show that HID blocks DIAP1's ability to inhibit caspase
activity and provide evidence suggesting that RPR and GRIM can act similarly.
Based on these results, we propose that RPR, HID, and GRIM promote apoptosis by
disrupting productive IAP-caspase interactions and that DIAP1 is required to
block apoptosis-inducing caspase activity. Supporting this hypothesis, we show
that elimination of DIAP1 function results in global early embryonic cell death
and a large increase in DIAP1-inhibitable caspase activity and that DIAP1 is
still required for cell survival when expression of rpr, hid, and grim is
eliminated.
PMID: 10481910 [PubMed - indexed for MEDLINE]
668: Mol Endocrinol 1999 Sep;13(9):1550-7
Coactivators for the orphan nuclear receptor RORalpha.
Atkins GB, Hu X, Guenther MG, Rachez C, Freedman LP, Lazar MA.
Department of Medicine, The Penn Diabetes Center, University of Pennsylvania
School of Medicine, Philadelphia 19104-6149, USA.
A mutation in the nuclear orphan receptor RORalpha results in a severe
impairment of cerebellar development by unknown mechanisms. We have shown
previously that RORalpha contains a strong constitutive activation domain in its
C terminus. We therefore searched for mammalian RORalpha coactivators using the
minimal activation domain as bait in a two-hybrid screen. Several known and
putative coactivators were isolated, including glucocorticoid
receptor-interacting protein-1 (GRIP-1) and peroxisome proliferator-activated
receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205). These interactions were
confirmed in vitro and require the intact activation domain of RORalpha although
different requirements for interaction with GRIP-1 and PBP were detected. Even
in the absence of exogenous ligand, RORalpha interacts with a complex or
complexes of endogenous proteins, similar to those that bind to ligand-occupied
thyroid hormone and vitamin D receptors. Both PBP and GRIP-1 were shown to be
present in these complexes. Thus we have identified several potential RORalpha
coactivators that, in contrast to the interactions with hormone receptors,
interact with RORalpha in yeast, in bacterial extracts, and in mammalian cells
in vivo and in vitro in the absence of exogenous ligand. GRIP-1 functioned as a
coactivator for the RORalpha both in yeast and in mammalian cells. Thus, GRIP-1
is the first proven coactivator for RORalpha.
PMID: 10478845 [PubMed - indexed for MEDLINE]
669: FEBS Lett 1999 Sep 3;457(3):363-8
Tpr1, a Schizosaccharomyces pombe protein involved in potassium transport.
Lichtenberg H, Heyer M, Hofer M.
Botanisches Institut der Universitat Bonn, Kirschallee 1, 53115, Bonn, Germany.
H.Lichtenberg@uni-bonn.de
The Schizosaccharomyces pombe Tpr1 was isolated as suppressor of the
Saccharomyces cerevisiae Delta trk1,2 potassium uptake deficient phenotype.
Tpr1, for tetratrico peptide repeat, encodes a 1039 amino acid residues protein
with several reiterated TPR units displaying significant homology to p150(TSP),
a recently identified phosphoprotein of mouse, to S. cerevisiae CTR9 and to
related sequences of human, Caenorhabditis elegans, Methanoccocus jannaschii and
Arabidopsis thaliana. Expression of Tpr1 restored growth on 0.2 mM K(+) media,
induced K(+) transport with a K(T) of 4.6 mM and resumed inward currents of -90
pA at -250 mV (pH 7.2) conducting K(+) and other alkali-metal ions. The
tetratrico peptide repeat is a degenerate motif of 34 amino acids that is
repeated several times within TPR-containing proteins and has been suggested to
mediate protein-protein interactions. The sequence and putative binding
properties of Tpr1 suggest the protein unlikely as transporter but involved in
the enhancement of K(+) uptake via conventional carriers.
PMID: 10471809 [PubMed - indexed for MEDLINE]
670: Genetics 1999 Sep;153(1):81-94
Genetic study of interactions between the cytoskeletal assembly protein sla1 and
prion-forming domain of the release factor Sup35 (eRF3) in Saccharomyces
cerevisiae.
Bailleul PA, Newnam GP, Steenbergen JN, Chernoff YO.
School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230,
USA.
Striking similarities between cytoskeletal assembly and the "nucleated
polymerization" model of prion propagation suggest that similar or overlapping
sets of proteins may assist in both processes. We show that the C-terminal
domain of the yeast cytoskeletal assembly protein Sla1 (Sla1C) specifically
interacts with the N-terminal prion-forming domain (Sup35N) of the yeast release
factor Sup35 (eRF3) in the two-hybrid system. Sla1C and several other
Sup35N-interacting proteins also exhibit two-hybrid interactions with the
poly-Gln-expanded N-proximal fragment of human huntingtin, which promotes
Huntington disease-associated aggregation. The Sup35N-Sla1C interaction is
inhibited by Sup35N alterations that make Sup35 unable to propagate the [PSI(+)]
state and by the absence of the chaperone protein Hsp104, which is essential for
[PSI] propagation. In a Sla1(-) background, [PSI] curing by dimethylsulfoxide or
excess Hsp104 is increased, while translational readthrough and de novo [PSI]
formation induced by excess Sup35 or Sup35N are decreased. These data show that,
in agreement with the proposed function of Sla1 during cytoskeletal formation,
Sla1 assists in [PSI] formation and propagation, but is not required for these
processes. Sla1(-) strains are sensitive to some translational inhibitors, and
some sup35 mutants, obtained in a Sla1(-) background, are sensitive to Sla1,
suggesting that the interaction between Sla1 and Sup35 proteins may play a role
in the normal function of the translational apparatus. We hypothesize that
Sup35N is involved in regulatory interactions with intracellular structural
networks, and [PSI] prion may be formed as a by-product of this process.
PMID: 10471702 [PubMed - indexed for MEDLINE]
671: Nat Genet 1999 Sep;23(1):81-5
Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and
recombination defective mutants.
Chen C, Kolodner RD.
Ludwig Institute for Cancer Research, Cancer Center and Department of Medicine,
University of California-San Diego School of Medicine, La Jolla, California
92093, USA.
Cancer progression is often associated with the accumulation of gross
chromosomal rearrangements (GCRs), such as translocations, deletion of a
chromosome arm, interstitial deletions or inversions. In many instances, GCRs
inactivate tumour-suppressor genes or generate novel fusion proteins that
initiate carcinogenesis. The mechanism underlying GCR formation appears to
involve interactions between DNA sequences of little or no homology. We
previously demonstrated that mutations in the gene encoding the largest subunit
of the Saccharomyces cerevisiae single-stranded DNA binding protein (RFA1)
increase microhomology-mediated GCR formation. To further our understanding of
GCR formation, we have developed a novel mutator assay in S. cerevisiae that
allows specific detection of such events. In this assay, the rate of GCR
formation was increased 600-5, 000-fold by mutations in RFA1, RAD27, MRE11, XRS2
and RAD50, but was minimally affected by mutations in RAD51, RAD54, RAD57,
YKU70, YKU80, LIG4 and POL30. Genetic analysis of these mutants suggested that
at least three distinct pathways can suppress GCRs: two that suppress
microhomology-mediated GCRs (RFA1 and RAD27) and one that suppresses
non-homology-mediated GCRs (RAD50/MRE11/XRS2).
PMID: 10471504 [PubMed - indexed for MEDLINE]
672: J Biol Chem 1999 Sep 3;274(36):25461-70
Helical interactions and membrane disposition of the 16-kDa proteolipid subunit
of the vacuolar H(+)-ATPase analyzed by cysteine replacement mutagenesis.
Harrison MA, Murray J, Powell B, Kim YI, Finbow ME, Findlay JB.
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2
9JT, United Kingdom. m.a.harrison@leeds.ac.uk
Theoretical mechanisms of proton translocation by the vacuolar H(+)-ATPase
require that a transmembrane acidic residue of the multicopy 16-kDa proteolipid
subunit be exposed at the exterior surface of the membrane sector of the enzyme,
contacting the lipid phase. However, structural support for this theoretical
mechanism is lacking. To address this, we have used cysteine mutagenesis to
produce a molecular model of the 16-kDa proteolipid complex. Transmembrane
helical contacts were determined using oxidative cysteine cross-linking, and
accessibility of cysteines to the lipid phase was determined by their reactivity
to the lipid-soluble probe N-(1-pyrenyl)maleimide. A single model for
organization of the four helices of each monomeric proteolipid was the best fit
to the experimental data, with helix 1 lining a central pore and helix 2 and
helix 3 immediately external to it and forming the principal intermolecular
contacts. Helix 4, containing the crucial acidic residue, is peripheral to the
complex. The model is consistent not only with theoretical proton transport
mechanisms, but has structural similarity to the dodecameric ring complex formed
by the related 8-kDa proteolipid of the F(1)F(0)-ATPase. This suggests some
commonality between the proton translocating mechanisms of the vacuolar and
F(1)F(0)-ATPases.
PMID: 10464277 [PubMed - indexed for MEDLINE]
673: J Cell Sci 1999 Sep;112 Pt 18:3103-14
The S. pombe zfs1 gene is required to prevent septation if mitotic progression
is inhibited.
Beltraminelli N, Murone M, Simanis V.
Cell Cycle Control Laboratory, ISREC, Chemin des Boveresses, 1066 Epalinges,
Switzerland.
Schizosaccharomyces pombe cdc16p is required to limit the cell to forming a
single division septum per cell cycle; the heat-sensitive loss-of-function
mutant cdc16-116 completes mitosis, and then undergoes multiple rounds of septum
formation without cell cleavage. cdc16p is a homologue of Saccharomyces
cerevisiae BUB2p, and has also been implicated in the spindle assembly
checkpoint function in S. pombe. To identify other proteins involved in
regulating septum formation, we have screened for multicopy suppressors of the
cdc16-116 mutation. In this paper, we describe one of these suppressors, zfs1.
The null allele (zfs1-D1) is viable. However, at low temperatures it divides at
a reduced size, while at higher temperatures, it partially suppresses heat
sensitive mutants in genes signalling the onset of septum formation. Zfs1-D1
cells show an increased rate of chromosome loss during exponential growth.
Moreover, if assembly of the spindle is prevented, zfs1-D1 cells do not arrest
normally, but the activity of cdc2p kinase decays, and cells form a division
septum without completing a normal mitosis. We conclude that zfs1 function is
required to prevent septum formation and exit from mitosis if the mitotic
spindle is not assembled. The suppression of cdc16-116 by zfs1 is independent of
dma1 function and the spindle assembly checkpoint genes mad2 and mph1. The
genetic interactions of zfs1 with genes regulating septum formation suggest that
it may be a modulator of the signal transduction network controlling the onset
of septum formation and exit from mitosis.
PMID: 10462526 [PubMed - indexed for MEDLINE]
674: Mol Cell Biol 1999 Sep;19(9):6441-7
Trithorax and ASH1 interact directly and associate with the trithorax
group-responsive bxd region of the Ultrabithorax promoter.
Rozovskaia T, Tillib S, Smith S, Sedkov Y, Rozenblatt-Rosen O, Petruk S, Yano T,
Nakamura T, Ben-Simchon L, Gildea J, Croce CM, Shearn A, Canaani E, Mazo A.
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot,
Israel.
Trithorax (TRX) and ASH1 belong to the trithorax group (trxG) of transcriptional
activator proteins, which maintains homeotic gene expression during Drosophila
development. TRX and ASH1 are localized on chromosomes and share several
homologous domains with other chromatin-associated proteins, including a highly
conserved SET domain and PHD fingers. Based on genetic interactions between trx
and ash1 and our previous observation that association of the TRX protein with
polytene chromosomes is ash1 dependent, we investigated the possibility of a
physical linkage between the two proteins. We found that the endogenous TRX and
ASH1 proteins coimmunoprecipitate from embryonic extracts and colocalize on
salivary gland polytene chromosomes. Furthermore, we demonstrated that TRX and
ASH1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic
gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis
of the effects of ash1 mutations on the activity of this regulatory region
indicates that it also contains ash1 response element(s). This suggests that
ASH1 and TRX act on Ubx in relatively close proximity to each other. Finally,
TRX and ASH1 appear to interact directly through their conserved SET domains,
based on binding assays in vitro and in yeast and on coimmunoprecipitation
assays with embryo extracts. Collectively, these results suggest that TRX and
ASH1 are components that interact either within trxG protein complexes or
between complexes that act in close proximity on regulatory DNA to maintain Ubx
transcription.
PMID: 10454589 [PubMed - indexed for MEDLINE]
675: Mol Cell Biol 1999 Sep;19(9):6110-9
The yeast trimeric guanine nucleotide-binding protein alpha subunit, Gpa2p,
controls the meiosis-specific kinase Ime2p activity in response to nutrients.
Donzeau M, Bandlow W.
Institut fur Genetik und Mikrobiologie, Ludwig-Maximilians-Universitat Munchen,
D-80638 Munich, Germany.
Saccharomyces cerevisiae Gpa2p, the alpha subunit of a heterotrimeric guanine
nucleotide-binding protein (G protein), is involved in the regulation of
vegetative growth and pseudohyphal development. Here we report that Gpa2p also
controls sporulation by interacting with the regulatory domain of Ime2p (Sme1p),
a protein kinase essential for entrance of meiosis and sporulation.
Protein-protein interactions between Gpa2p and Ime2p depend on the GTP-bound
state of Gpa2p and correlate with down-regulation of Ime2p kinase activity in
vitro. Overexpression of Ime2p inhibits pseudohyphal development and enables
diploid cells to sporulate even in the presence of glucose or nitrogen. In
contrast, overexpression of Gpa2p in cells simultaneously overproducing Ime2p
results in a drastic reduction of sporulation efficiency, demonstrating an
inhibitory effect of Gpa2p on Ime2p function. Furthermore, deletion of GPA2
accelerates sporulation on low-nitrogen medium. These observations are
consistent with the following model. In glucose-containing medium, diploid cells
do not sporulate because Ime2p is inactive or expressed at low levels. Upon
starvation, expression of Gpa2p and Ime2p is induced but sporulation is
prevented as long as nitrogen is present in the medium. The negative control of
Ime2p kinase activity is exerted at least in part through the activated form of
Gpa2p and is released as soon as nutrients are exhausted. This model attributes
a switch function to Gpa2p in the meiosis-pseudohyphal growth decision.
PMID: 10454558 [PubMed - indexed for MEDLINE]
676: Mol Cell Biol 1999 Sep;19(9):6065-75
Interactions of TLC1 (which encodes the RNA subunit of telomerase), TEL1, and
MEC1 in regulating telomere length in the yeast Saccharomyces cerevisiae.
Ritchie KB, Mallory JC, Petes TD.
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina 27599-3280, USA.
In the yeast Saccharomyces cerevisiae, chromosomes terminate with a repetitive
sequence [poly(TG(1-3))] 350 to 500 bp in length. Strains with a mutation of
TEL1, a homolog of the human gene (ATM) mutated in patients with ataxia
telangiectasia, have short but stable telomeric repeats. Mutations of TLC1
(encoding the RNA subunit of telomerase) result in strains that have continually
shortening telomeres and a gradual loss of cell viability; survivors of
senescence arise as a consequence of a Rad52p-dependent recombination events
that amplify telomeric and subtelomeric repeats. We show that a mutation in MEC1
(a gene related in sequence to TEL1 and ATM) reduces telomere length and that
tel1 mec1 double mutant strains have a senescent phenotype similar to that found
in tlc1 strains. As observed in tlc1 strains, survivors of senescence in the
tel1 mec1 strains occur by a Rad52p-dependent amplification of telomeric and
subtelomeric repeats. In addition, we find that strains with both tel1 and tlc1
mutations have a delayed loss of cell viability compared to strains with the
single tlc1 mutation. This result argues that the role of Tel1p in telomere
maintenance is not solely a direct activation of telomerase.
PMID: 10454554 [PubMed - indexed for MEDLINE]
677: J Mol Biol 1999 Aug 27;291(4):761-73
Yeast aspartyl-tRNA synthetase residues interacting with tRNA(Asp) identity
bases connectively contribute to tRNA(Asp) binding in the ground and
transition-state complex and discriminate against non-cognate tRNAs.
Eriani G, Gangloff J.
UPR 9002 SMBMR du CNRS, Institut de Biologie Moleculaire et Cellulaire, 15, rue
Rene Descartes, Strasbourg, 67084, France.
Crystallographic studies of the aspartyl-tRNA synthetase-tRNA(Asp)complex from
yeast identified on the enzyme a number of residues potentially able to interact
with tRNA(Asp). Alanine replacement of these residues (thought to disrupt the
interactions) was used in the present study to evaluate their importance in
tRNA(Asp)recognition and acylation. The results showed that contacts with the
acceptor A of tRNA(Asp)by amino acid residues interacting through their
side-chain occur only in the acylation transition state, whereas those located
near the G73 discriminator base occur also during initial binding of tRNA(Asp).
Interactions with the anticodon bases provide the largest free energy
contribution to stability of the enzyme-tRNA complex in its ground state. These
contacts also favour catalysis, by acting connectively with each other and with
those of G73, as shown by multiple mutant analysis. This implies structural
communication transmitting the anticodon recognition signal to the distally
located acylation site. This signal might be conveyed via tRNA(Asp)as suggested
by the observed conformational change of this molecule upon interaction with
AspRS. From binding free energy values corresponding to the different
AspRS-tRNA(Asp)interaction domains, it might be concluded that upon complex
formation, the anticodon interacts first. Finally, acylation efficiencies of
AspRS mutants in the presence of pure tRNA(Asp)and non-fractionated tRNAs
indicate that residues involved in the binding of identity bases also
discriminate against non-cognate tRNAs. Copyright 1999 Academic Press.
PMID: 10452887 [PubMed - indexed for MEDLINE]
678: Cell Biol Int 1998;22(9-10):709-14
Resumption of rapid proliferation from lag phase in cultures of Saccharomyces
cerevisiae in poor nutrient conditions. Effect of surface and intracellular
signalling mechanisms.
Overgaard AK, Kierklo L, Christiansen H, Chrois P, Rasmussen L, Friis J.
Department of Anatomy and Cytology, Odense University, Odense M, 5230, Denmark.
Saccharomyces cerevisiae was inoculated into a dilute synthetic minimal medium
with glycerol as the carbon source. The number of live cells in the cultures was
determined by colony counts on agar plates. Untreated control cells had doubled
in number about once at the end of the first week and had gone through eight
doublings by the end of the second week. Addition of either 8-bromo-cyclic
guanosine monophosphate (8-bromo-cGMP) or human recombinant insulin, made the
cells go through 12 and 10 doublings, respectively, by the end of the first
week. In contrast, 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP) had
only slight stimulating effects on cell multiplication, but if it was combined
with phorbol-12-myristate-13-acetate (PMA) the cells went through about 12
doublings during the first week. Addition of LY 83583, an inhibitor of soluble
guanylate cyclase, prevented cell proliferation. Further addition of
8-bromo-cGMP bypassed this inhibition. Singly, bradykinin or PMA did not affect
cell multiplication. However, when these two compounds were combined, the cells
went through about 10 doublings during the first week. Neither bradykinin, nor
PMA had any releasing effect on the inhibition of LY 83583. These results
indicate the existence of several routes leading to cell proliferation in
wildtype S. cerevisiae cells. Copyright 1998 Academic Press.
PMID: 10452842 [PubMed - indexed for MEDLINE]
679: J Mol Biol 1999 Aug 20;291(3):715-25
Proteins can adopt totally different folded conformations.
Damaschun G, Damaschun H, Gast K, Zirwer D.
Humboldt-Universitat zu Berlin, Institut fur Biologie, c/o Max-Delbruck-Centrum
fur Molekulare Medizin, Robert-Rossle-Strasse 10, Berlin, PF 740238, D-13092,
Germany. gdamasc@mdc-berlin.de
The three-dimensional structure of a protein is determined by interactions
between its amino acids and by interactions of the amino acids with molecules of
the environment. The great influence of the latter interactions is demonstrated
for the enzyme phosphoglycerate kinase from yeast (PGK). In the native state,
PGK is a compact, bilobal molecule; 35% and 13% of its amino acids are organised
in the form of alpha-helices and beta-sheets, respectively. The molecules unfold
at acidic pH and low ionic strength forming random-walk structures with a
persistence length of 3 nm. More than 90% of the amino acid residues of the
ensemble have phi,psi-angles corresponding to those of a straight beta-chain.
Upon addition of 50% (v/v) trifluoroethanol to the acid-unfolded protein, the
entire molecule is transformed into a rod-like, flexible alpha-helix. Addition
of anions, such as chloride or trichloroacetate, to the acid-unfolded protein
leads to the formation of amyloid-like fibres over a period of many hours when
the anion concentration exceeds a critical limit. Half of the amino acid
residues are then organised in beta-sheets. Both of the non-natively folded
states of PGK contain more regular secondary structure than the native one. The
misfolding starts in both cases from the acid-unfolded state, in which the
molecules are essentially more expanded than in other denatured states, e.g.
those effected by temperature or guanidine hydrochloride. Copyright 1999
Academic Press.
PMID: 10448049 [PubMed - indexed for MEDLINE]
680: J Bioenerg Biomembr 1999 Apr;31(2):95-104
The role of the amino-terminal beta-barrel domain of the alpha and beta subunits
in the yeast F1-ATPase.
Yao B, Mueller DM.
Department of Biochemistry and Molecular Biology, The Chicago Medical School,
Illinois 60064, USA.
The crystal structure of mitochondrial F1-ATPase indicates that the alpha and
beta subunits fold into a structure defined by three domains: the top
beta-barrel domain, the middle nucleotide-binding domain, and the C-terminal
alpha-helix bundle domain (Abrahams et al., 1994); Bianchet et al., 1998). The
beta-barrel domains of the alpha and beta subunits form a crown structure at the
top of F1, which was suggested to stabilize it (Abrahams et al. 1994). In this
study, the role of the beta-barrel domain in the alpha and beta subunits of the
yeast Saccharomyces cerevisiae F1, with regard to its folding and assembly, was
investigated. The beta-barrel domains of yeast F1alpha and beta subunits were
expressed individually and together in Escherichia coli. When expressed
separately, the beta-barrel domain of the beta subunit formed a large aggregate
structure, while the domain of the alpha subunit was predominately a monomer or
dimer. However, coexpression of the beta-barrel domain of alpha subunit with the
beta-barrel domain of beta subunit, greatly reduced the aggregation of the beta
subunit domain. Furthermore, the two domains copurified in complexes with the
major portion of the complex found in a small molecular weight form. These
results indicate that the beta-barrel domain of the alpha and beta subunits
interact specifically with each other and that these interactions prevent the
aggregation of the beta-barrel domain of the beta subunit. These results mimic
in vivo results and suggest that the interactions of the beta-barrel domains may
be critical during the folding and assembly of F1.
PMID: 10449236 [PubMed - indexed for MEDLINE]
681: Curr Genet 1999 Aug;36(1-2):13-20
Characterization of the role played by the RAD59 gene of Saccharomyces
cerevisiae in ectopic recombination.
Jablonovich Z, Liefshitz B, Steinlauf R, Kupiec M.
Department of Molecular Microbiology and Biotechnology,
The RAD52 group of genes in the yeast Saccharomyces cerevisiae controls the
repair of DNA damage by a recombinational mechanism. Despite the growing
evidence for physical and biochemical interactions between the proteins of this
repair group, mutations in individual genes show very different effects on
various types of recombination. The RAD59 gene encodes a protein with similarity
to Rad52p which plays a role in the repair of damage caused by ionizing
radiation. In the present study we have examined the role played by the Rad59
protein in mitotic ectopic recombination and analyzed the genetic interactions
with other members of the repair group. We found that Rad59p plays a role in
ectopic gene conversion that depends on the presence of Rad52p but is
independent of the function of the RecA homologue Rad51p and of the Rad57
protein. The RAD59 gene product also participates in the RAD1-dependent pathway
of recombination between direct repeats. We propose that Rad59p may act in a
salvage mechanism that operates when the Rad51 filament is not functional.
PMID: 10447590 [PubMed - indexed for MEDLINE]
682: J Biol Chem 1999 Aug 20;274(34):23794-801
Half of Saccharomyces cerevisiae carbamoyl phosphate synthetase produces and
channels carbamoyl phosphate to the fused aspartate transcarbamoylase domain.
Serre V, Guy H, Penverne B, Lux M, Rotgeri A, Evans D, Herve G.
Laboratoire de Biochimie des Signaux Regulateurs Cellulaires et Moleculaires,
UMR 7631 CNRS-Universite Pierre et Marie Curie, 96 Bd Raspail 75006 Paris,
France.
The first two steps of the de novo pyrimidine biosynthetic pathway in
Saccharomyces cerevisiae are catalyzed by a 240-kDa bifunctional protein encoded
by the ura2 locus. Although the constituent enzymes, carbamoyl phosphate
synthetase (CPSase) and aspartate transcarbamoylase (ATCase) function
independently, there are interdomain interactions uniquely associated with the
multifunctional protein. Both CPSase and ATCase are feedback inhibited by UTP.
Moreover, the intermediate carbamoyl phosphate is channeled from the CPSase
domain where it is synthesized to the ATCase domain where it is used in the
synthesis of carbamoyl aspartate. To better understand these processes, a
recombinant plasmid was constructed that encoded a protein lacking the
amidotransferase domain and the amino half of the CPSase domain, a 100-kDa chain
segment. The truncated complex consisted of the carboxyl half of the CPSase
domain fused to the ATCase domain via the pDHO domain, an inactive
dihydroorotase homologue that bridges the two functional domains in the native
molecule. Not only was the "half CPSase" catalytically active, but it was
regulated by UTP to the same extent as the parent molecule. In contrast, the
ATCase domain was no longer sensitive to the nucleotide, suggesting that the two
catalytic activities are controlled by distinct mechanisms. Most remarkably,
isotope dilution and transient time measurements showed that the truncated
complex channels carbamoyl phosphate. The overall CPSase-ATCase reaction is much
less sensitive than the parent molecule to the ATCase bisubstrate analogue,
N-phosphonacetyl-L-aspartate (PALA), providing evidence that the endogenously
produced carbamoyl phosphate is sequestered and channeled to the ATCase active
site.
PMID: 10446140 [PubMed - indexed for MEDLINE]
683: J Enzyme Inhib 1999;14(3):175-92
Molecular modelling of lanosterol 14 alpha-demethylase (CYP51) from
Saccharomyces cerevisiae via homology with CYP102, a unique bacterial cytochrome
P450 isoform: quantitative structure-activity relationships (QSARs) within two
related series of antifungal azole derivatives.
Lewis DF, Wiseman A, Tarbit MH.
School of Biological Sciences, University of Surrey, Guildford, UK.
d.lewis@surrey.ac.uk
The construction of a three-dimensional molecular model of the fungal form of
cytochrome P450 (CYP51) from Saccharomyces cerevisiae, based on homology with
the haemoprotein domain of CYP102 from Bacillus megaterium (a unique bacterial
P450 of known crystal structure) is described. It is found that the endogenous
substrate, lanosterol, can readily occupy the putative active site of the CYP51
model such that the known mono-oxygenation reaction, leading to
C14-demethylation of lanosterol, is the preferred route of metabolism for this
particular substrate. Key amino acid contacts within the CYP51 active site
appear to orientate lanosterol for oxidative attack at the C14-methyl group, and
the position of the substrate relative to the haem moiety is consistent with the
phenyl-iron complexation studies reported by Tuck et al. [J. Biol. Chem., 267,
13175-13179 (1992)]. Typical azole inhibitors, such as ketoconazole, are able to
fit the putative active site of CYP51 by a combination of haem ligation,
hydrogen bonding, pi-pi stacking and hydrophobic interactions within the
enzyme's haem environment. The mode of action of azole antifungals, as described
by the modelling studies, is supported by quantitative structure-activity
relationship (QSAR) analyses on two groups of structurally related fungal
inhibitors. Moreover, the results of molecular electrostatic isopotential (EIP)
energy calculations are compatible with the proposed mode of binding between
azole antifungal agents and the putative active site of CYP51, although membrane
interactions may also have a role in the antifungal activity of azole
derivatives.
PMID: 10445042 [PubMed - indexed for MEDLINE]
684: Genes Dev 1999 Aug 1;13(15):1983-93
Functional interactions of Prp8 with both splice sites at the spliceosomal
catalytic center.
Siatecka M, Reyes JL, Konarska MM.
The Rockefeller University, New York, New York 10021, USA.
A U5 snRNP protein, hPrp8, interacts closely with the GU dinucleotide at the 5'
splice site (5'SS), forming a specific UV-inducible cross-link. To test if this
physical contact between the 5'SS and the carboxy-terminal region of Prp8
reflects a functional recognition of the 5'SS during spliceosome assembly, we
mutagenized the corresponding region of yeast Prp8 and screened the resulting
mutants for suppression of 5'SS mutations in vivo. All of the isolated prp8
alleles not only suppress 5'SS but also 3'SS mutations, affecting the second
catalytic step. Suppression of the 5'SS mutations by prp8 alleles was also
tested in the presence of U1-7U snRNA, a predicted suppressor of the U+2A
mutation. As expected, U1-7U efficiently suppresses prespliceosome formation,
and the first, but not the second, step of U+2A pre-mRNA splicing.
Independently, Prp8 functionally interacts with both splice sites at the later
stage of splicing, affecting the efficiency of the second catalytic step. The
striking proximity of two of the prp8 suppressor mutations to the site of the
5'SS:hPrp8 cross-link suggests that some protein:5'SS contacts made before the
first step may be subsequently extended to accommodate the 3'SS for the second
catalytic step. Together, these results strongly implicate Prp8 in specific
interactions at the catalytic center of the spliceosome.
PMID: 10444596 [PubMed - indexed for MEDLINE]
685: Genes Dev 1999 Aug 1;13(15):1970-82
Allele-specific genetic interactions between Prp8 and RNA active site residues
suggest a function for Prp8 at the catalytic core of the spliceosome.
Collins CA, Guthrie C.
Graduate Group in Biophysics, University of California San Francisco (UCSF), San
Francisco, California 94143-0448, USA.
The highly conserved spliceosomal protein Prp8 is known to cross-link the
critical sequences at both the 5' (GU) and 3' (YAG) ends of the intron. We have
identified prp8 mutants with the remarkable property of suppressing exon
ligation defects due to mutations in position 2 of the 5' GU, and all positions
of the 3' YAG. The prp8 mutants also suppress mutations in position A51 of the
critical ACAGAG motif in U6 snRNA, which has been observed previously to
cross-link position 2 of the 5' GU. Other mutations in the 5' splice site,
branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed.
Notably, the suppressed residues are specifically conserved from yeast to man,
and from U2- to U12-dependent spliceosomes. We propose that Prp8 participates in
a previously unrecognized tertiary interaction between U6 snRNA and both the 5'
and 3' ends of the intron. This model suggests a mechanism for positioning the
3' splice site for catalysis, and assigns a fundamental role for Prp8 in
pre-mRNA splicing.
PMID: 10444595 [PubMed - indexed for MEDLINE]
686: Dev Genet 1999;25(2):168-79
Enhancer of split [E(spl)(D)] is a gro-independent, hypermorphic mutation in
Drosophila.
Nagel AC, Yu Y, Preiss A.
Universitat Hohenheim, Institut fur Genetik, Stuttgart, Germany.
Enhancer of split [E(spl)] refers to a gene complex in Drosophila melanogaster,
which contains a number of target genes of the Notch signaling pathway. The
complex was originally identified by a dominant mutation E(spl)(D) that displays
allele-specific interactions with a recessive mutation in the Notch locus called
split (N(spl)). The spl phenotype is characterized by smaller eyes with
irregularly spaced ommatidia, and it is strongly enhanced by E(spl)(D). This
enhancement is correlated with a truncation of one of the E(spl) bHLH genes, m8,
causing an increased stability of the mutant transcripts and an altered
C-terminus in the mutant M8* protein. Concurrently, an insertion of a middle
repetitive element in the adjacent groucho (gro) gene was observed. In this
work, three different E(spl)(D) revertants (BE22, BE25, BX37), which have lost
the ability to enhance N(spl) completely, were analyzed at the molecular level.
In each case, the structure of the mutant M8* protein was affected, suggesting a
specific involvement of the aberrant protein in the enhancement of the spl
phenotype. This hypothesis is supported by the finding that a perfect phenocopy
of spl enhancement can be achieved with hybrid constructs, where the altered
C-terminus of M8* was fused to other E(spl) bHLH proteins. Thus, the ability to
interact with N(spl) is not restricted to M8* but instead can be induced by an
appropriate mutation in other E(spl) bHLH genes within the context of N(spl). In
a N(spl) background, E(spl)(D) behaves like a hyperactive M8 mutation. However,
the mutant M8* protein has lost the ability of binding to the corepressor Gro,
which is an essential feature for normal E(spl) activity. Yet, other protein
interactions, notably those with other bHLH proteins of either E(spl) or
proneural family, are still observed. These findings suggest that the structural
changes associated with the E(spl)(D) mutant protein are the primary cause for
the phenotypic interactions with the recessive Notch mutation N(spl). Copyright
1999 Wiley-Liss, Inc.
PMID: 10440851 [PubMed - indexed for MEDLINE]
687: Solid State Nucl Magn Reson 1999 Jul;14(2):117-36
Long-distance rotational echo double resonance measurements for the
determination of secondary structure and conformational heterogeneity in
peptides.
Arshava B, Breslav M, Antohi O, Stark RE, Garbow JR, Becker JM, Naider F.
Department of Chemistry, College of Staten Island and the Graduate School of the
City University of New York, 10314, USA.
The utility of rotational echo double resonance (REDOR) NMR spectroscopy for
determining the conformations of linear peptides has been examined critically
using a series of crystalline and amorphous samples. The focus of the present
work was the evaluation of long-distance (> 5 A) interactions using 13C-15N
dephasing. Detailed studies of specifically labeled melanostatin and synthetic
analogs of the alpha-factor yeast mating hormone show that nitrogen-dephased,
carbon-observe REDOR measurements are reliable for distances up to 6.0 A, and
that dipolar interactions can be detected for distances up to 7 A. By contrast,
nitrogen-observe REDOR gives reliable results only for distances shorter than
5.0 A. To measure distances accurately, REDOR data must be corrected for the
effects of natural-abundance spins. These corrections are particularly important
for measuring long distances, which are of the greatest value for determining
peptide secondary structure. We have developed a spherical shell model for
calculating the effect of these background spins. The REDOR studies also
indicate that in a lyophilized powder, the tridecapeptide alpha-factor mating
pheromone from Saccharomyces cerevisiae (WHWLQLKPGQPMY) probably exists as a
distribution of different turn structures around the KPGQ region. This finding
revises previous solid-state NMR studies on this peptide, which concluded
alpha-factor assumes a distorted type-I beta-turn in the Pro-Gly central region
of the molecule [J.R. Garbow, M. Breslav, O. Antohi, F. Naider, Biochemistry, 33
(1994) 10094].
PMID: 10437665 [PubMed - indexed for MEDLINE]
688: Mol Biol Cell 1999 Aug;10(8):2559-72
Multiple sex pheromones and receptors of a mushroom-producing fungus elicit
mating in yeast.
Fowler TJ, DeSimone SM, Mitton MF, Kurjan J, Raper CA.
Department of Microbiology and Molecular Genetics, University of Vermont,
Burlington, Vermont 05405, USA.
The mushroom-producing fungus Schizophyllum commune has thousands of mating
types defined, in part, by numerous lipopeptide pheromones and their G
protein-linked receptors. Compatible combinations of pheromones and receptors
encoded by different mating types regulate a pathway of sexual development
leading to mushroom formation and meiosis. A complex set of pheromone-receptor
interactions maximizes the likelihood of outbreeding; for example, a single
pheromone can activate more than one receptor and a single receptor can be
activated by more than one pheromone. The current study demonstrates that the
sex pheromones and receptors of Schizophyllum, when expressed in Saccharomyces
cerevisiae, can substitute for endogenous pheromone and receptor and induce the
yeast pheromone response pathway through the yeast G protein. Secretion of
active Schizophyllum pheromone requires some, but not all, of the biosynthetic
machinery used by the yeast lipopeptide pheromone a-factor. The specificity of
interaction among pheromone-receptor pairs in Schizophyllum was reproduced in
yeast, thus providing a powerful system for exploring molecular aspects of
pheromone-receptor interactions for a class of seven-transmembrane-domain
receptors common to a wide range of organisms.
PMID: 10436012 [PubMed - indexed for MEDLINE]
689: Biochemistry 1999 Aug 3;38(31):9992-10003
Role of glutamate 91 in information transfer during substrate activation of
yeast pyruvate decarboxylase.
Li H, Furey W, Jordan F.
Department of Chemistry and Biological Sciences and Program in Cellular and
Molecular Biodynamics, Rutgers, the State University, Newark, New Jersey 07102,
USA.
Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate
decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at E91, located on the
putative substrate activation pathway and linking the alpha and gamma domains of
the enzyme. While C221 on the beta domain is the residue at which substrate
activation is triggered [Baburina, I., et al. (1994) Biochemistry 33, 5630-5635;
Baburina, I., et al. (1996) Biochemistry 35, 10249-10255], that information, via
the substrate bound at C221, is transmitted to H92 on the alpha domain, across
the domain divide from C221 [Baburina, I. , et al. (1998) Biochemistry 37,
1235-1244], thence to E91 on the alpha domain, and then on to W412 on the gamma
domain [Li, H., and Jordan, F. (1999) Biochemistry 38, 10004-10012] and to the
active site thiamin diphosphate located at the interface of the alpha and gamma
domains [Arjunan, D., et al. (1996) J. Mol. Biol. 256, 590-600]. Substitution at
E91 with Q, D, or A led to modest reductions in the specific activity (4-, 5-,
and 30-fold), as well as in both the turnover number and the catalytic
efficiency, in that order. Interestingly, the Hill coefficient was only slightly
reduced for the E91D variant, but cooperativity was virtually abolished for the
E91Q and E91A variants. The thermal stability of the variants was reduced in the
following order: wild type > E91Q > E91D > E91A; circular dichroism and
fluorescence experiments also demonstrated that the tertiary structure of the
enzyme was affected by these substitutions. The variants could be purified as
apoenzymes, demonstrating their impaired ability to bind thiamin diphosphate.
Apparently, the charge at residue 91 is quite important for maintaining optimal
cooperativity. To maintain strong domain-domain interactions, the length of the
side chain at position 91 with hydrogen bonding potential to W412 is sufficient.
PMID: 10433706 [PubMed - indexed for MEDLINE]
690: Biochem J 1999 Aug 15;342 ( Pt 1):27-32
Production in vitro by the cytochrome P450 CYP94A1 of major C18 cutin monomers
and potential messengers in plant-pathogen interactions: enantioselectivity
studies.
Pinot F, Benveniste I, Salaun JP, Loreau O, Noel JP, Schreiber L, Durst F.
Institut de Biologie Moleculaire des Plantes-CNRS UPR406, Departement
d'Enzymologie Cellulaire et Moleculaire, 28 rue Goethe, F-67083 Strasbourg
Cedex, France. franck.pinot@bota-ulp.u-strasbg.fr
The major C(18) cutin monomers are 18-hydroxy-9,10-epoxystearic and
9,10,18-trihydroxystearic acids. These compounds are also known messengers in
plant-pathogen interactions. We have previously shown that their common
precursor 9,10-epoxystearic acid was formed by the epoxidation of oleic acid in
Vicia sativa microsomes (Pinot, Salaun, Bosch, Lesot, Mioskowski and Durst
(1992) Biochem. Biophys. Res. Commun. 184, 183-193). Here we determine the
chirality of the epoxide produced as (9R,10S) and (9S,10R) in the ratio 90:10
respectively. We further show that microsomes from yeast expressing the
cytochrome P450 CYP94A1 are capable of hydroxylating the methyl terminus of
9,10-epoxystearic and 9,10-dihydroxystearic acids in the presence of NADPH to
form the corresponding 18-hydroxy derivatives. The reactions were not catalysed
by microsomes from yeast transformed with a void plasmid or in absence of NADPH.
After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid with
microsomes of yeast expressing CYP94A1, the chirality of the residual epoxide
was shifted to 66:34 in favour of the (9S,10R) enantiomer. Both enantiomers were
incubated separately and V(max)/K(m) values of 16 and 3.42 ml/min per nmol of
P450 for (9R, 10S) and (9S,10R) respectively were determined, demonstrating that
CYP94A1 is enantioselective for the (9R,10S) enantiomer, which is preferentially
formed in V. sativa microsomes. Compared with the epoxide, the diol
9,10-dihydroxystearic acid was a much poorer substrate for the
omega-hydroxylase, with a measured V(max)/K(m) of 0.33 ml/min per nmol of P450.
Our results indicate that the activity of CYP94A1 is strongly influenced by the
stereochemistry of the 9, 10-epoxide and the nature of substituents on carbons 9
and 10, with V(max)/K(m) values for epoxide>>oleic acid>diol.
PMID: 10432296 [PubMed - indexed for MEDLINE]
691: Proc Natl Acad Sci U S A 1999 Aug 3;96(16):9033-8
Binding of elongin A or a von Hippel-Lindau peptide stabilizes the structure of
yeast elongin C.
Botuyan MV, Koth CM, Mer G, Chakrabartty A, Conaway JW, Conaway RC, Edwards AM,
Arrowsmith CH, Chazin WJ.
Division of Molecular and Structural Biology, Ontario Cancer Institute and
Department of Medical Biophysics, University of Toronto, 610 University Avenue,
Toronto, ON, Canada M5G 2M9.
Elongin is a heterotrimeric transcription elongation factor composed of subunits
A, B, and C in mammals. Elongin A and C are F-box-containing and SKP1 homologue
proteins, respectively, and are therefore of interest for their potential roles
in cell cycle-dependent proteolysis. Mammalian elongin C interacts with both
elongin A and elongin B, as well as with the von Hippel-Lindau tumor suppressor
protein VHL. To investigate the corresponding interactions in yeast, we have
utilized NMR spectroscopy combined with ultracentrifugal sedimentation
experiments to examine complexes of yeast elongin C (Elc1) with yeast elongin A
(Ela1) and two peptides from homologous regions of Ela1 and human VHL. Elc1
alone is a homotetramer composed of subunits with a structured N-terminal region
and a dynamically unstable C-terminal region. Binding of a peptide fragment of
the Elc1-interaction domain of Ela1 or with a homologous peptide from VHL
promotes folding of the C-terminal region of Elc1 into two regular helical
structures and dissociates Elc1 into homodimers. Moreover, analysis of the
complex of Elc1 with the full Elc1-interaction domain of Ela1 reveals that the
Elc1 homodimer is dissociated to preferentially form an Ela1/Elc1 heterodimer.
Thus, elongin C is found to oligomerize in solution and to undergo significant
structural rearrangements upon binding of two different partner proteins. These
results suggest a structural basis for the interaction of an F-box-containing
protein with a SKP1 homologue and the modulation of this interaction by the
tumor suppressor VHL.
PMID: 10430890 [PubMed - indexed for MEDLINE]
692: Proc Natl Acad Sci U S A 1999 Aug 3;96(16):8890-4
Domain structure and lipid interaction of recombinant yeast Tim44.
Weiss C, Oppliger W, Vergeres G, Demel R, Jeno P, Horst M, de Kruijff B, Schatz
G, Azem A.
Department of Biochemistry, Tel-Aviv University, Tel-Aviv 69978, Israel.
Tim44 is an essential component of the machinery that mediates the translocation
of nuclear-encoded proteins across the mitochondrial inner membrane. It
functions as a membrane anchor for the ATP-driven protein import motor whose
other subunits are the mitochondrial 70-kDa heat-shock protein (mhsp70) and its
nucleotide exchange factor, mGrpE. To understand how this motor is anchored to
the inner membrane, we have overexpressed Tim44 in Escherichia coli and studied
the properties of the pure protein and its interaction with model lipid
membranes. Limited proteolysis and analytical ultracentrifugation indicate that
Tim44 is an elongated monomer with a stably folded C-terminal domain. The
protein binds strongly to liposomes composed of phosphatidylcholine and
cardiolipin but only weakly to liposomes containing phosphatidylcholine alone.
Studies with phospholipid monolayers suggest that Tim44 binds to phospholipids
of the mitochondrial inner membrane both by electrostatic interactions and by
penetrating the polar head group region.
PMID: 10430866 [PubMed - indexed for MEDLINE]
693: Genetics 1999 Aug;152(4):1543-56
Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide
exchangers.
Jones S, Jedd G, Kahn RA, Franzusoff A, Bartolini F, Segev N.
Department of Pharmacological and Physiological Sciences, The University of
Chicago, Chicago, Illinois 60637, USA.
Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular
transport. While Arf proteins are implicated in vesicle budding from the donor
compartment, Ypt/rab proteins are involved in the targeting of vesicles to the
acceptor compartment. Recently, we have shown a role for Ypt31/32p in exit from
the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in
vesicle budding as well. Here we report the identification of a new member of
the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation.
Several proteins that belong to the Sec7-domain family, including the yeast
Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases.
Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound
form, is thought to be crucial for their function. Sec7p itself has an important
role in the yeast secretory pathway. However, its mechanism of action is not yet
understood. We show that all members of the Sec7-domain family exhibit distinct
genetic interactions with the YPT genes. Biochemical assays demonstrate that,
although the homology between the members of the Sec7-domain family is
relatively low (20-35%) and limited to a small domain, they all can act as
guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt
GTPases. The Sec7-domain of Sec7p is sufficient for this activity.
Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a
fungal metabolite that inhibits some of the Arf-GEFs, indicating that this
domain is a target for BFA. These results demonstrate that the ability to act as
Arf-GEFs is a general property of all Sec7-domain proteins in yeast. The genetic
interactions observed between Arf GEFs and Ypt GTPases suggest the existence of
a Ypt-Arf GTPase cascade in the secretory pathway.
PMID: 10430582 [PubMed - indexed for MEDLINE]
694: Eur J Biochem 1999 Jul;263(1):118-27
ATP-regulation of cytochrome oxidase in yeast mitochondria: role of subunit VIa.
Beauvoit B, Bunoust O, Guerin B, Rigoulet M.
Institut de Biochimie et Genetique Cellulaires du CNRS, Universite Victor
Segalen, Bordeaux, France. bertrand.beauvoit@ibgc.u-bordeaux2.fr
The role of the nuclear-encoded subunit VIa in the regulation of cytochrome
oxidase by ATP was investigated in isolated yeast mitochondria. As the subunit
VIa-null strain possesses a fully active and assembled cytochrome oxidase,
multiple ATP-regulating sites were characterized with respect to their location
and their kinetic effect: (a) intra-mitochondrial ATP inhibited the complex IV
activity of the null strain, whereas the prevailing effect of ATP on the
wild-type strain, at low ionic strength, was activation on the cytosolic side of
complex IV, mediated by subunit VIa. However, at physiological ionic strength
(i.e. approximately 200 mM), activation by ATP was absent but inhibition was not
impaired; (b) in ethanol-respiring mitochondria, when the electron flux was
modulated using a protonophoric uncoupler, the redox state of aa3 cytochromes
varied with respect to activation (wild-type) or inhibition (null-mutant) of the
cytochrome oxidase by ATP; (c) consequently, the control coefficient of
cytochrome oxidase on respiratory flux, decreased (wild-type) or increased
(null-mutant) in the presence of ATP; (d) considering electron transport from
cytochrome c to oxygen, the response of cytochrome oxidase to its thermodynamic
driving force was increased by ATP for the wild-type but not for the mutant
subunit. Taken together, these findings indicate that at physiological
concentration, ATP regulates yeast cytochrome oxidase via subunit-mediated
interactions on both sides of the inner membrane, thus subtly tuning the
thermodynamic and kinetic control of respiration. This study opens up new
prospects for understanding the feedback regulation of the respiratory chain by
ATP.
PMID: 10429195 [PubMed - indexed for MEDLINE]
695: Plant J 1999 Jun;18(5):541-50
Arabidopsis thaliana proteins related to the yeast SIP and SNF4 interact with
AKINalpha1, an SNF1-like protein kinase.
Bouly JP, Gissot L, Lessard P, Kreis M, Thomas M.
Laboratoire de Biologie du Dveloppement des Plantes, Institut de Biotechnologie
des Plantes, UMR CNRS 8618, Universite de Paris-Sud, Orsay, France.
AKINalpha1, a Ser/Thr kinase from Arabidopsis thaliana belongs to the highly
conserved SNF1 family of protein kinases in eukaryotes. Recent data suggest that
the plant SNF1-related kinases (SnRK1 family) are key enzymes implicated in the
regulation of carbohydrate and lipid metabolism. In Saccharomyces cerevisiae and
mammals, the SNF1 and AMPKalpha protein kinases interact with two other families
of proteins, namely SNF4/AMPKgamma and SIP1/SIP2/GAL83/AMPKbeta, to form active
heterotrimeric complexes. In this paper, we describe the characterisation of
three novel cDNAs. AKINbeta1 and AKINbeta2 encode proteins similar to SIP1, SIP2
and GAL83 and AKINgamma codes for a protein showing similarity with SNF4. Using
the two-hybrid system, specific interactions have been shown between A. thaliana
AKINbeta1/beta2, AKINgamma and AKINgamma as well as between the A. thaliana and
S. cerevisiae subunits. Interestingly, AKINbeta1, AKINbeta2 and AKINgamma mRNAs
accumulate differentially in A. thaliana tissues and are modulated during
development and under different growth conditions. These data suggest the
presence in higher plants of a conserved heterotrimeric complex. Moreover, the
differential transcription of different non-catalytic subunits can constitute a
first level of regulation of the SNF1-like complex in plants.
PMID: 10417704 [PubMed - indexed for MEDLINE]
696: Biochemistry 1999 Jul 20;38(29):9242-53
Ubiquitin binding interface mapping on yeast ubiquitin hydrolase by NMR chemical
shift perturbation.
Rajesh S, Sakamoto T, Iwamoto-Sugai M, Shibata T, Kohno T, Ito Y.
Laboratory of Cellular and Molecular Biology, Institute of Physical and Chemical
Research (RIKEN), Saitama, Japan.
The interaction between the 26 kDa yeast ubiquitin hydrolase (YUH1), involved in
maintaining the monomeric ubiquitin pool in cells, and the 8.5 kDa yeast
ubiquitin protein has been studied by heteronuclear multidimensional NMR
spectroscopy. Chemical shift perturbation of backbone (1)H(N), (15)N, and
(13)C(alpha) resonances of YUH1, in a YUH1-ubiquitin mixture and in a 35 kDa
covalent complex with ubiquitin (a stable analogue of the tetrahedral reaction
intermediate), was employed to identify the ubiquitin binding interface of YUH1.
This interface mapped on the secondary structure of YUH1 suggests a wide area of
contact for ubiquitin, encompassing the N-terminus, alpha1, alpha4, beta2,
beta3, and beta6, coincident with the high specificity of YUH1 for ubiquitin.
The presence of several hydrophobic clusters in the ubiquitin binding interface
of YUH1 suggests that hydrophobic interactions are equally important as ionic
interactions in contacting ubiquitin. The residues in the binding interface
exhibit a high percentage of homology among the members of the ubiquitin
C-terminal hydrolase family, indicating the well-conserved nature of the
ubiquitin binding interface reported in this study. The secondary structure of
YUH1, from our NMR studies, was similar to the recently determined structure of
its human homologue ubiquitin C-terminal hydrolase L3 (UCH-L3), except for the
absence of the helix H3 of UCH-L3. This region in YUH1 (helix H3 of UCH-L3) was
least perturbed upon ubiquitin binding. Therefore, the binding interface was
mapped onto the corresponding residues in the UCH-L3 crystal structure. A model
for ubiquitin binding to YUH1 is proposed, in which a good correlation was
observed for the lateral binding of ubiquitin to UCH-L3 (YUH1), stabilized by
the electrostatic and hydrophobic interactions.
PMID: 10413498 [PubMed - indexed for MEDLINE]
697: Biochemistry 1999 Jul 20;38(29):9198-208
Glutamic acid 472 and lysine 480 of the sodium pump alpha 1 subunit are
essential for activity. Their conservation in pyrophosphatases suggests their
involvement in recognition of ATP phosphates.
Scheiner-Bobis G, Schreiber S.
Institut fur Biochemie und Endokrinologie, Fachbereich Veterinarmedizin,
Justus-Liebig-Universitat Giessen, Germany.
Georgios.Scheiner-Bobis@vetmed.uni-giessen.de
P-type ATPases such as the Na+,K+-ATPase (sodium pump) hydrolyze ATP to pump
ions through biological membranes against their electrochemical gradients. The
mechanisms that couple ATP hydrolysis to the vectorial ion transport are not yet
understood, but unveiling structures that participate in ATP binding and in the
formation of the ionophore might help to gain insight into this process. Looking
at the alpha- and beta-phosphates of ATP as a pyrophosphate molecule, we found
that peptides highly conserved among all soluble inorganic pyrophosphatases are
also present in ion-transporting ATPases. Included therein are Glu48 and Lys56
of the Saccharomyces cerevisiae pyrophosphatase (SCE1-PPase) that are essential
for the activity of this enzyme and have been shown in crystallographic analysis
to interact with phosphate molecules. To test the hypothesis that equivalent
amino acids are also essential for the activity of ion-transporting ATPases,
Glu472 and Lys480 of the sodium pump alpha 1 subunit corresponding to Glu48 and
Lys56 of SCE1-PPase were mutated to various amino acids. Mutants of the sodium
pump alpha1 subunit were expressed in yeast and analyzed for their ATPase
activity and their ability to bind ouabain in the presence of either ATP, Mg2+,
and Na+ or phosphate and Mg2+. All four mutants investigated, Glu472Ala,
Glu472Asp, Lys480Ala, and Lys480Arg, display only a fraction of the ATPase
activity obtained with the wild-type enzyme. The same applies with respect to
their ability to bind ouabain, where maximum ouabain binding to the mutants
accounts for only about 10% of the binding obtained with the wild-type enzyme.
On the basis of our results, we conclude that Glu472 and Lys480 are essential
for the activity of the sodium pump. Their function is probably to arrest the
alpha- and beta-phosphate groups of ATP in a proper position prior to hydrolysis
of the gamma-phosphate group. The identification of these amino acids as
essential components of the ATP-recognizing mechanism of the pump has resulted
in a testable hypothesis for the initial interactions of the sodium pump, and
possibly of other P-type ATPases, with ATP.
PMID: 10413494 [PubMed - indexed for MEDLINE]
698: Biochemistry 1999 Jul 13;38(28):8961-71
Spt16 and Pob3 of Saccharomyces cerevisiae form an essential, abundant
heterodimer that is nuclear, chromatin-associated, and copurifies with DNA
polymerase alpha.
Wittmeyer J, Joss L, Formosa T.
Department of Biochemistry, University of Utah, Salt Lake City 84132, USA.
Previously we showed that the yeast proteins Spt16 (Cdc68) and Pob3 are
physically associated, and interact physically and genetically with the
catalytic subunit of DNA polymerase alpha, Pol1 [Wittmeyer and Formosa (1997)
Mol. Cell. Biol. 17, 4178-4190]. Here we show that purified Spt16 and Pob3 form
a stable, abundant, elongated heterodimer and provide evidence that this is the
functional form of these proteins. Genetic interactions between mutations in
SPT16 and POB3 support the importance of the Spt16-Pob3 interaction in vivo.
Spt16, Pob3, and Pol1 proteins were all found to localize to the nucleus in S.
cerevisiae. A portion of the total cellular Spt16-Pob3 was found to be
chromatin-associated, consistent with the proposed roles in modulating chromatin
function. Some of the Spt16-Pob3 complex was found to copurify with the yeast
DNA polymerase alpha/primase complex, further supporting a connection between
Spt16-Pob3 and DNA replication.
PMID: 10413469 [PubMed - indexed for MEDLINE]
699: Proc Natl Acad Sci U S A 1999 Jul 20;96(15):8567-72
"Mutagenesis" by peptide aptamers identifies genetic network members and pathway
connections.
Geyer CR, Colman-Lerner A, Brent R.
The Molecular Sciences Institute, 2168 Shattuck Avenue, Berkeley, CA 94704, USA.
We selected peptide aptamers from combinatorial libraries that disrupted
cell-cycle arrest caused by mating pheromone in yeast. We used these aptamers as
baits in two-hybrid hunts to identify genes involved in cell-cycle arrest. These
experiments identified genes known to function in the pathway, as well as a
protein kinase, the CBK1 product, whose function was not known. We used a
modified two-hybrid system to identify specific interactions disrupted by these
aptamers. These experiments demonstrate a means to perform "genetics" on the
protein complement of a cell without altering its genetic material. Peptide
aptamers can be identified that disrupt a process. These aptamers can then be
used as affinity reagents to identify individual proteins and protein
interactions needed for the process. Forward genetic analysis with peptide
aptamer "mutagens" should be particularly useful in elucidating genetic networks
in organisms and processes for which classical genetics is not feasible.
PMID: 10411916 [PubMed - indexed for MEDLINE]
700: Mol Cell Biol 1999 Aug;19(8):5417-28
Hec1p, an evolutionarily conserved coiled-coil protein, modulates chromosome
segregation through interaction with SMC proteins.
Zheng L, Chen Y, Lee WH.
Department of Molecular Medicine, Institute of Biotechnology, University of
Texas Health Science Center San Antonio, San Antonio, Texas 78245, USA.
hsHec1p, a Homo sapiens coiled-coil-enriched protein, plays an important role in
M-phase progression in mammalian cells. A Saccharomyces cerevisiae protein,
identical to Tid3p/Ndc80p and here designated scHec1p, has similarities in
structure and biological function to hsHec1p. Budding yeast cells deleted in the
scHEC1/NDC80 allele are not viable, but this lethal phenotype can be rescued by
hsHEC1 under control of the endogenous scHEC1 promoter. At the nonpermissive
temperature, significant mitotic delay, chromosomal missegregation, and
decreased viability were observed in yeast cells with temperature-sensitive (ts)
alleles of hsHEC1. In the hshec1-113 ts mutant, we found a single-point mutation
changing Trp395 to a stop codon, which resulted in the expression of a
C-terminally truncated 45-kDa protein. The binding of this mutated protein,
hshec1-113p, to five identified hsHec1p-associated proteins was unchanged, while
its binding to human SMC1 protein and yeast Smc1p was ts. Hec1p also interacts
with Smc2p, and the binding of the mutated hshec1-113p to Smc2p was not ts.
Overexpression of either hsHEC1 or scHEC1 suppressed the lethal phenotype of
smc1-2 and smc2-6 at nonpermissive temperatures, suggesting that the
interactions between Hec1p and Smc1p and -2p are biologically significant. These
results suggest that Hec1 proteins play a critical role in modulating
chromosomal segregation, in part, through their interactions with SMC proteins.
PMID: 10409732 [PubMed - indexed for MEDLINE]
701: Mol Cell Biol 1999 Aug;19(8):5279-88
Chromatin opening and transactivator potentiation by RAP1 in Saccharomyces
cerevisiae.
Yu L, Morse RH.
Molecular Genetics Program, Wadsworth Center, New York State Department of
Health, and State University of New York School of Public Health, Albany, New
York 12201-2002, USA.
Transcriptional activators function in vivo via binding sites that may be
packaged into chromatin. Here we show that whereas the transcriptional activator
GAL4 is strongly able to perturb chromatin structure via a nucleosomal binding
site in yeast, GCN4 does so poorly. Correspondingly, GCN4 requires assistance
from an accessory protein, RAP1, for activation of the HIS4 promoter, whereas
GAL4 does not. The requirement for RAP1 for GCN4-mediated HIS4 activation is
dictated by the DNA-binding domain of GCN4 and not the activation domain,
suggesting that RAP1 assists GCN4 in gaining access to its binding site.
Consistent with this, overexpression of GCN4 partially alleviates the
requirement for RAP1, whereas HIS4 activation via a weak GAL4 binding site
requires RAP1. RAP1 is extremely effective at interfering with positioning of a
nucleosome containing its binding site, consistent with a role in opening
chromatin at the HIS4 promoter. Furthermore, increasing the spacing between
binding sites for RAP1 and GCN4 by 5 or 10 bp does not impair HIS4 activation,
indicating that cooperative protein-protein interactions are not involved in
transcriptional facilitation by RAP1. We conclude that an important role of RAP1
is to assist activator binding by opening chromatin.
PMID: 10409719 [PubMed - indexed for MEDLINE]
702: J Biol Chem 1999 Jul 23;274(30):21297-304
The Cap-binding protein eIF4E promotes folding of a functional domain of yeast
translation initiation factor eIF4G1.
Hershey PE, McWhirter SM, Gross JD, Wagner G, Alber T, Sachs AB.
Department of Molecular and Cell Biology, University of California at Berkeley,
Berkeley, California 94720, USA.
The association of eucaryotic translation initiation factor eIF4G with the
cap-binding protein eIF4E establishes a critical link between the mRNA and the
ribosome during translation initiation. This association requires a conserved
seven amino acid peptide within eIF4G that binds to eIF4E. Here we report that a
98-amino acid fragment of S. cerevisiae eIF4G1 that contains this eIF4E binding
peptide undergoes an unfolded to folded transition upon binding to eIF4E. The
folding of the eIF4G1 domain was evidenced by the eIF4E-dependent changes in its
protease sensitivity and (1)H-(15)N HSQC NMR spectrum. Analysis of a series of
charge-to-alanine mutations throughout the essential 55.4-kDa core of yeast
eIF4G1 also revealed substitutions within this 98-amino acid region that led to
reduced eIF4E binding in vivo and in vitro. These data suggest that the
association of yeast eIF4E with eIF4G1 leads to the formation of a structured
domain within eIF4G1 that could serve as a specific site for interactions with
other components of the translational apparatus. They also suggest that the
stability of the native eIF4E-eIF4G complex is determined by amino acid residues
outside of the conserved seven-residue consensus sequence.
PMID: 10409688 [PubMed - indexed for MEDLINE]
703: Biotechniques 1999 Jul;27(1):86-8, 92-4
Application of the green fluorescent protein as a reporter for Ace1-based,
two-hybrid studies.
Mayer G, Launhardt H, Munder T.
Hans-Knoll-Institut fur Naturstoff-Forschung e.V. Jena, Germany.
The two-hybrid system in Saccharomyces cerevisiae is a genetic approach for the
detection of of protein-protein interactions in vivo. This technology relies on
the the activity of separated DNA-binding and transactivation domains of
specific transcription factors to reconstitute an active transcription factor
complex if interacting proteins are fused to these domains. Interactions are
consequently detected through the activity of reporter genes. The two-hybrid
technology has been successfully applied for the determination of interactions
between numerous proteins of several organisms. Conventional reporter systems,
such as the beta-galacatosidase from Escherichia coli, suffer from a variety of
drawbacks, including the requirement for external substrates. In this report, we
describe an alternative version of the two hybrid system using the combined
advantages of the copper-inducible transcription factor Acel together with the
yeast metallothionein gene CUP1 and the green fluorescence protein from aquatic
invertebrates as reporters. This technique allows the copper-dependent
monitoring of protein-protein interactions in living yeast cells.
PMID: 10407670 [PubMed - indexed for MEDLINE]
704: Yeast 1999 Jul;15(10B):963-72
Epitope tagging of yeast genes using a PCR-based strategy: more tags and
improved practical routines.
Knop M, Siegers K, Pereira G, Zachariae W, Winsor B, Nasmyth K, Schiebel E.
The Beatson Institute for Cancer Research, CRC Beatson Laboratories, Garscube
Estate, Switchback Road, Bearsden, Glasgow G61 1BD, U.K.
Epitope tagging of proteins as a strategy for the analysis of function,
interactions and the subcellular distribution of proteins has become widely
used. In the yeast Saccharomyces cerevisiae, molecular biological techniques
have been developed that use a simple PCR-based strategy to introduce epitope
tags to chromosomal loci (Wach et al., 1994). To further employ the power of
this strategy, a variety of novel tags was constructed. These tags were combined
with different selectable marker genes, resulting in PCR amplificable modules.
Only one set of primers is required for the amplification of any module.
Furthermore, convenient laboratory techniques are described that facilitate the
genetic manipulations of yeast strains, as well as the analysis of the
epitope-tagged proteins. Copyright 1999 John Wiley & Sons, Ltd.
PMID: 10407276 [PubMed - indexed for MEDLINE]
705: Yeast 1999 Jul;15(10A):865-72
A systematic nomenclature for new translation initiation factor genes from S.
pombe and other fungi.
Linder P, Vornlocher HP, Hershey JW, McCarthy JE.
Departement de Biochimie Medicale, Centre Medical Universitaire, 1, rue Michel
Servet 1211 Geneve 4, Switzerland.
Eukaryotic translation initiation factors and their corresponding genes have
been characterized using biochemical and genetic methods from a variety of
different organisms. The designations of the factors relate to their apparent
roles in the biochemical process. Many gene names indicate genetic interactions
with other genes or the functional attributes used to identify them. On the
other hand, progress in systematic sequencing of the genomes of organisms like
Saccharomyces cerevisiae and Schizosaccharomyces pombe has revealed many genes
homologous to known translation initiation factor genes. The genes defined by
the systematic sequencing approach are assigned numerical designations
completely unrelated to their biological function. So far there have been
publications on only three genes encoding translation initiation factors from
Schizosaccharomyces pombe. We therefore see this an an ideal opportunity to
propose a systematic and logical nomenclature for genes encoding translation
initiation factor genes that can be applied to all further genes of this type
that are characterized in this fission yeast. Copyright 1999 John Wiley & Sons,
Ltd.
PMID: 10407266 [PubMed - indexed for MEDLINE]
706: EMBO J 1999 Jul 15;18(14):4068-75
Repressor binding to a dorsal regulatory site traps human eIF4E in a high
cap-affinity state.
Ptushkina M, von der Haar T, Karim MM, Hughes JM, McCarthy JE.
Posttranscriptional Control Group, Department of Biomolecular Sciences, UMIST,
PO Box 88, Manchester M60 1QD, UK.
Eukaryotic translation initiation involves recognition of the 5' end of cellular
mRNA by the cap-binding complex known as eukaryotic initiation factor 4F
(eIF4F). Initiation is a key point of regulation in gene expression in response
to mechanisms mediated by signal transduction pathways. We have investigated the
molecular interactions underlying inhibition of human eIF4E function by
regulatable repressors called 4E-binding proteins (4E-BPs). Two essential
components of eIF4F are the cap-binding protein eIF4E, and eIF4G, a
multi-functional protein that binds both eIF4E and other essential eIFs. We show
that the 4E-BPs 1 and 2 block the interaction between eIF4G and eIF4E by
competing for binding to a dorsal site on eIF4E. Remarkably, binding of the
4E-BPs at this dorsal site enhances cap-binding via the ventral cap-binding
slot, thus trapping eIF4E in inactive complexes with high affinity for capped
mRNA. The binding contacts and affinities for the interactions between 4E-BP1/2
and eIF4E are distinct (estimated K(d) values of 10(-8) and 3x10(-9) for 4E-BP1
and 2, respectively), and the differences in these properties are determined by
three amino acids within an otherwise conserved motif. These data provide a
quantitative framework for a new molecular model of translational regulation.
PMID: 10406811 [PubMed - indexed for MEDLINE]
707: Biochem Biophys Res Commun 1999 Jul 14;260(3):799-805
Oligomerized Ced-4 kills budding yeast through a caspase-independent mechanism.
Tao W, Walke DW, Morgan JI.
Department of Developmental Neurobiology, St. Jude Children's Research Hospital,
332 North Lauderdale Street, Memphis, Tennessee, 38105-2794, USA.
In Caenorhabdtis elegans, Ced-3, Ced-4, and Ced-9 are components of a cell
suicide program. Ced-4 facilitates the proteolytic activation of the caspase,
Ced-3, while Ced-9 opposes Ced-3/Ced-4 killing. To examine the interactions
among these proteins they were expressed in Saccharomyces cerevisiae. Ced-3 and
Ced-4 were lethal when expressed alone, revealing an intrinsic Ced-4 killing
activity. Coexpression of Ced-9 blocked Ced-3- and Ced-4-induced killing,
showing Ced-9 can independently antagonize the action of both proteins. Ced-3-
but not Ced-4-toxicity was attenuated by coexpression of the caspase inhibitors,
CrmA and p35. Thus, besides its Ced-3- and Ced-9-dependent action in C. elegans,
Ced-4 has an additional Ced-9-dependent, Ced-3-independent killing mechanism in
yeast. Two-hybrid analysis confirmed that Ced-4 formed heteromers with Ced-9. In
addition, Ced-4 formed homomers and mutation of its nucleoside triphosphate
binding motif eliminated both homomerization and cell killing. We suggest the
caspase-independent lethality of Ced-4 in yeast is mediated by a Ced-4 homomer.
Copyright 1999 Academic Press.
PMID: 10403845 [PubMed - indexed for MEDLINE]
708: J Biol Chem 1999 Jul 16;274(29):20235-43
A small region in HMG I(Y) is critical for cooperation with NF-kappaB on DNA.
Zhang XM, Verdine GL.
Department of Molecular and Cellular Biology, Harvard University, Cambridge,
Massachusetts 02138, USA.
The high mobility group HMG I(Y) protein has been reported to promote the
expression of several NF-kappaB-dependent genes by enhancing the binding of
NF-kappaB to DNA. The molecular origins of cooperativity in the binding of
NF-kappaB and HMG I(Y) to DNA are not well understood. Here we have examined the
determinants of specificity in the binding of HMG I(Y), both alone and in
cooperation with NF-kappaB, to two different DNA elements, PRDII from the
interferon-beta enhancer and IgkappaB from the immunoglobulin kappa light chain
enhancer. Of particular interest was the influence of a flanking AT-rich
sequence on binding by HMG I(Y). Utilizing yeast one-hybrid screening assays
together with alanine-scanning mutagenesis, we have identified mutations of
residues in HMG I(Y) that decrease cooperative binding of NF-kappaB to PRDII and
IgkappaB sites. These same mutations similarly decreased the binding of HMG I(Y)
alone to DNA, and paradoxically, decreased the strength of protein-protein
interactions between HMG I(Y) and NF-kappaB. Of the three tandemly repeated
basic regions that represent putative DNA-binding motifs in HMG I(Y), the
residues within the second repeat are most important for recognition of core
NF-kappaB sites, whereas the second and third repeats both appear to be involved
in binding to sites that are flanked by AT-rich sequences. Overall, the second
repeat of HMG I(Y) is primarily responsible for the stimulatory effect of this
protein on the binding of NF-kappaB to PRDII and IgkappaB elements.
PMID: 10400641 [PubMed - indexed for MEDLINE]
709: Yeast 1999 Jun 30;15(9):721-40
Specific negative effects resulting from elevated levels of the recombinational
repair protein Rad54p in Saccharomyces cerevisiae.
Clever B, Schmuckli-Maurer J, Sigrist M, Glassner BJ, Heyer WD.
Institute for General Microbiology, University of Bern, Bern, Switzerland.
RAD54 is an important gene in the RAD52 group that controls recombinational
repair of DNA damage in Saccharomyces cerevisiae. Rad54p is a DNA-dependent
ATPase and shares seven conserved sequence motifs with proteins of the
Swi2p/Snf2p family. Genetic analysis of mutations in motif IA, the putative
ATP-binding fold of Rad54p, demonstrated the functional importance of this
motif. Overexpression of these mutant proteins resulted in strong,
dominant-negative effects on cell survival. High levels of full-length wild-type
Rad54p or specific parts of Rad54p also resulted in negative effects, dependent
on the ploidy of the host cell. This differential effect was not under a/alpha
mating-type control. Deletion of the RAD54 gene led to a small but significant
increase in the mutation rate. However, the negative overexpression effects in
haploid cells could not be explained by an accumulation of (recessive) lethal
mutations. All negative overexpression effects were found to be enhanced under
genotoxic stress. We suggest that the negative overexpression effects are the
result of unbalanced protein-protein interactions, indicating that Rad54p is
involved in multiple interactions, dependent on the physiological situation.
Diploid wild-type cells contained an estimated 7000 Rad54p molecules/cell,
whereas haploid cells about 3500/cell. Rad54p levels were highest in actively
growing cells compared to stationary phase cells. Rad54 protein levels were
found to be elevated after DNA damage. Copyright 1999 John Wiley & Sons, Ltd.
PMID: 10398342 [PubMed - indexed for MEDLINE]
710: Mol Gen Genet 1999 Jun;261(4-5):788-95
Genetic evidence for interactions between yeast importin alpha (Srp1p) and its
nuclear export receptor, Cse1p.
Schroeder AJ, Chen XH, Xiao Z, Fitzgerald-Hayes M.
Department of Biochemistry and Molecular Biology, University of Massachusetts,
Amherst 01003, USA.
The yeast Srp1p protein functions as an import receptor for proteins bearing
basic nuclear localization signals. Cse1p, the yeast homolog of mammalian CAS,
recycles Srp1p back to the cytoplasm after import substrates have been released
into the nucleoplasm. In this report we describe genetic interactions between
SRP1 and CSE1. Results from genetic suppression and synthetic lethality studies
demonstrate that these gene products interact to ensure accurate chromosome
segregation. We also describe new mutant alleles of CSE1 and analyze a new
temperature-sensitive allele of CSE1, cse1-2. This allele causes high levels of
chromosome missegregation and cell cycle arrest during mitosis at the
nonpermissive temperature.
PMID: 10394916 [PubMed - indexed for MEDLINE]
711: Proc Natl Acad Sci U S A 1999 Jul 6;96(14):7791-6
Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter
proximal RNA target is both necessary and sufficient for full activation of
transcription.
Bieniasz PD, Grdina TA, Bogerd HP, Cullen BR.
Howard Hughes Medical Institute and Department of Genetics, Box 3025, Duke
University Medical Center, Durham, NC 27710, USA.
Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR)
promoter element by the viral Tat protein is an essential step in the HIV-1 life
cycle. Tat function is mediated by the TAR RNA target element encoded within the
LTR and is known to require the recruitment of a complex consisting of Tat and
the cyclin T1 (CycT1) component of positive transcription elongation factor b
(P-TEFb) to TAR. Here, we demonstrate that both TAR and Tat become entirely
dispensable for activation of the HIV-1 LTR promoter when CycT1/P-TEFb is
artificially recruited to a heterologous promoter proximal RNA target. The level
of activation observed was indistinguishable from the level induced by Tat and
was neither inhibited nor increased when Tat was expressed in trans. Activation
by artificially recruited CycT1 depended on the ability to bind the CDK9
component of P-TEFb. In contrast, although binding to both Tat and TAR was
essential for the ability of CycT1 to act as a Tat cofactor, these interactions
became dispensable when CycT1 was directly recruited to the LTR. Importantly,
activation of the LTR both by Tat and by directly recruited CycT1 was found to
be at the level of transcription elongation. Together, these data demonstrate
that recruitment of CycT1/P-TEFb to the HIV-1 LTR is fully sufficient to
activate this promoter element and imply that the sole role of the Tat/TAR axis
in viral transcription is to permit the recruitment of CycT1/P-TEFb.
PMID: 10393900 [PubMed - indexed for MEDLINE]
712: J Biol Chem 1999 Jul 9;274(28):19617-22
N-terminal tail export from the mitochondrial matrix. Adherence to the
prokaryotic "positive-inside" rule of membrane protein topology.
Rojo EE, Guiard B, Neupert W, Stuart RA.
Institut fur Physiologische Chemie der Universitat Munchen, Goethestrasse 33,
80336 Munchen, Germany.
Export of N-terminal tails of mitochondrial inner membrane proteins from the
mitochondrial matrix is a membrane potential-dependent process, mediated by the
Oxa1p translocation machinery. The hydrophilic segments of these membrane
proteins, which undergo export, display a characteristic charge profile where
intermembrane space-localized segments bear a net negative charge, whereas those
remaining in the matrix have a net positive one. Using a model protein,
preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated
N-tail export, we demonstrate here that the net charge of N- and C-flanking
regions of the transmembrane domain play a critical role in determining the
orientation of the insertion process. The N-tail must bear a net negative charge
to be exported to the intermembrane space. Furthermore, a net positive charge of
the C-terminal region supports this N-tail export event. These data provide
experimental evidence that protein export in mitochondria adheres to the
"positive-inside" rule, described for sec-independent sorting of membrane
proteins in prokaryotes. We propose here that the importance of a charge profile
reflects a need for specific protein-protein interactions to occur in the export
reaction, presumably at the level of the Oxa1p export machinery.
PMID: 10391898 [PubMed - indexed for MEDLINE]
713: Genetics 1999 Jul;152(3):881-93
In vivo analysis of the domains of yeast Rvs167p suggests Rvs167p function is
mediated through multiple protein interactions.
Colwill K, Field D, Moore L, Friesen J, Andrews B.
Department of Molecular and Medical Genetics, University of Toronto, Toronto,
Ontario M5S 1A8, Canada.
Morphological changes during cell division in the yeast Saccharomyces cerevisiae
are controlled by cell-cycle regulators. The Pcl-Pho85p kinase complex has been
implicated in the regulation of the actin cytoskeleton at least in part through
Rvs167p. Rvs167p consists of three domains called BAR, GPA, and SH3. Using a
two-hybrid assay, we demonstrated that each region of Rvs167p participates in
protein-protein interactions: the BAR domain bound the BAR domain of another
Rvs167p protein and that of Rvs161p, the GPA region bound Pcl2p, and the SH3
domain bound Abp1p. We identified Rvs167p as a Las17p/Bee1p-interacting protein
in a two-hybrid screen and showed that Las17p/Bee1p bound the SH3 domain of
Rvs167p. We tested the extent to which the Rvs167p protein domains rescued
phenotypes associated with deletion of RVS167: salt sensitivity, random budding,
and endocytosis and sporulation defects. The BAR domain was sufficient for full
or partial rescue of all rvs167 mutant phenotypes tested but not required for
the sporulation defect for which the SH3 domain was also sufficient.
Overexpression of Rvs167p inhibits cell growth. The BAR domain was essential for
this inhibition and the SH3 domain had only a minor effect. Rvs167p may link the
cell cycle regulator Pcl-Pho85p kinase and the actin cytoskeleton. We propose
that Rvs167p is activated by phosphorylation in its GPA region by the Pcl-Pho85p
kinase. Upon activation, Rvs167p enters a multiprotein complex, making critical
contacts in its BAR domain and redundant or minor contacts with its SH3 domain.
PMID: 10388809 [PubMed - indexed for MEDLINE]
714: J Mol Biol 1999 Jul 2;290(1):331-45
Equilibrium folding properties of the yeast prion protein determinant Ure2.
Perrett S, Freeman SJ, Butler PJ, Fersht AR.
Centre for Protein Engineering, Department of Chemistry, University of
Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
The yeast non-Mendelian factor [URE3] propagates by a prion-like mechanism,
involving aggregation of the chromosomally encoded protein Ure2. The [URE3]
phenotype is equivalent to loss of function of Ure2, a protein involved in
regulation of nitrogen metabolism. The prion-like behaviour of Ure2 in vivo is
dependent on the first 65 amino acid residues of its N-terminal region which
contains a highly repetitive sequence rich in asparagine. This region has been
termed the prion-determining domain (PrD). Removal of as little as residues 2-20
of the protein is sufficient to prevent occurrence of the [URE3] phenotype.
Removal of the PrD does not affect the regulatory activity of Ure2. The
C-terminal portion of the protein has homology to glutathione S -transferases,
which are dimeric proteins. We have produced the Ure2 protein to high yield in
Escherichia coli from a synthetic gene. The recombinant purified protein is
shown to be a dimer. The stability, folding and oligomeric state of Ure2 and a
series of N-terminally truncated or deleted variants were studied and compared.
The stability of Ure2, DeltaGD-N, H2O, determined by chemical denaturation and
monitored by fluorescence, is 12.1(+/-0.4) kcal mol-1at 25 degrees C and pH 8.4.
A range of structural probes show a single, coincident unfolding transition,
which is invariant over a 550-fold change in protein concentration. The
stability is the same within error for Ure2 variants lacking all or part of the
prion-determining domain. The data indicate that in the folded protein the PrD
is in an unstructured conformation and does not form specific intra- or
intermolecular interactions at micromolar protein concentrations. This suggests
that the C-terminal domain may stabilise the PrD against prion formation by
steric means, and implies that the PrD does not induce prion formation by
altering the thermodynamic stability of the folded protein. Copyright 1999
Academic Press.
PMID: 10388576 [PubMed - indexed for MEDLINE]
715: Biochemistry 1999 Jun 22;38(25):8138-49
Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of
the contributions from charge-charge interactions to protein stability.
Ibarra-Molero B, Loladze VV, Makhatadze GI, Sanchez-Ruiz JM.
Facultad de Ciencias, Departamento de Quimica Fisica, Universidad de Granada,
Spain.
We have characterized the guanidine-induced unfolding of both yeast and bovine
ubiquitin at 25 degrees C and in the acidic pH range on the basis of
fluorescence and circular dichroism measurements. Unfolding Gibbs energy changes
calculated by linear extrapolation from high guanidine unfolding data are found
to depend very weakly on pH. A simple explanation for this result involves the
two following assumptions: (1) charged atoms of ionizable groups are exposed to
the solvent in native ubiquitin (as supported by accessible surface area
calculations), and Gibbs energy contributions associated with charge desolvation
upon folding (a source of pK shifts) are small; (2) charge-charge interactions
(another source of pK shifts upon folding) are screened out in concentrated
guanidinium chloride solutions. We have also characterized the thermal unfolding
of both proteins using differential scanning calorimetry. Unfolding Gibbs energy
changes calculated from the calorimetric data do depend strongly on pH, a result
that we attribute to the pH dependence of charge-charge interactions (not
eliminated in the absence of guanidine). In fact, we find good agreement between
the difference between the two series of experimental unfolding Gibbs energy
changes (determined from high guanidine unfolding data by linear extrapolation
and from thermal denaturation data in the absence of guanidine) and the
theoretical estimates of the contribution from charge-charge interactions to the
Gibbs energy change for ubiquitin unfolding obtained by using the
solvent-accessibility-corrected Tanford-Kirkwood model, together with the
Bashford-Karplus (reduced-set-of-sites) approximation. This contribution is
found to be stabilizing at neutral pH, because most charged groups on the native
protein interact mainly with groups of the opposite charge, a fact that,
together with the absence of large charge-desolvation contributions, may explain
the high stability of ubiquitin at neutral pH. In general, our analysis suggests
the possibility of enhancing protein thermal stability by adequately redesigning
the distribution of solvent-exposed, charged residues on the native protein
surface.
PMID: 10387059 [PubMed - indexed for MEDLINE]
716: Protein Sci 1999 Jun;8(6):1250-6
The Schiff base complex of yeast 5-aminolaevulinic acid dehydratase with
laevulinic acid.
Erskine PT, Newbold R, Roper J, Coker A, Warren MJ, Shoolingin-Jordan PM, Wood
SP, Cooper JB.
Division of Biochemistry and Molecular Biology, School of Biological Sciences,
University of Southampton, United Kingdom.
The X-ray structure of the complex formed between yeast 5-aminolaevulinic acid
dehydratase (ALAD) and the inhibitor laevulinic acid has been determined at 2.15
A resolution. The inhibitor binds by forming a Schiff base link with one of the
two invariant lysines at the catalytic center: Lys263. It is known that this
lysine forms a Schiff base link with substrate bound at the enzyme's so-called
P-site. The carboxyl group of laevulinic acid makes hydrogen bonds with the
side-chain-OH groups of Tyr329 and Ser290, as well as with the main-chain >NH
group of Ser290. The aliphatic moiety of the inhibitor makes hydrophobic
interactions with surrounding aromatic residues in the protein including Phe219,
which resides in the flap covering the active site. Our analysis strongly
suggests that the same interactions will be made by P-side substrate and also
indicates that the substrate that binds at the enzyme's A-site will interact
with the enzyme's zinc ion bound by three cysteines (133, 135, and 143).
Inhibitor binding caused a substantial ordering of the active site flap
(residues 217-235), which was largely invisible in the native electron density
map and indicates that this highly conserved yet flexible region has a specific
role in substrate binding during catalysis.
PMID: 10386874 [PubMed - indexed for MEDLINE]
717: Neuroreport 1999 May 14;10(7):1409-15
A novel candidate presenilin-1 interacting protein containing tetratricopeptide
repeats.
Prihar G, Gonzalez de Chavez F, Baker M, Crook R, McGowan E, Grover A, Hardy J,
Hutton M.
Neurogenetics Laboratory, The Mayo Clinic, Jacksonville, FL 32224, USA.
The yeast two-hybrid system, immunofluorescence and co-immunoprecipitation
techniques were used to identify a novel candidate protein with which
presenilin-1 (PS-1) interacts. This interacting protein, the gene of which is
encoded on chromosome 16, contains two tetratricopeptide repeats (TPR) that are
known to mediate interactions between proteins, appears to be primarily
localized to the cytoplasm of transfected HEK293 cells, and is expressed in
brain. Preliminary yeast two-hybrid data suggests this candidate may interact
with both heat shock protein-90 and heat shock protein-70 and thus may be a
novel member of TPR-containing proteins which interact with this complex.
PMID: 10380955 [PubMed - indexed for MEDLINE]
718: Microbiology 1999 May;145 ( Pt 5):1115-22
Changes in Aspergillus nidulans gene expression induced by bafilomycin, a
Streptomyces-produced antibiotic.
Melin P, Schnurer J, Wagner EG.
Department of Microbiology, Swedish University of Agricultural Sciences,
Uppsala. Petter.Melin@mikrob.slu.se
In natural environments bacteria and filamentous fungi often compete for the
same resources. Consequently, production of antibiotic secondary metabolites and
defence mechanisms against these compounds have evolved in these organisms. An
experimental model has been developed to study the response in fungi exposed to
one such antibiotic. The filamentous fungus Aspergillus nidulans was treated
with bafilomycin B1, a Streptomyces-produced antibiotic which reduces radial
growth rate and induces morphological changes in fungi. mRNA differential
display was used to study changes in fungal gene expression. For five genes,
changes in abundance of the corresponding mRNAs, directly or indirectly caused
by bafilomycin, were observed. Of these, three were up-regulated and two
repressed. With four of these the change in mRNA abundance measured ranged from
10- to 60-fold. However, for one gene the mRNA was only detected after
bafilomycin treatment. One of the down-regulated mRNAs encodes ASPND1, a
glycoprotein that belongs to a known family of antigens identified in
aspergilloma patients. One up-regulated mRNA shows sequence similarities, at the
amino acid level, with a cell-wall protein of Saccharomyces cerevisiae. The
remaining three genes were also cloned and sequenced; their sequences do not
correspond to known genes in A. nidulans, and no similarities with published
nucleotide or protein sequences in other organisms were found. These results
indicate the feasibility of using mRNA differential display to study
interactions between bacteria and filamentous fungi.
PMID: 10376827 [PubMed - indexed for MEDLINE]
719: Mol Cell Biol 1999 Jul;19(7):4561-71
Std1 and Mth1 proteins interact with the glucose sensors to control
glucose-regulated gene expression in Saccharomyces cerevisiae.
Schmidt MC, McCartney RR, Zhang X, Tillman TS, Solimeo H, Wolfl S, Almonte C,
Watkins SC.
Department of Molecular Genetics and Biochemistry, University of Pittsburgh
School of Medicine, Pittsburgh, Pennsylvania 15261, USA. mcs2@pop.pitt.edu
The Std1 protein modulates the expression of glucose-regulated genes, but its
exact molecular role in this process is unclear. A two-hybrid screen for
Std1-interacting proteins identified the hydrophilic C-terminal domains of the
glucose sensors, Snf3 and Rgt2. The homologue of Std1, Mth1, behaves differently
from Std1 in this assay by interacting with Snf3 but not Rgt2. Genetic
interactions between STD1, MTH1, SNF3, and RGT2 suggest that the glucose
signaling is mediated, at least in part, through interactions of the products of
these four genes. Mutations in MTH1 can suppress the raffinose growth defect of
a snf3 mutant as well as the glucose fermentation defect present in cells
lacking both glucose sensors (snf3 rgt2). Genetic suppression by mutations in
MTH1 is likely to be due to the increased and unregulated expression of hexose
transporter genes. In media lacking glucose or with low levels of glucose, the
hexose transporter genes are subject to repression by a mechanism that requires
the Std1 and Mth1 proteins. An additional mechanism for glucose sensing must
exist since a strain lacking all four genes (snf3 rgt2 std1 mth1) is still able
to regulate SUC2 gene expression in response to changes in glucose
concentration. Finally, studies with green fluorescent protein fusions indicate
that Std1 is localized to the cell periphery and the cell nucleus, supporting
the idea that it may transduce signals from the plasma membrane to the nucleus.
PMID: 10373505 [PubMed - indexed for MEDLINE]
720: FEMS Microbiol Rev 1999 Jun;23(3):277-95
3'-End processing of pre-mRNA in eukaryotes.
Wahle E, Ruegsegger U.
Institut fur Biochemie, Martin-Luther-Universitat Halle-Wittenberg, Germany.
ewahle@biochemtech.uni-halle.de
3'-Ends of almost all eukaryotic mRNAs are generated by endonucleolytic cleavage
and addition of a poly(A) tail. In mammalian cells, the reaction depends on the
sequence AAUAAA upstream of the cleavage site, a degenerate GU-rich sequence
element downstream of the cleavage site and stimulatory sequences upstream of
AAUAAA. Six factors have been identified that carry out the two reactions. With
a single exception, they have been purified to homogeneity and cDNAs for 11
subunits have been cloned. Some of the cooperative RNA-protein and
protein-protein interactions within the processing complex have been analyzed,
but many details, including the identity of the endonuclease, remain unknown.
Several examples of regulated polyadenylation are being analyzed at the
molecular level. In the yeast Saccharomyces cerevisiae, sequences directing
cleavage and polyadenylation are more degenerate than in metazoans, and a
downstream element has not been identified. The list of processing factors may
be complete now with approximately a dozen polypeptides, but their functions in
the reaction are largely unknown. 3'-Processing is known to be coupled to
transcription. This connection is thought to involve interactions of processing
factors with the mRNA cap as well as with RNA polymerase II.
Publication Types:
Review
Review, Tutorial
PMID: 10371034 [PubMed - indexed for MEDLINE]
721: J Mol Biol 1999 Jun 18;289(4):691-9
Polyanionic inhibitors of phosphoglycerate mutase: combined structural and
biochemical analysis.
Rigden DJ, Walter RA, Phillips SE, Fothergill-Gilmore LA.
School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2
9JT, England.
The effects that the inhibitors inositol hexakisphosphate and benzene tri-,
tetra- and hexacarboxylates have on the phosphoglycerate mutases from
Saccharomyces cerevisiae and Schizosaccharomyces pombe have been determined.
Their Kivalues have been calculated, and the ability of the inhibitors to
protect the enzymes against limited proteolysis investigated. These biochemical
data have been placed in a structural context by the solution of the crystal
structures of S. cerevisiae phosphoglycerate mutase soaked with inositol
hexakisphosphate or benzene hexacarboxylate. These large polyanionic compounds
bind to the enzyme so as to block the entrance to the active-site cleft. They
form multiple interactions with the enzyme, consistent with their low Kivalues,
and afford good protection against limited proteolysis of the C-terminal region
by thermolysin. The inositol compound is more efficacious because of its greater
number of negative charges. The S. pombe phosphoglycerate mutase that is
inherently lacking a comparable C-terminal region has higher Kivalues for the
compounds tested. Moreover, the S. pombe enzyme is less sensititive to
proteolysis, and the presence or absence of the inhibitor molecules has little
effect on susceptibility to proteolysis. Copyright 1999 Academic Press.
PMID: 10369755 [PubMed - indexed for MEDLINE]
722: Cell 1999 May 28;97(5):657-66
Crystal structure of a phosphatidylinositol 3-phosphate-specific
membrane-targeting motif, the FYVE domain of Vps27p.
Misra S, Hurley JH.
Laboratory of Molecular Biology, National Institute of Digestive, Diabetes, and
Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0580,
USA.
Phosphatidylinositol 3-phosphate regulates membrane trafficking and signaling
pathways by interacting with the FYVE domains of target proteins. The 1.15 A
structure of the Vps27p FYVE domain reveals two antiparallel beta sheets and an
alpha helix stabilized by two Zn2+-binding clusters. The core secondary
structures are similar to a rabphilin-3A Zn2+-binding domain and to the C1 and
LIM domains. Phosphatidylinositol 3-phosphate binds to a pocket formed by the
(R/K)(R/K)HHCR motif. A lattice contact shows how anionic ligands can interact
with the phosphatidylinositol 3-phosphate-binding site. The tip of the FYVE
domain has basic and hydrophobic surfaces positioned so that nonspecific
interactions with the phospholipid bilayer can abet specific binding to
phosphatidylinositol 3-phosphate.
PMID: 10367894 [PubMed - indexed for MEDLINE]
723: Virology 1999 Jun 5;258(2):271-81
Activation of Ste20 by Nef from human immunodeficiency virus induces
cytoskeletal rearrangements and downstream effector functions in Saccharomyces
cerevisiae.
Plemenitas A, Lu X, Geyer M, Veranic P, Simon MN, Peterlin BM.
Institute of Biochemistry, Medical Faculty, Vrazov trg2, Ljubljana, 1000,
Slovenia.
The negative factor (Nef) from human and simian immunodeficiency viruses is
important for the pathogenesis of acquired immune deficiency syndrome. Among
other targets, it activates the Nef-associated kinase, which is related to the
p21-activated kinase. In this study, we demonstrate that Nef activates Ste20,
the homolog of p21-activated kinase in Saccharomyces cerevisiae. Nef binds to
the adaptor proteins Bem1 and Ste20 via its proline-rich (PXXP) and diarginine
(RR) motifs, respectively. These interactions induce the mitogen-activated
protein kinase and increase the rates of budding, sizes of cells, and patterns
of mating projections. These effects of Nef depend on the small GTPase Cdc42 and
guanine nucleotide exchange factor Cdc24. Thus, studies in S. cerevisiae
identified specific interactions between Nef and cellular proteins and their
associated signaling cascade. Copyright 1999 Academic Press.
PMID: 10366564 [PubMed - indexed for MEDLINE]
724: Biochem Biophys Res Commun 1999 Jun 7;259(2):391-400
RNase treatment of yeast and mammalian cell extracts affects in vitro substrate
methylation by type I protein arginine N-methyltransferases.
Frankel A, Clarke S.
Department of Chemistry & Biochemistry and Molecular Biology Institute, UCLA,
Los Angeles, California 90095-1569, USA.
Type I protein arginine N-methyltransferases catalyze the formation of
omega-NG-monomethylarginine and asymmetric omega-NG, NG-dimethylarginine
residues using S-adenosyl-l-methionine as the methyl donor. In vitro these
enzymes can modify a number of soluble methyl-accepting substrates in yeast and
mammalian cell extracts including several species that interact with RNA. We
treated normal and hypomethylated Saccharomyces cerevisiae and RAT1 cell
extracts with RNase prior to in vitro methylation by recombinant protein
N-arginine methyltransferases and found that the methylation of certain
polypeptides is enhanced up to 12-fold whereas that of others is diminished. 2-D
gel electrophoresis of RNase-treated yeast extracts allowed us to tentatively
identify the glycine- and arginine-rich (GAR) domain-containing proteins Gar1,
Nop1, Sbp1, and Npl3 as major methyl-acceptors based on their known isoelectric
points and apparent molecular weights. These results suggest that the
methylation and RNA-binding of GAR domain-containing proteins in vivo may
regulate protein-nucleic acid or protein-protein interactions. Copyright 1999
Academic Press.
PMID: 10362520 [PubMed - indexed for MEDLINE]
725: J Biol Chem 1999 Jun 11;274(24):17219-25
Modulation of human heat shock factor trimerization by the linker domain.
Liu PC, Thiele DJ.
Department of Biological Chemistry, University of Michigan Medical School, Ann
Arbor, Michigan 48109-0606, USA. dthiele@umich.edu
Heat shock transcription factors (HSFs) are stress-responsive proteins that
activate the expression of heat shock genes and are highly conserved from
bakers' yeast to humans. Under basal conditions, the human HSF1 protein is
maintained as an inactive monomer through intramolecular interactions between
two coiled-coil domains and interactions with heat shock proteins; upon
environmental, pharmacological, or physiological stress, HSF1 is converted to a
homotrimer that binds to its cognate DNA binding site with high affinity. To
dissect regions of HSF1 that make important contributions to the stability of
the monomer under unstressed conditions, we have used functional complementation
in bakers' yeast as a facile assay system. Whereas wild-type human HSF1 is
restrained as an inactive monomer in yeast that is unable to substitute for the
essential yeast HSF protein, mutations in the linker region between the DNA
binding domain and the first coiled-coil allow HSF1 to homotrimerize and rescue
the viability defect of a hsfDelta strain. Fine mapping by functional analysis
of HSF1-HSF2 chimeras and point mutagenesis revealed that a small region in the
amino-terminal portion of the HSF1 linker is required for maintenance of HSF1 in
the monomeric state in both yeast and in transfected human 293 cells. Although
linker regions in transcription factors are known to modulate DNA binding
specificity, our studies suggest that the human HSF1 linker plays no role in
determining HSF1 binding preferences in vivo but is a critical determinant in
regulating the HSF1 monomer-trimer equilibrium.
PMID: 10358080 [PubMed - indexed for MEDLINE]
726: J Biol Chem 1999 Jun 11;274(24):17080-7
A two-hybrid dual bait system to discriminate specificity of protein
interactions.
Serebriiskii I, Khazak V, Golemis EA.
Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania
19111, USA.
Biological regulatory systems require the specific organization of proteins into
multicomponent complexes. Two hybrid systems have been used to identify novel
components of signaling networks based on interactions with defined partner
proteins. An important issue in the use of two-hybrid systems has been the
degree to which interacting proteins distinguish their biological partner from
evolutionarily conserved related proteins and the degree to which observed
interactions are specific. We adapted the basic two-hybrid strategy to create a
novel dual bait system designed to allow single-step screening of libraries for
proteins that interact with protein 1 of interest, fused to DNA binding domain A
(LexA), but do not interact with protein 2, fused to DNA binding domain B
(lambda cI). Using the selective interactions of Ras and Krev-1(Rap1A) with Raf,
RalGDS, and Krit1 as a model, we systematically compared LexA- and cI-fused
baits and reporters. The LexA and cI baitr reporter systems are well matched for
level of bait expression and sensitivity range for interaction detection and
allow effective isolation of specifically interacting protein pairs against a
nonspecific background. These reagents should prove useful to refine the
selectivity of library screens, to reduce the isolation of false positives in
such screens, and to perform directed analyses of sequence elements governing
the interaction of a single protein with multiple partners.
PMID: 10358061 [PubMed - indexed for MEDLINE]
727: J Biol Chem 1999 Jun 11;274(24):16861-70
The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating the
apical-isotropic switch and nuclear division in yeast.
Richman TJ, Sawyer MM, Johnson DI.
Department of Microbiology and Molecular Genetics and the Markey Center for
Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA.
The Cdc42p GTPase is involved in the signal transduction cascades controlling
bud emergence and polarized cell growth in S. cerevisiae. Cells expressing the
cdc42(V44A) effector domain mutant allele displayed morphological defects of
highly elongated and multielongated budded cells indicative of a defect in the
apical-isotropic switch in bud growth. In addition, these cells contained one,
two, or multiple nuclei indicative of a G2/M delay in nuclear division and also
a defect in cytokinesis and/or cell separation. Actin and chitin were
delocalized, and septin ring structure was aberrant and partially delocalized to
the tips of elongated cdc42(V44A) cells; however, Cdc42(V44A)p localization was
normal. Two-hybrid protein analyses showed that the V44A mutation interfered
with Cdc42p's interactions with Cla4p, a p21(Cdc42/Rac)-activated kinase
(PAK)-like kinase, and the novel effectors Gic1p and Gic2p, but not with the
Ste20p or Skm1p PAK-like kinases, the Bni1p formin, or the Iqg1p IQGAP homolog.
Furthermore, the cdc42(V44A) morphological defects were suppressed by deletion
of the Swe1p cyclin-dependent kinase inhibitory kinase and by overexpression of
Cla4p, Ste20p, the Cdc12 septin protein, or the guanine nucleotide exchange
factor Cdc24p. In sum, these results suggest that proper Cdc42p function is
essential for timely progression through the apical-isotropic switch and G2/M
transition and that Cdc42(V44A)p differentially interacts with a number of
effectors and regulators.
PMID: 10358031 [PubMed - indexed for MEDLINE]
728: J Biol Chem 1999 Jun 11;274(24):16747-53
Homo- and heterodimerization of synapsins.
Hosaka M, Sudhof TC.
Center for Basic Neuroscience and Department of Molecular Genetics, Howard
Hughes Medical Institute, The University of Texas Southwestern Medical School,
Dallas, Texas 75235, USA.
In vertebrates, synapsins constitute a family of synaptic vesicle proteins
encoded by three genes. Synapsins contain a central ATP-binding domain, the
C-domain, that is highly homologous between synapsins and evolutionarily
conserved in invertebrates. The crystal structure of the C-domain from synapsin
I revealed that it constitutes a large (>300 amino acids), independently folded
domain that forms a tight dimer with or without bound ATP. We now show that the
C-domains of all synapsins form homodimers, and that in addition, C-domains from
different synapsins associate into heterodimers. This conclusion is based on
four findings: 1) in yeast two-hybrid screens with full-length synapsin IIa as a
bait, the most frequently isolated prey cDNAs encoded the C-domain of synapsins;
2) quantitative yeast two-hybrid protein-protein binding assays demonstrated
pairwise strong interactions between all synapsins; 3) immunoprecipitations from
transfected COS cells confirmed that synapsin II heteromultimerizes with
synapsins I and III in intact cells, and similar results were obtained with
bacterial expression systems; and 4) quantification of the synapsin III level in
synapsin I/II double knockout mice showed that the level of synapsin III is
decreased by 50%, indicating that heteromultimerization of synapsin III with
synapsins I or II occurs in vivo and is required for protein stabilization.
These data suggest that synapsins coat the surface of synaptic vesicles as homo-
and heterodimers in which the C-domains of the various subunits have distinct
regulatory properties and are flanked by variable C-terminal sequences. The data
also imply that synapsin III does not compensate for the loss of synapsins I and
II in the double knockout mice.
PMID: 10358015 [PubMed - indexed for MEDLINE]
729: EMBO J 1999 Jun 1;18(11):3139-52
Post-termination ribosome interactions with the 5'UTR modulate yeast mRNA
stability.
Vilela C, Ramirez CV, Linz B, Rodrigues-Pousada C, McCarthy JE.
Post-transcriptional Control Group, Department of Biomolecular Sciences,
University of Manchester Institute of Science and Technology (UMIST), PO Box 88,
Manchester M60 1QD, UK.
A novel form of post-transcriptional control is described. The 5' untranslated
region (5'UTR) of the Saccharomyces cerevisiae gene encoding the AP1-like
transcription factor Yap2 contains two upstream open reading frames (uORF1 and
uORF2). The YAP2-type of uORF functions as a cis-acting element that attenuates
gene expression at the level of mRNA turnover via termination-dependent decay.
Release of post-termination ribosomes from the YAP2 5'UTR causes accelerated
decay which is largely independent of the termination modulator gene UPF1. Both
of the YAP2 uORFs contribute to the destabilization effect. A G/C-rich stop
codon context, which seems to promote ribosome release, allows an uORF to act as
a transferable 5'UTR-destabilizing element. Moreover, termination-dependent
destabilization is potentiated by stable secondary structure 3' of the uORF stop
codon. The potentiation of uORF-mediated destabilization is eliminated if the
secondary structure is located further downstream of the uORF, and is also
influenced by a modulatory mechanism involving eIF2. Destabilization is
therefore linked to the kinetics of acquisition of reinitiation-competence by
post-termination ribosomes in the 5'UTR. Our data explain the destabilizing
properties of YAP2-type uORFs and also support a more general model for the mode
of action of other known uORFs, such as those in the GCN4 mRNA.
PMID: 10357825 [PubMed - indexed for MEDLINE]
730: Scand J Immunol 1999 Jun;49(6):620-8
Protein-protein interactions between native Ro52 and immunoglobulin G heavy
chain.
Yang Y, Eversole T, Lee DJ, Sontheimer RD, Capra JD.
Department of Internal Medicine, Pulmonary and Critical Care Division, UT
Southwestern Medical Center, 5323 Harry Hines Blvd. Dallas, TX 75235-9034, USA.
Using a yeast two-hybrid system to search for proteins interacting with Ro52
autoantigen, we identified a novel protein-protein interaction. Two different
cDNA clones, which interacted with Ro52 in the yeast two-hybrid system, were
identified and isolated from a human B-cell library. Surprisingly, both clones
encoded the heavy chain of human IgG1. The expression of both HIS3 and
beta-galactosidase reporter genes in yeast suggested that the interaction
between Ro52 and IgG occurred in vivo. In vitro studies utilizing recombinant
Ro52 and purified immunoglobulins indicated that the interaction was
immunoglobulin class and subclass specific. Ro52 interacted with IgG1 and IgG4,
but not with IgG2, IgG3, IgA or IgM. Ro52 could also precipitate IgG directly
from serum. The identified cDNA clones did not include the variable region of
IgG, which suggested a non-classical interaction independent of antibody
specificity. We further mapped the domain of Ro52 responsible for this
interaction to the C-terminus rfp-like region. In conclusion, our data support
an unusual interaction between native Ro52 and IgG. The potential biological
significance of this unusual protein-protein interaction is discussed.
PMID: 10354373 [PubMed - indexed for MEDLINE]
731: J Cell Biol 1999 May 31;145(5):933-50
The unstable F-box protein p58-Ctf13 forms the structural core of the CBF3
kinetochore complex.
Russell ID, Grancell AS, Sorger PK.
Massachusetts Institute of Technology, Department of Biology, Cambridge,
Massachusetts 02139, USA.
Kinetochores are smaller and more accessible experimentally in budding yeast
than in any other eukaryote. Believing that simple and complex kinetochores have
important structural and functional properties in common, we characterized the
structure of CBF3, the essential centromere-binding complex that initiates
kinetochore formation in Saccharomyces cerevisiae. We find that the four
subunits of CBF3 are multimeric in solution: p23(Skp1) and p58(Ctf13) form a
heterodimer, and p64(Cep3) and p110(Ndc10) form homodimers. Subcomplexes
involving p58 and each of the other CBF3 subunits can assemble in the absence of
centromeric DNA. In these subcomplexes, p58 appears to function as a structural
core mediating stable interactions among other CBF3 proteins. p58 has a short
half-life in yeast, being subject to ubiquitin-dependent proteolysis, but we
find that it is much more stable following association with p64. We propose that
p23(Skp1)-p58-p64 complexes constitute the primary pool of active p58 in yeast
cells. These complexes can either dissociate, reexposing p58 to the degradation
pathway, or can bind to p110 and centromeric DNA, forming a functional CBF3
complex in which p58 is fully protected from degradation. This pathway may
constitute an editing mechanism preventing the formation of ectopic kinetochores
and ensuring the fidelity of chromosome segregation.
PMID: 10352012 [PubMed - indexed for MEDLINE]
732: Methods Enzymol 1999;303:422-50
Screening for protein-protein interactions.
Germino FJ, Moskowitz NK.
University of Medical Dentistry of New Jersey, Clinical Institute of New Jersey,
New Brunswick 08901, USA.
The assays described above can be used to screen for cellular proteins that can
interact with a protein of interest, to screen for mutant proteins that retain
the ability to bind to its partner, and to identify the domains and amino acids
involved in known protein-protein interactions. Nonetheless, the biological
significance of some of these interactions needs to be confirmed by appropriate
cellular studies.
PMID: 10349658 [PubMed - indexed for MEDLINE]
733: J Biol Chem 1999 Jun 4;274(23):16508-12
Species barrier to RNA recognition overcome with nonspecific RNA binding
domains.
Wang CC, Schimmel P.
The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La
Jolla, California 92037, USA.
We show here that nonspecific RNA-protein interactions can significantly enhance
the biological activity of an essential RNA. protein complex. Bacterial
glutaminyl-tRNA synthetase poorly aminoacylates yeast tRNA and, as a
consequence, cannot rescue a knockout allele of the gene for the yeast
homologue. In contrast to the bacterial protein, the yeast enzyme has an extra
appended domain at the N terminus. Previously, we showed that fusion of this
yeast-specific domain to the bacterial protein enabled it to function as a yeast
enzyme in vivo and in vitro. We suggested that the novel yeast-specific domain
contributed to RNA interactions in a way that compensated for the poor fit
between the yeast tRNA and bacterial enzyme. Here we establish that the novel
appended domain by itself binds nonspecifically to different RNA structures. In
addition, we show that fusion of an unrelated yeast protein, Arc1p, to the
bacterial enzyme also converts it into a functional yeast enzyme in vivo and in
vitro. A small C-terminal segment of Arc1p is necessary and sufficient for this
conversion. This segment was shown by others to have nonspecific tRNA binding
properties. Thus, nonspecific RNA binding interactions in general can compensate
for barriers to formation of a specific and essential RNA.protein complex.
PMID: 10347214 [PubMed - indexed for MEDLINE]
734: J Biol Chem 1999 Jun 4;274(23):16363-9
Structure/function of the beta-barrel domain of F1-ATPase in the yeast
Saccharomyces cerevisiae.
Bakhtiari N, Lai-Zhang J, Yao B, Mueller DM.
Department of Biochemistry and Molecular Biology, Chicago Medical School, North
Chicago, Illinois 60064, USA.
The first 90 amino acids of the alpha- and beta-subunits of mitochondrial
F1-ATPase are folded into beta-barrel domains and were postulated to be
important for stabilizing the enzyme (Abrahams, J. P., Leslie, A. G., Lutter,
R., and Walker, J. E. (1994) Nature 370, 621-628). The role of the domains was
studied by making chimeric enzymes, replacing the domains from the yeast
Saccharomyces cerevisiae enzyme with the corresponding domains from the enzyme
of the thermophilic bacterium Bacillus PS3. The enzymes containing the chimeric
alpha-, beta-, or alpha- and beta-subunits were not functional. However,
gain-of-function mutations were obtained from the strain containing the enzyme
with the chimeric PS3/yeast beta-subunit. The gain-of-function mutations were
all in codons encoding the beta-barrel domain of the beta-subunit, and the
residues appear to map out a region of subunit-subunit interactions.
Gain-of-function mutations were also obtained that provided functional
expression of the chimeric PS3/yeast alpha- and beta-subunits together.
Biochemical analysis of this active chimeric enzyme indicated that it was not
significantly more thermostable or labile than the wild type. The results of
this study indicate that the beta-barrel domains form critical contacts
(distinct from those between the alpha- and beta-subunits) that are important
for the assembly of the ATP synthase.
PMID: 10347195 [PubMed - indexed for MEDLINE]
735: J Biol Chem 1999 Jun 4;274(23):16242-8
Physical and functional interactions of neuronal growth suppressor necdin with
p53.
Taniura H, Matsumoto K, Yoshikawa K.
Division of Regulation of Macromolecular Functions, Institute for Protein
Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
hideo@protein.osaka-u.ac.jp
Necdin is expressed in virtually all postmitotic neurons, and ectopic expression
of this protein suppresses cell proliferation. Necdin, like the retinoblastoma
protein, interacts with cell cycle promoting proteins such as simian virus 40
large T antigen, adenovirus E1A, and the transcription factor E2F1. Here we
demonstrate that necdin interacts with the tumor suppressor protein p53 as well.
The yeast two-hybrid and in vitro binding analyses revealed that necdin bound to
a narrow region (amino acids 35-62) located between the MDM2-binding site and
the proline-rich region in the amino-terminal domain of p53. The electrophoretic
mobility shift assay showed that necdin supershifted a complex between p53 and
its binding DNA, implying that the p53-necdin complex is competent for DNA
binding. In p53-deficient osteosarcoma SAOS-2 cells, necdin markedly suppressed
p53-dependent activation of the p21/WAF promoter. Necdin and p53 inhibited cell
growth in an additive manner as assessed by the colony formation of SAOS-2
cells, suggesting that necdin does not affect p53-mediated growth suppression.
On the other hand, necdin inhibited p53-induced apoptosis of osteosarcoma U2OS
cells. Thus, necdin can be a growth suppressor that targets p53 and modulates
its biological functions in postmitotic neurons.
PMID: 10347180 [PubMed - indexed for MEDLINE]
736: Proc Natl Acad Sci U S A 1999 May 25;96(11):6523-8
Interaction of NPR1 with basic leucine zipper protein transcription factors that
bind sequences required for salicylic acid induction of the PR-1 gene.
Zhang Y, Fan W, Kinkema M, Li X, Dong X.
Developmental, Cell, and Molecular Biology Group, Department of Botany, Box
91000, Duke University, Durham, NC 27708-1000, USA.
The Arabidopsis thaliana NPR1 has been shown to be a key regulator of gene
expression during the onset of a plant disease-resistance response known as
systemic acquired resistance. The npr1 mutant plants fail to respond to systemic
acquired resistance-inducing signals such as salicylic acid (SA), or express
SA-induced pathogenesis-related (PR) genes. Using NPR1 as bait in a yeast
two-hybrid screen, we identified a subclass of transcription factors in the
basic leucine zipper protein family (AHBP-1b and TGA6) and showed that they
interact specifically in yeast and in vitro with NPR1. Point mutations that
abolish the NPR1 function in A. thaliana also impair the interactions between
NPR1 and the transcription factors in the yeast two-hybrid assay. Furthermore, a
gel mobility shift assay showed that the purified transcription factor protein,
AHBP-1b, binds specifically to an SA-responsive promoter element of the A.
thaliana PR-1 gene. These data suggest that NPR1 may regulate PR-1 gene
expression by interacting with a subclass of basic leucine zipper protein
transcription factors.
PMID: 10339621 [PubMed - indexed for MEDLINE]
737: Genes Genet Syst 1998 Dec;73(6):365-75
Isolation and characterization of the yeast las21 mutants, which are sensitive
to a local anestheticum, tetracaine.
Tohe A, Oguchi T.
Department of Biological Sciences, Graduate School of Science, University of
Tokyo, Japan.
We isolated and characterized yeast mutants whose growth is sensitive to a local
anestheticum tetracaine and, at the same time, temperature sensitive. These
mutants were collectively called las mutants (local anestheticum sensitive). The
las21 mutants were analyzed in this study. The wild type LAS21 gene was cloned
by exploiting temperature sensitivity of the las21 mutants and we found that
LAS21 encodes ORF YJL062w which has not been analyzed before. Las21p is putative
membrane protein belonging to the major facilitator super family containing
plural membrane spanning domains. Complete elimination of the LAS21 ORF did not
kill the cells but made their growth temperature sensitive. Interestingly, the
complete loss of the LAS21 gene canceled the sensitivity to tetracaine. The
ability of the las21 mutants to grow at a higher temperature was recovered in
the various media containing an osmotic stabilizer or salts. Furthermore,
temperature sensitivity of the las21 mutants was partially suppressed by
introduction of PKC1, encoding protein kinase C, on a high copy vector. We found
some genetic interactions between LAS21 and Ras/cAMP cascade genes. These
results suggest that LAS21 defines unknown pathway regulating the stress
response of yeast.
PMID: 10333567 [PubMed - indexed for MEDLINE]
738: Hum Mol Genet 1999 Jun;8(6):947-57
Dentatorubral-pallidoluysian atrophy protein interacts through a proline-rich
region near polyglutamine with the SH3 domain of an insulin receptor tyrosine
kinase substrate.
Okamura-Oho Y, Miyashita T, Ohmi K, Yamada M.
Department of Genetics, National Children's Memorial Medical Research Center,
Taishido, Setagaya, Tokyo, Japan.
Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neuro
degrees enerative disorder associated with CAG/glutamine repeat expansion. While
the DRPLA gene is ubiquitously expressed, neuron death occurs in specific
anatomical areas of the brain. This predicts that the DRPLA protein interacts
with other proteins and that these interactions may play a role in pathogenesis.
Here, we describe a protein that binds to the DRPLA product. One of the clones
isolated with a yeast two-hybrid system was identified as a human homolog of the
insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). The gene
produced two mRNA forms by differential splicing and encoded 552 and 521 amino
acids, respectively. The longer form was mainly expressed in the brain and the
shorter one in other tissues. The products were phosphorylated upon stimulation
of cultured cells with insulin or insulin-like growth factor 1. Binding of the
DRPLA protein to IRSp53 was ascertained by co-immunoprecipitation with
antibodies and also by co-localization in perinuclear oval dots in cells
expressing engineered constructs. A proline-rich region near the polyglutamine
tract of the DRPLA protein and the SH3 domain of IRSp53 were involved in the
binding. An extended polyglutamine tract significantly reduced binding ability
in yeast cells, but not in in vitro binding assays. The identification of IRSp53
and other proteins detected by the yeast hybrid system predicts that DRPLA
functions in a signal transduction pathway coupled with insulin/IGF-1.
PMID: 10332026 [PubMed - indexed for MEDLINE]
739: J Cell Biol 1999 May 17;145(4):659-72
LST1 is a SEC24 homologue used for selective export of the plasma membrane
ATPase from the endoplasmic reticulum.
Roberg KJ, Crotwell M, Espenshade P, Gimeno R, Kaiser CA.
Department of Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, USA.
In Saccharomyces cerevisiae, vesicles that carry proteins from the ER to the
Golgi compartment are encapsulated by COPII coat proteins. We identified
mutations in ten genes, designated LST (lethal with sec-thirteen), that were
lethal in combination with the COPII mutation sec13-1. LST1 showed
synthetic-lethal interactions with the complete set of COPII genes, indicating
that LST1 encodes a new COPII function. LST1 codes for a protein similar in
sequence to the COPII subunit Sec24p. Like Sec24p, Lst1p is a peripheral ER
membrane protein that binds to the COPII subunit Sec23p. Chromosomal deletion of
LST1 is not lethal, but inhibits transport of the plasma membrane proton-ATPase
(Pma1p) to the cell surface, causing poor growth on media of low pH.
Localization by both immunofluorescence microscopy and cell fractionation shows
that the export of Pma1p from the ER is impaired in lst1Delta mutants. Transport
of other proteins from the ER was not affected by lst1Delta, nor was Pma1p
transport found to be particularly sensitive to other COPII defects. Together,
these findings suggest that a specialized form of the COPII coat subunit, with
Lst1p in place of Sec24p, is used for the efficient packaging of Pma1p into
vesicles derived from the ER.
PMID: 10330397 [PubMed - indexed for MEDLINE]
740: Mol Cell Biol 1999 Jun;19(6):4414-22
DIX domains of Dvl and axin are necessary for protein interactions and their
ability to regulate beta-catenin stability.
Kishida S, Yamamoto H, Hino S, Ikeda S, Kishida M, Kikuchi A.
Department of Biochemistry, Hiroshima University School of Medicine, Minami-ku,
Hiroshima 734-8551, Japan.
The N-terminal region of Dvl-1 (a mammalian Dishevelled homolog) shares 37%
identity with the C-terminal region of Axin, and this related region is named
the DIX domain. The functions of the DIX domains of Dvl-1 and Axin were
investigated. By yeast two-hybrid screening, the DIX domain of Dvl-1 was found
to interact with Dvl-3, a second mammalian Dishevelled relative. The DIX domains
of Dvl-1 and Dvl-3 directly bound one another. Furthermore, Dvl-1 formed a
homo-oligomer. Axin also formed a homo-oligomer, and its DIX domain was
necessary. The N-terminal region of Dvl-1, including its DIX domain, bound to
Axin directly. Dvl-1 inhibited Axin-promoted glycogen synthase kinase
3beta-dependent phosphorylation of beta-catenin, and the DIX domain of Dvl-1 was
required for this inhibitory activity. Expression of Dvl-1 in L cells induced
the nuclear accumulation of beta-catenin, and deletion of the DIX domain
abolished this activity. Although expression of Axin in SW480 cells caused the
degradation of beta-catenin and reduced the cell growth rate, expression of an
Axin mutant that lacks the DIX domain did not affect the level of beta-catenin
or the growth rate. These results indicate that the DIX domains of Dvl-1 and
Axin are important for protein-protein interactions and that they are necessary
for the ability of Dvl-1 and Axin to regulate the stability of beta-catenin.
PMID: 10330181 [PubMed - indexed for MEDLINE]
741: Mol Cell Biol 1999 Jun;19(6):4324-33
A new class of repression modules is critical for heme regulation of the yeast
transcriptional activator Hap1.
Hach A, Hon T, Zhang L.
Department of Biochemistry, NYU Medical Center, New York, New York 10016, USA.
Heme plays key regulatory roles in numerous molecular and cellular processes for
systems that sense or use oxygen. In the yeast Saccharomyces cerevisiae, oxygen
sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1
contains seven heme-responsive motifs (HRMs): six are clustered in the heme
domain, and a seventh is near the activation domain. To determine the functional
role of HRMs and to define which parts of Hap1 mediate heme regulation, we
carried out a systematic analysis of Hap1 mutants with various regions deleted
or mutated. Strikingly, the data show that HRM1 to -6, located in the previously
designated Hap1 heme domain, have little impact on heme regulation. All seven
HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is
required for Hap1 activation by heme. More importantly, we show that a novel
class of repression modules-RPM1, encompassing residues 245 to 278; RPM2,
encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to
244-is critical for Hap1 repression in the absence of heme. Biochemical analysis
indicates that RPMs mediate Hap1 repression, at least partly, by the formation
of a previously identified higher-order complex termed the high-molecular-weight
complex (HMC), while HRMs mediate heme activation by permitting heme binding and
the disassembly of the HMC. These findings provide significant new insights into
the molecular interactions critical for Hap1 repression in the absence of heme
and Hap1 activation by heme.
PMID: 10330173 [PubMed - indexed for MEDLINE]
742: Mol Cell Biol 1999 Jun;19(6):4167-81
GCD14p, a repressor of GCN4 translation, cooperates with Gcd10p and Lhp1p in the
maturation of initiator methionyl-tRNA in Saccharomyces cerevisiae.
Calvo O, Cuesta R, Anderson J, Gutierrez N, Garcia-Barrio MT, Hinnebusch AG,
Tamame M.
Instituto de Microbiologia Bioquimica del CSIC/Universidad de Salamanca, 37007
Salamanca, Spain.
Gcd10p and Gcd14p were first identified genetically as repressors of GCN4 mRNA
translation in Saccharomyces cerevisiae. Recent findings indicate that Gcd10p
and Gcd14p reside in a nuclear complex required for the presence of
1-methyladenosine in tRNAs. Here we show that Gcd14p is an essential protein
with predicted binding motifs for S-adenosylmethionine, consistent with a direct
function in tRNA methylation. Two different gcd14 mutants exhibit defects in
cell growth and accumulate high levels of initiator methionyl-tRNA (tRNAiMet)
precursors containing 5' and 3' extensions, suggesting a defect in processing of
the primary transcript. Dosage suppressors of gcd10 mutations, encoding tRNAiMet
(hcIMT1 to hcIMT4; hc indicates that the gene is carried on a high-copy-number
plasmid) or a homologue of human La protein implicated in tRNA 3'-end formation
(hcLHP1), also suppressed gcd14 mutations. In fact, the lethality of a GCD14
deletion was suppressed by hcIMT4, indicating that the essential function of
Gcd14p is required for biogenesis of tRNAiMet. A mutation in GCD10 or deletion
of LHP1 exacerbated the defects in cell growth and expression of mature tRNAiMet
in gcd14 mutants, consistent with functional interactions between Gcd14p,
Gcd10p, and Lhp1p in vivo. Surprisingly, the amounts of NME1 and RPR1, the RNA
components of RNases P and MRP, were substantially lower in gcd14 lhp1::LEU2
double mutants than in the corresponding single mutants, whereas 5S rRNA was
present at wild-type levels. Our findings suggest that Gcd14p and Lhp1p
cooperate in the maturation of a subset of RNA polymerase III transcripts.
PMID: 10330157 [PubMed - indexed for MEDLINE]
743: Mol Cell Biol 1999 Jun;19(6):4101-12
Rapamycin antifungal action is mediated via conserved complexes with FKBP12 and
TOR kinase homologs in Cryptococcus neoformans.
Cruz MC, Cavallo LM, Gorlach JM, Cox G, Perfect JR, Cardenas ME, Heitman J.
Departments of Genetics, Duke University Medical Center, Durham, North Carolina
27710, USA.
Cryptococcus neoformans is a fungal pathogen that causes meningitis in patients
immunocompromised by AIDS, chemotherapy, organ transplantation, or high-dose
steroids. Current antifungal drug therapies are limited and suffer from toxic
side effects and drug resistance. Here, we defined the targets and mechanisms of
antifungal action of the immunosuppressant rapamycin in C. neoformans. In the
yeast Saccharomyces cerevisiae and in T cells, rapamycin forms complexes with
the FKBP12 prolyl isomerase that block cell cycle progression by inhibiting the
TOR kinases. We identified the gene encoding a C. neoformans TOR1 homolog. Using
a novel two-hybrid screen for rapamycin-dependent TOR-binding proteins, we
identified the C. neoformans FKBP12 homolog, encoded by the FRR1 gene.
Disruption of the FKBP12 gene conferred rapamycin and FK506 resistance but had
no effect on growth, differentiation, or virulence of C. neoformans. Two
spontaneous mutations that confer rapamycin resistance alter conserved residues
on TOR1 or FKBP12 that are required for FKBP12-rapamycin-TOR1 interactions or
FKBP12 stability. Two other spontaneous mutations result from insertion of novel
DNA sequences into the FKBP12 gene. Our observations reveal that the antifungal
activities of rapamycin and FK506 are mediated via FKBP12 and TOR homologs and
that a high proportion of spontaneous mutants in C. neoformans result from
insertion of novel DNA sequences, and they suggest that nonimmunosuppressive
rapamycin analogs have potential as antifungal agents.
PMID: 10330150 [PubMed - indexed for MEDLINE]
744: J Biol Chem 1999 May 21;274(21):15262-70
Interaction of insulin receptor substrate 3 with insulin receptor, insulin
receptor-related receptor, insulin-like growth factor-1 receptor, and downstream
signaling proteins.
Xu P, Jacobs AR, Taylor SI.
Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892,
USA.
Insulin receptor substrates (IRS) mediate biological actions of insulin, growth
factors, and cytokines. All four mammalian IRS proteins contain pleckstrin
homology (PH) and phosphotyrosine binding (PTB) domains at their N termini.
However, the molecules diverge in their C-terminal sequences. IRS3 is
considerably shorter than IRS1, IRS2, and IRS4, and is predicted to interact
with a distinct group of downstream signaling molecules. In the present study,
we investigated interactions of IRS3 with various signaling molecules. The PTB
domain of mIRS3 is necessary and sufficient for binding to the juxtamembrane
NPXpY motif of the insulin receptor in the yeast two-hybrid system. This
interaction is stronger if the PH domain or the C-terminal phosphorylation
domain is retained in the construct. As determined in a modified yeast
two-hybrid system, mIRS3 bound strongly to the p85 subunit of
phosphatidylinositol 3-kinase. Although high affinity interaction required the
presence of at least two of the four YXXM motifs in mIRS3, there was not a
requirement for specific YXXM motifs. mIRS3 also bound to SHP2, Grb2, Nck, and
Shc, but less strongly than to p85. Studies in COS-7 cells demonstrated that
deletion of either the PH or the PTB domain abolished insulin-stimulated
phosphorylation of mIRS3. Insulin stimulation promoted the association of mIRS3
with p85, SHP2, Nck, and Shc. Despite weak association between mIRS3 and Grb2,
this interaction was not increased by insulin, and may not be mediated by the
SH2 domain of Grb2. Thus, in contrast to other IRS proteins, mIRS3 appears to
have greater specificity for activation of the phosphatidylinositol 3-kinase
pathway rather than the Grb2/Ras pathway.
PMID: 10329736 [PubMed - indexed for MEDLINE]
745: J Biol Chem 1999 May 21;274(21):14685-91
Identification of rabbit reticulocyte E217K as a UBC7 homologue and functional
characterization of its core domain loop.
Lin H, Wing SS.
Department of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada.
The structural basis by which ubiquitin (Ub)-conjugating enzymes (E2s) determine
substrate specificity remains unclear. We cloned rabbit reticulocyte E217K
because unlike the similarly sized class I E2s, E214K and UBC4, it is unable to
support ubiquitin-protein ligase (E3)-dependent conjugation to endogenous
proteins. RNA analysis revealed that this E2 was expressed in all tissues
tested, with higher levels in the testis. Analysis of testis RNA from rats of
different ages showed that E217K mRNA was induced from days 15 to 30. The
predicted amino acid sequence indicates that E217K is a 19. 5-kDa class I E2 but
differs from other class I enzymes in possessing an insertion of 13 amino acids
distal to the active site cysteine. E217K shows 74% amino acid identity with
Saccharomyces cerevisiae UBC7, and therefore, we rename it mammalian UBC7. Yeast
UBC7 crystal structure indicates that this insertion forms a loop out of the
otherwise conserved folding structure. Sequence analysis of E2s had previously
suggested that this loop is a hypervariable region and may play a role in
substrate specificity. We created mutant UBC7 lacking the loop (ubc7Deltaloop)
and a mutant E214k with an inserted loop (E214k+loop) and characterized their
biochemical functions. Ubc7Deltaloop had higher affinity for the E1-Ub thiol
ester than native UBC7 and permitted conjugation of Ub to selected proteins in
the testis but did not permit the broad spectrum E3-dependent conjugation to
endogenous reticulocyte proteins. Surprisingly, E214k+loop was unable to accept
Ub from ubiquitin-activating enzyme (E1) but was able to accept NEDD8 from E1.
E214k+loop was able to support conjugation of NEDD8 to endogenous reticulocyte
proteins but with much lower efficiency than E214k. Thus, the loop can influence
interactions of the E2 with charged E1 as well as with E3s or substrates, but
the exact nature of these interactions depends on divergent sequences in the
remaining conserved core domain.
PMID: 10329663 [PubMed - indexed for MEDLINE]
746: Virology 1999 May 25;258(1):95-9
Potyvirus helper component-proteinase self-interaction in the yeast two-hybrid
system and delineation of the interaction domain involved.
Urcuqui-Inchima S, Walter J, Drugeon G, German-Retana S, Haenni AL, Candresse T,
Bernardi F, Le Gall O.
Institut Jacques Monod, 2 place Jussieu-Tour 43, Paris Cedex 05, 75251, France.
Using the yeast two-hybrid system, a screen was performed for possible
interactions between the proteins encoded by the 5' region of potyviral genomes
[P1, helper component-proteinase (HC-Pro), and P3]. A positive self-interaction
involving HC-Pro was detected with lettuce mosaic virus (LMV) and potato virus Y
(PVY). The possibility of heterologous interaction between the HC-Pro of LMV and
of PVY was also demonstrated. No interaction involving either the P1 or the P3
proteins was detected. A series of ordered deletions from either the N- or
C-terminal end of the LMV HC-Pro was used to map the domain involved in
interaction to the 72 N-terminal amino acids of the protein, a region known to
be dispensable for virus viability but necessary for aphid transmission. A
similar but less detailed analysis mapped the interacting domain to the
N-terminal half of the PVY HC-Pro. Copyright 1999 Academic Press.
PMID: 10329571 [PubMed - indexed for MEDLINE]
747: J Mol Biol 1999 May 7;288(3):337-52
Characterization of KLBCK1, encoding a MAP kinase kinase kinase of Kluyveromyces
lactis.
Jacoby JJ, Kirchrath L, Gengenbacher U, Heinisch JJ.
Institut fur Mikrobiologie, Heinrich-Heine-Universitat Dusseldorf,
Universitatsstr.1 Geb.: 26.12, Dusseldorf, D-40225, FRG.
The cellular integrity and response to hypoosmotic conditions in the yeast
Saccharomyces cerevisiae are ensured by a MAP kinase signal transduction pathway
mediated by the yeast homolog of mammalian protein kinase C. Bck1p functions as
the MAP kinase kinase kinase of this pathway. Here we report on the cloning and
analysis of the BCK1 homolog from the milk yeast Kluyveromyces lactis (KlBCK1).
The deduced protein sequences display three highly conserved domains with the
serine/threonine kinase domain containing 89 % identical amino acid residues.
Interestingly, a region identified in KlBck1p as a putative SAM domain,
mediating protein-protein interactions, is also conserved in ScBck1p. Yet,
two-hybrid analyses indicate that this region may not be involved in
dimerization of KlBck1p in contrast to its S. cerevisiae counterpart. Expression
of KlBCK1 fully complements the defects in a Scbck1 null mutant and is capable
of activating the pathway as indicated by a reporter system based on the
transcription factor Rlm1p. However, deletion from the haploid K. lactis genome
does not result in a loss of cellular integrity under a variety of conditions
tested. Thus, despite the functional conservation in this component of the MAP
kinase pathway in both yeast, cellular integrity in K. lactis may depend at
least in part on different signalling mechanisms when compared with S.
cerevisiae. Copyright 1999 Academic Press.
PMID: 10329146 [PubMed - indexed for MEDLINE]
748: Trends Biochem Sci 1999 Apr;24(4):146-50
Interactions among pathways for phosphatidylcholine metabolism, CTP synthesis
and secretion through the Golgi apparatus.
Kent C, Carman GM.
Dept of Biological Chemistry, University of Michigan Medical School, Ann Arbor,
MI 48109, USA.
Phosphatidylcholine is the major phospholipid in eukaryotic cells. It serves as
a structural component of cell membranes and a reservoir of several lipid
messengers. Recent studies in yeast and mammalian systems have revealed
interrelationships between the two pathways of phosphatidylcholine metabolism,
and between these pathways and those for CTP synthesis and secretion via the
Golgi. These processes involve the regulation of the CDP-choline and
phosphatidylethanolamine-methylation pathways of phosphatidylcholine synthesis,
CTP synthetase, phospholipase D and the phospholipid-transfer protein Sec14p.
Publication Types:
Review
Review, Tutorial
PMID: 10322420 [PubMed - indexed for MEDLINE]
749: J Cell Biochem 1999 Jun 1;73(3):390-9
Human p120ctn catenin: tissue-specific expression of isoforms and molecular
interactions with BP180/type XVII collagen.
Aho S, Rothenberger K, Uitto J.
Department of Dermatology, Jefferson Medical College, and Jefferson Institute of
Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania
19107, USA. aho1@mail.tju.edu
Catenins, a family of structurally related proteins, are involved in epidermal
keratinocyte cell-cell adhesion by interacting through their central Armadillo
repeats with the intracellular domains of cadherins, transmembrane components of
the adhesion junctions. p120ctn is a catenin expressed in different isoforms due
to alternative splicing and multiple translation start sites. BP180 is a
collagenous transmembrane protein (type XVII collagen) localized to
hemidesmosomal attachment complexes in basal keratinocytes. In this study, we
have delineated the molecular interaction between these two proteins utilizing
the yeast two-hybrid system, which was confirmed by an in vitro protein-protein
interaction assay. Specifically, it was shown that an amino-terminal segment of
BP180 (aa. 13-25) contains the information necessary for binding to p120ctn
isoforms 1-3, but not to the isoform 4, suggesting that the interacting domain
is located immediately upstream from the Armadillo repeats and is encoded by
exons 5 and 6, which are subject to alternative splicing only in a minority of
transcripts. In addition to epidermal keratinocytes, p120ctn was shown to be
expressed in a variety of adult and fetal tissues as well as in a number of
human tumors. The expression pattern of various p120ctn transcripts, reflecting
alternative splicing of the 5' exons, was strikingly similar between the
corresponding adult and fetal tissues, while the expression patterns were
discordant between certain tumors and their normal parental tissues, suggesting
a functional role for the tissue-specific expression of the p120ctn isoforms.
Finally, the tissue-specific expression of BP180 was shown to partially overlap
with that of p120ctn, suggesting that the interaction of these two proteins may
contribute to the modulation of cell-cell/matrix interactions in such tissues.
PMID: 10321838 [PubMed - indexed for MEDLINE]
750: Yeast 1999 Apr;15(6):481-96
Analysis of genetic interactions between DHH1, SSD1 and ELM1 indicates their
involvement in cellular morphology determination in Saccharomyces cerevisiae.
Moriya H, Isono K.
Graduate School of Science and Technology, Kobe University, Japan.
The DHH1 gene of Saccharomyces cerevisiae belongs to a family of genes that
encode highly conserved DEAD-box proteins commonly present in various eukaryotic
organisms. Its precise function in yeast has not yet been well documented. To
investigate its role in vivo, we constructed a DHH1 disruptant, characterized it
genetically and searched for genes the mutations in which would cause synthetic
lethality in combination with the DHH1 disruption. CDC28, ELM1 and SSD1 were
thus found to be such candidates and we subsequently analysed their
interactions. Mutations in ELM1 were previously reported to result in the
elongation of cells. We confirmed this phenotype and observed in addition
elongated bud formation in an Elm1p overproducing strain. Also, Elm1p fused with
the green fluorescent protein (GFP) was found to be localized at the bud neck.
These and other observations seem to suggest that Elm1p plays a role during
cytokinesis in S. cerevisiae. The phenotypes of strains harbouring either delta
dhh1 delta elm1 or ssd1-d delta elm1 were very similar to each other, showing
abnormal cellular morphology and defects in cytokinesis and mitosis.
Furthermore, DHH1 and SSD1 could functionally complement each other in the ade2
red colour pigment formation, hypersensitivity to SDS, growth on synthetic media
and at high temperature. A triple mutant, delta dhh1 ssd1-d delta elm1,
apparently had very fragile cell walls and could grow only in a medium
supplemented with 1 M sorbitol.
PMID: 10234786 [PubMed - indexed for MEDLINE]
751: EMBO J 1999 May 4;18(9):2424-34
A Bub2p-dependent spindle checkpoint pathway regulates the Dbf2p kinase in
budding yeast.
Fesquet D, Fitzpatrick PJ, Johnson AL, Kramer KM, Toyn JH, Johnston LH.
Division of Yeast Genetics, National Institute for Medical Research, The
Ridgeway, Mill Hill, London NW7 1AA, UK.
Exit from mitosis in all eukaroytes requires inactivation of the mitotic kinase.
This occurs principally by ubiquitin-mediated proteolysis of the cyclin subunit
controlled by the anaphase-promoting complex (APC). However, an abnormal spindle
and/or unattached kinetochores activates a conserved spindle checkpoint that
blocks APC function. This leads to high mitotic kinase activity and prevents
mitotic exit. DBF2 belongs to a group of budding yeast cell cycle genes that
when mutated prevent cyclin degradation and block exit from mitosis. DBF2
encodes a protein kinase which is cell cycle regulated, peaking in
metaphase-anaphase B/telophase, but its function remains unknown. Here, we show
the Dbf2p kinase activity to be a target of the spindle checkpoint. It is
controlled specifically by Bub2p, one of the checkpoint components that is
conserved in fission yeast and higher eukaroytic cells. Significantly, in
budding yeast, Bub2p shows few genetic or biochemical interactions with other
members of the spindle checkpoint. Our data now point to the protein kinase
Mps1p triggering a new parallel branch of the spindle checkpoint in which Bub2p
blocks Dbf2p function.
PMID: 10228157 [PubMed - indexed for MEDLINE]
752: Anticancer Res 1999 Jan-Feb;19(1A):317-27
Expression, purification and DNA-cleavage activity of recombinant 68-kDa human
topoisomerase I-target for antitumor drugs.
Bronstein IB, Wynne-Jones A, Sukhanova A, Fleury F, Ianoul A, Holden JA, Alix
AJ, Dodson GG, Jardillier JC, Nabiev I, Wilkinson AJ.
Department of Chemistry, University of York, United Kingdom.
igor@yorvic.york.ac.uk
The gene encoding human DNA topoisomerase (topo) I, the target of numerous
anticancer drugs, has been subcloned into bacterial, yeast and baculovirus-based
expression systems in attempts to overexpress the enzyme for extensive
structural and functional characterisation. Expression in E.coli produced a
protein which was not suitable for structural studies. Expression in the yeast
system was more successful enabling the enzyme to be purified and characterised.
However, the resulting yield was modest for our requirements and the full-length
protein was found to be susceptible to proteolysis when expressed in this
system. As it is known that topo I from human placental tissue contains
significant quantities of a 68kDa proteolytic fragment which retains both DNA
relaxation and cleavage activity, we have isolated this fragment and shown by
N-terminal sequence analysis that it starts at Lysine-191. This information was
used to construct vectors which direct the overexpression of this fragment in
baculovirus infected insect cells. The recombinant protein has been purified to
homogeneity in a yield of 5-10mg/l of cell culture. The fragment is stable and
retains all of the DNA driving activities of the intact enzyme. We have
characterised the interactions of the topo I fragment with synthetic DNA
substrates and identified oligonucleotides and conditions that allow covalent
complexes between 68kDa topo I and DNA to be formed with high efficiency and in
large quantity. A flow linear dichroism technique has been further developed and
applied for real-time monitoring of supercoiled (sc) DNA relaxation by the
enzyme and for comparative analysis of inhibition of 68kDa topo I by
camptothecin (CPT).
PMID: 10226561 [PubMed - indexed for MEDLINE]
753: J Cell Biol 1999 May 3;145(3):447-55
Phosphorylation regulates in vivo interaction and molecular targeting of
serine/arginine-rich pre-mRNA splicing factors.
Yeakley JM, Tronchere H, Olesen J, Dyck JA, Wang HY, Fu XD.
Division of Cellular and Molecular Medicine, Department and School of Medicine,
University of California, San Diego, La Jolla, California 92093-0651, USA.
The SR superfamily of splicing factors and regulators is characterized by
arginine/serine (RS)-rich domains, which are extensively modified by
phosphorylation in cells. In vitro binding studies revealed that RS
domain-mediated protein interactions can be differentially affected by
phosphorylation. Taking advantage of the single nonessential SR protein-specific
kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions
in vivo using the two-hybrid assay. Strikingly, all RS domain-mediated
interactions were abolished by SKY1 deletion and were rescuable by yeast or
mammalian SR protein-specific kinases, indicating that phosphorylation has a far
greater impact on RS domain interactions in vivo than in vitro. To understand
this dramatic effect, we examined the localization of SR proteins and found that
SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon
was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may
be differentially regulated by phosphorylation. Using a transcriptional
repression assay, we further showed that most LexA-SR fusion proteins depend on
Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting
that molecular targeting of RS domain-containing proteins within the nucleus was
also affected. Together, these results reveal multiple phosphorylation-dependent
steps for SR proteins to interact with one another efficiently and specifically,
which may ultimately determine the splicing activity and specificity of these
factors in mammalian cells.
PMID: 10225947 [PubMed - indexed for MEDLINE]
754: Genetics 1999 May;152(1):153-66
Genetic and biochemical interactions involving tricarboxylic acid cycle (TCA)
function using a collection of mutants defective in all TCA cycle genes.
Przybyla-Zawislak B, Gadde DM, Ducharme K, McCammon MT.
Department of Biochemistry and Molecular Biology, University of Arkansas for
Medical Sciences, Little Rock, Arkansas 72205, USA.
The eight enzymes of the tricarboxylic acid (TCA) cycle are encoded by at least
15 different nuclear genes in Saccharomyces cerevisiae. We have constructed a
set of yeast strains defective in these genes as part of a comprehensive
analysis of the interactions among the TCA cycle proteins. The 15 major TCA
cycle genes can be sorted into five phenotypic categories on the basis of their
growth on nonfermentable carbon sources. We have previously reported a novel
phenotype associated with mutants defective in the IDH2 gene encoding the Idh2p
subunit of the NAD+-dependent isocitrate dehydrogenase (NAD-IDH). Null and
nonsense idh2 mutants grow poorly on glycerol, but growth can be enhanced by
extragenic mutations, termed glycerol suppressors, in the CIT1 gene encoding the
TCA cycle citrate synthase and in other genes of oxidative metabolism. The TCA
cycle mutant collection was utilized to search for other genes that can suppress
idh2 mutants and to identify TCA cycle genes that display a similar suppressible
growth phenotype on glycerol. Mutations in 7 TCA cycle genes were capable of
functioning as suppressors for growth of idh2 mutants on glycerol. The only
other TCA cycle gene to display the glycerol-suppressor-accumulation phenotype
was IDH1, which encodes the companion Idh1p subunit of NAD-IDH. These results
provide genetic evidence that NAD-IDH plays a unique role in TCA cycle function.
PMID: 10224250 [PubMed - indexed for MEDLINE]
755: Trends Cell Biol 1999 Apr;9(4):150-3
Targeting vesicles to specific sites on the plasma membrane: the role of the
sec6/8 complex.
Hsu SC, Hazuka CD, Foletti DL, Scheller RH.
Howard Hughes Medical Institute and the Dept of Molecular and Cellular
Physiology, Stanford University Medical School, Stanford, CA 94305, USA.
The delivery of secretory vesicles to appropriate docking and fusion sites on
the plasma membrane is crucial for many cellular functions, including formation
of synapses, exocytosis of neurotransmitter, establishment and maintenance of
cell polarity, cell growth and plasma membrane wound healing. Cell-biological,
genetic and biochemical approaches have identified crucial proteins and protein
interactions important for vesicle docking and fusion. However, a description of
the molecular mechanisms underlying vesicle targeting to specific
membrane-fusion sites remains elusive. This review discusses a set of proteins
that might direct vesicles to specific domains of the plasma membrane.
Publication Types:
Review
Review, Tutorial
PMID: 10203793 [PubMed - indexed for MEDLINE]
756: Biochemistry 1999 Apr 27;38(17):5401-11
Assembly of the type 1 procollagen molecule: selectivity of the interactions
between the alpha 1(I)- and alpha 2(I)-carboxyl propeptides.
Alvares K, Siddiqui F, Malone J, Veis A.
Division of Oral Biology, Northwestern University Dental School, Chicago,
Illinois 60611, USA.
Assembly of the heterotrimeric procollagen I molecule is initiated by
interactions between the carboxyl propeptide domains of the completed nascent
pro alpha chains. The [pro alpha 1(I)]2[pro alpha 2(I)] heterotrimer is the
predominant molecule, with much smaller amounts of stable [pro alpha 1(I)]3
homotrimer also being formed. However, the [pro alpha 2(1)]3 homotrimer has not
been detected, raising questions as to the mechanism of chain assembly and why
[pro alpha2(1)]3 homotrimers are not formed. These questions have been examined
here by expressing the intact and amino- or carboxyl-terminal truncated
C-propeptides of the pro alpha chains recombinantly in bacteria and in a coupled
transcription/translation reticulocyte lysate system. Their interactions were
studied in vitro by binding analyses and in vivo by using the yeast two-hybrid
system. The C-pro alpha 1(I) interacted with itself, and with C-pro alpha 2(I),
as expected. Surprisingly, the C-pro alpha 2(I) also interacted with itself,
both in vitro and in vivo. While the interaction of C-pro alpha 2(I) with itself
and C-pro alpha 1(I) in vitro was strong, these interactions were weaker in
vivo. Deletion of 36 amino acids from the C-terminal domain of C-pro alpha 1 had
no effect on its binding to intact self or intact C-pro alpha 2, but the same
deletion in C-pro alpha 2 completely abolished its binding to intact C-pro alpha
2 and to C-pro alpha 1. Comparable N-terminal deletions in C-pro alpha 1 or
C-pro alpha 2 diminished, but did not abolish, their binding to intact C-pro
alpha 1 and C-pro alpha 2. In the yeast two-hybrid system, C-pro alpha 2
interacted with itself more weakly than with C-pro alpha 1. Molecular modeling
and circular dichroism analyses showed that C-pro alpha 1 and C-pro alpha 2 have
different folded structures and stability. Studies with antibodies specific to
the C-pro alpha1 and alpha2 peptides showed them to precipitate different,
specific, and distinct cell proteins from fibroblast lysates. The C-pro alpha
2(I) antibody complexed with more cell proteins. We hypothesize that the lack of
pro alpha 2(I) homotrimers is not due to the inability of the C-pro alpha 2(I)
to interact with itself, but rather to the competing presence of other cell
proteins. The specificity of these interactions may reside in conformational
differences in N- and C-terminal sequences of the two propeptides or in their
different folding patterns.
PMID: 10220327 [PubMed - indexed for MEDLINE]
757: Nucleic Acids Res 1999 May 15;27(10):2181-8
A Saccharomyces cerevisiae RNA 5'-triphosphatase related to mRNA capping enzyme.
Rodriguez CR, Takagi T, Cho EJ, Buratowski S.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, 240 Longwood Avenue, Boston, MA 02115, USA.
The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: the
RNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Using
computer homology searching, a S. cerevisiae gene was identified that encodes a
protein resembling the C-terminal region of Cet1. Accordingly, we designated
this gene CTL1 (capping enzyme RNAtriphosphatase-like 1). CTL1 is not essential
for cell viability and no genetic or physical interactions with the capping
enzyme genes were observed. The protein is found in both the nucleus and
cytoplasm. Recombinant Ctl1 protein releases gamma-phosphate from the 5'-end of
RNA to produce a diphosphate terminus. The enzyme is specific for polynucleotide
RNA in the presence of magnesium, but becomes specific for nucleotide
triphosphates in the presence of manganese. Ctl1 is the second member of the
yeast RNA triphosphatase family, but is probably involved in an RNA processing
event other than mRNA capping.
PMID: 10219091 [PubMed - indexed for MEDLINE]
758: Nucleic Acids Res 1999 May 15;27(10):2165-74
Isolation of Ku70-binding proteins (KUBs).
Yang CR, Yeh S, Leskov K, Odegaard E, Hsu HL, Chang C, Kinsella TJ, Chen DJ,
Boothman DA.
Department of Radiation Oncology and Department of Pharmacology and the Ireland
Cancer Center,Laboratory of Molecular Stress Responses, Case Western Reserve
University, BRB-326 East,10900 Euclid Avenue, Cleveland, OH 44106-4942, USA.
DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA
double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and
DNA ligase IV, other proteins which play a role(s) in this repair pathway remain
unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding
subunit (Ku70 and Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using
yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein
J (apoJ), also known as X-ray-inducible transcript 8 (XIP8),
testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80.
KUB3 and KUB4 are unknown, >10 kb trans-cripts. Interactions of apoJ/XIP8 or
KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8
breast cancer or IMR-90 normal lung fibroblast cells, respectively. The
interaction of apoJ/XIP8 with Ku70 was confirmed by far-western analyses. Stable
over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80
DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role
of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under
investigation.
PMID: 10219089 [PubMed - indexed for MEDLINE]
759: Nucleic Acids Res 1999 May 15;27(10):2126-34
A role for Ctr9p and Paf1p in the regulation G1 cyclin expression in yeast.
Koch C, Wollmann P, Dahl M, Lottspeich F.
Institut fur Genetik der Universitat Munchen, Maria-Ward-Strasse 1a, D-80638
Munchen, Germany. c.koch@lrz.uni-muenchen.de
Entry into the cell cycle in budding yeast involves transcriptional activation
of G1cyclin genes and DNA synthesis genes when cells reach a critical size in
late G1. Expression of G1cyclins CLN1 and CLN2 is regulated by the transcription
factor SBF (composed of Swi4p and Swi6p) and depends on the cyclin-dependent
Cdc28 protein kinase and cyclin Cln3p. To identify novel regulators of
SBF-dependent gene expression we screened for mutants that fail to activate
transcription of G1cyclins. We found mutations in a gene called CTR9. ctr9
mutants are inviable at 37 degrees C and accumulate large cells. CTR9 is
identical to CDP1. CTR9 encodes a conserved nuclear protein of 125 kDa
containing several TPR repeats implicated in protein-protein interactions. We
show that Ctr9p is a component of a high molecular weight protein complex. Using
immuno-affinity chromatography we found that Ctr9p associates with polypeptides
of 50 and 65 kDa. By mass spectrometry these were identified as Cdc73p and
Paf1p. We show that Paf1p, like Ctr9p, is required for efficient CLN2
transcription, whereas Cdc73p is not. Paf1p and Cdc73p were previously reported
to be RNA poly-merase II-associated proteins, suggesting that the Ctr9p complex
may interact with the general transcription apparatus.
PMID: 10219085 [PubMed - indexed for MEDLINE]
760: Nucleic Acids Res 1999 May 15;27(10):2072-9
Formation of the yeast Mre11-Rad50-Xrs2 complex is correlated with DNA repair
and telomere maintenance.
Chamankhah M, Xiao W.
Department of Microbiology and Immunology, University of Saskatchewan, 107
Wiggins Road, Saskatoon, SK S7N 5E5, Canada.
The yeast Mre11 is a multi-functional protein and is known to form a protein
complex with Rad50 and Xrs2. In order to elucidate the relationship between
Mre11 complex formation and its mitotic functions, and to determine domain(s)
required for Mre11 protein interactions, we performed yeast two-hybrid and
functional analyses with respect to Mre11 DNA repair and telomere maintenance.
Evidence presented in this study indicates that the N-terminal region of Mre11
constitutes the core homo-dimerization and hetero-dimerization domain and is
sufficient for Mre11 DNA repair and maintaining the wild-type telomere length.
In contrast, a stretch of 134 amino acids from the extreme C-terminus, although
essential for achieving a full level of self-association, is not required for
the aforementioned Mre11 mitotic functions. Interestingly, deletion of these
same 134 amino acids enhanced the interaction of Mre11 with Rad50 and Xrs2,
which is consistent with the notion that this region is specific for meiotic
functions. While Mre11 self-association alone is insufficient to provide the
above mitotic activities, our results are consistent with a strong correlation
between Mre11-Rad50-Xrs2 complex formation, mitotic DNA repair and telomere
maintenance. This correlation was further strengthened by analyzing two mre11
phosphoesterase motif mutants ( mre11-2 and rad58S ), which are defective in DNA
repair, telomere maintenance and protein interactions, and a rad50S mutant,
which is normal in both complex formation and mitotic functions. Together, these
results support and extend a current model regarding Mre11 structure and
functions in mitosis and meiosis.
PMID: 10219078 [PubMed - indexed for MEDLINE]
761: FEBS Lett 1999 Mar 19;447(1):115-20
The presumed potassium carrier Trk2p in Saccharomyces cerevisiae determines an
H+-dependent, K+-independent current.
Bihler H, Gaber RF, Slayman CL, Bertl A.
Department of Cellular and Molecular Physiology, Yale School of Medicine, New
Haven, CT 06510, USA.
Ionic currents related to the major potassium uptake systems in Saccharomyces
cerevisiae were examined by whole cell patch-clamping, under K+ replete
conditions. Those currents have the following properties. They (1) are inward
under all conditions investigated, (2) arise instantaneously with appropriate
voltage steps, (3) depend solely upon the moderate affinity transporter Trk2p,
not upon the high affinity transporter Trk1p. They (4) appear to be independent
of the extracellular K+ concentration, (5) are also independent of extracellular
Ca2+, Mg2+ and Cl- but (6) are strongly dependent on extracellular pH, being
large at low pH (up to several hundred pA at -200 mV and pH 4) and near zero at
high pH (above 7.5). They (7) increase in proportion to log[H+]o, rather than
directly in proportion to the proton concentration and (8) behave kinetically as
if each transporter cycle moved one proton plus one (high pH) or two (low pH)
other ions, as yet unidentified. In view of background knowledge on K+ transport
related to Trk2p, the new results suggest that the K+ status of yeast cells
modulates both the kinetics of Trk2p-mediated transport and the identity of ions
involved. That modulation could act either on the Trk2 protein itself or on
interactions of Trk2 with other proteins in a hypothetical transporter complex.
Structural considerations suggest a strong analogy to the KtrAB system in Vibrio
alginolyticus and/or the TrkH system in Escherichia coli.
PMID: 10218594 [PubMed - indexed for MEDLINE]
762: J Bacteriol 1999 May;181(9):2963-5
The C-terminal domain of dnaQ contains the polymerase binding site.
Taft-Benz SA, Schaaper RM.
Laboratory of Molecular Genetics, National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina 27709, USA.
The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading
(epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ
mutants has suggested that epsilon might consist of two domains, an N-terminal
domain containing the exonuclease and a C-terminal domain essential for binding
the polymerase (alpha) subunit. We have created truncated forms of dnaQ
resulting in epsilon subunits that contain either the N-terminal or the
C-terminal domain. Using the yeast two-hybrid system, we analyzed the
interactions of the single-domain epsilon subunits with the alpha and theta
subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal
186 amino acids, was defective in binding to the alpha subunit while retaining
normal binding to the theta subunit. In contrast, the NDelta186 protein,
consisting of the C-terminal 57 amino acids, exhibited normal binding to the
alpha subunit but was defective in binding to the theta subunit. A strain
carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as
expected from a defective alpha binding mutant. The data are consistent with the
existence of two functional domains in epsilon, with the C-terminal domain
responsible for polymerase binding.
PMID: 10217794 [PubMed - indexed for MEDLINE]
763: J Biomol Struct Dyn 1999 Feb;16(4):757-74
The effect of queuosine on tRNA structure and function.
Morris RC, Brown KG, Elliott MS.
Department of Biochemistry and Chemistry, Old Dominion University, Norfolk, VA
23529, USA.
Computational modeling was performed to determine the potential function of the
queuosine modification of tRNA found in wobble position 34 of tRNAasp, tRNAasn,
tRNAhis, and tRNAtyr. Using the crystal structure of tRNAasp and a
tRNA-tRNA-mRNA complex model, we show that the queuosine modification serves as
a structurally restrictive base for tRNA anticodon loop flexibility. An extended
intraresidue and intramolecular hydrogen bonding network is established by
queuosine. The quaternary amine of the 7-aminomethyl side chain hydrogen bonds
with the base's carbonyl oxygen. This positions the dihydroxycyclopentenediol
ring of queuosine in proper orientation for hydrogen bonding with the backbone
of the neighboring uridine 33 residue. The interresidue association stabilizes
the formation of a cross-loop hydrogen bond between the uridine 33 base and the
phosphoribosyl backbone of the cytosine at position 36. Additional interactions
between RNAs in the translation complex were studied with regard to potential
codon context and codon bias effects. Neither steric nor electrostatic
interaction occurs between aminoacyl- and peptidyl-site tRNA anticodon loops
that are modified with queuosine. However, there is a difference in the strength
of anticodon/codon associations (codon bias) based on the presence or lack of
queuosine in the wobble position of the tRNA. Unmodified (guanosine-containing)
tRNAasp forms a very stable association with cytosine (GAC), but is much less
stable in complex with a uridine-containing codon (GAU). Queuosine-modified
tRNAasp exhibits no bias for either of cognate codons GAC or GAU and
demonstrates a lower binding energy similar to the wobble pairing of
guanosine-containing tRNA with a GAU codon. This is proposed to be due to the
inflexibility of the queuosine-modified anticodon loop to accommodate proper
positioning for optimal Watson-Crick type associations. A preliminary survey of
codon usage patterns in oncodevelopmental versus housekeeping gene transcripts
suggests a significant difference in bias for the queuosine-associated codons.
Therefore, the queuosine modification may have the potential to influence
cellular growth and differentiation by codon bias-based regulation of protein
synthesis for discrete mRNA transcripts.
PMID: 10217448 [PubMed - indexed for MEDLINE]
764: Biochimie 1999 Jan-Feb;81(1-2):87-105
The contribution of homologous recombination in preserving genome integrity in
mammalian cells.
Thompson LH, Schild D.
Biology and Biotechnology Research Program, Lawrence Livermore National
Laboratory, Livermore, CA 94551-0808, USA.
Although it is clear that mammalian somatic cells possess the enzymatic
machinery to perform homologous recombination of DNA molecules, the importance
of this process in mitigating DNA damage has been uncertain. An initial genetic
framework for studying homologous recombinational repair (HRR) has come from
identifying relevant genes by homology or by their ability to correct mutants
whose phenotypes are suggestive of recombinational defects. While yeast has been
an invaluable guide, higher eukaryotes diverge in the details and complexity of
HRR. For eliminating DSBs, HRR and end-joining pathways share the burden, with
HRR contributing critically during S and G2 phases. It is likely that the
removal of interstrand cross-links is absolutely dependent on efficient HRR, as
suggested by the extraordinary sensitivity of the ercc1, xpf/ercc4, xrcc2, and
xrcc3 mutants to cross-linking chemicals. Similarly, chromosome stability in
untreated cells requires intact HRR, which may eliminate DSBs arising during DNA
replication and thereby prevent chromosome aberrations. Complex regulation of
HRR by cell cycle checkpoint and surveillance functions is suggested not only by
direct interactions between human Rad51 and p53, c-Abl, and BRCA2, but also by
very high recombination rates in p53-deficient cells.
Publication Types:
Review
Review, Academic
PMID: 10214914 [PubMed - indexed for MEDLINE]
765: J Biol Chem 1999 Apr 30;274(18):12480-7
Genetic analysis and enzyme activity suggest the existence of more than one
minimal functional unit capable of synthesizing phosphoribosyl pyrophosphate in
Saccharomyces cerevisiae.
Hernando Y, Carter AT, Parr A, Hove-Jensen B, Schweizer M.
Genetics and Microbiology Department, Institute of Food Research, Norwich
Research Park, Colney, Norwich NR4 7UA, United Kingdom.
The PRS gene family in Saccharomyces cerevisiae consists of five genes each
capable of encoding a 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase
polypeptide. To gain insight into the functional organization of this gene
family we have constructed a collection of strains containing all possible
combinations of disruptions in the five PRS genes. Phenotypically these deletant
strains can be classified into three groups: (i) a lethal phenotype that
corresponds to strains containing a double disruption in PRS2 and PRS4 in
combination with a disruption in either PRS1 or PRS3; simultaneous deletion of
PRS1 and PRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second
phenotype that is encountered in strains containing disruptions in PRS1 and PRS3
together or in combination with any of the other PRS genes manifests itself as a
reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third
phenotype that corresponds to strains that, although affected in their
phosphoribosyl pyrophosphate-synthesizing ability, are unimpaired for growth and
have nucleotide profiles virtually the same as the wild type. Deletions of PRS2,
PRS4, and PRS5 or combinations thereof cause this phenotype. These results
suggest that the polypeptides encoded by the members of the PRS gene family may
be organized into two functional entities. Evidence that these polypeptides
interact with each other in vivo was obtained using the yeast two-hybrid system.
Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and
there are significant interactions between the PRS5 polypeptide and either the
PRS2 or PRS4 polypeptides. These data suggest that yeast phosphoribosyl
pyrophosphate synthetase exists in vivo as multimeric complex(es).
PMID: 10212224 [PubMed - indexed for MEDLINE]
766: Curr Biol 1999 Mar 25;9(6):325-8
Mouse Rad54 affects DNA conformation and DNA-damage-induced Rad51 foci
formation.
Tan TL, Essers J, Citterio E, Swagemakers SM, de Wit J, Benson FE, Hoeijmakers
JH, Kanaar R.
Department of Cell Biology and Genetics, Erasmus University, Rotterdam, PO Box
1738, 3000 DR Rotterdam, The Netherlands.
Error-free repair by homologous recombination of DNA double-strand breaks
induced by ionizing radiation (IR) requires the Rad52 group proteins, including
Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a
'joint' molecule between the damaged DNA and the homologous repair template is a
key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4]
[5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6].
Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci
that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced
by genotoxic stress, but only when lesions that required mRad54 for their repair
were formed. Interestingly, mRad54 was essential for the formation of IR-induced
mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which
modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a
topological assay suggested that purified human Rad54 (hRad54) protein can
unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of
the homologous repair template could promote the formation or stabilization of
hRad51-mediated joint molecules. Rad54 appears to be required downstream of
other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that
assist Rad51 in interacting with the broken DNA [2] [3] [4].
PMID: 10209103 [PubMed - indexed for MEDLINE]
767: Mol Cell Biol 1999 May;19(5):3580-7
Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and
exocytosis, is a GTPase which interacts with Myo2 and Exo70.
Robinson NG, Guo L, Imai J, Toh-E A, Matsui Y, Tamanoi F.
Department of Microbiology and Molecular Genetics, Molecular Biology Institute,
University of California, Los Angeles, California 90095-1489, USA.
The Rho3 protein plays a critical role in the budding yeast Saccharomyces
cerevisiae by directing proper cell growth. Rho3 appears to influence cell
growth by regulating polarized secretion and the actin cytoskeleton, since rho3
mutants exhibit large rounded cells with an aberrant actin cytoskeleton. To gain
insights into how Rho3 influences these events, we have carried out a yeast
two-hybrid screen using an S. cerevisiae cDNA library to identify proteins
interacting with Rho3. Two proteins, Exo70 and Myo2, were identified in this
screen. Interactions with these two proteins are greatly reduced or abolished
when mutations are introduced into the Rho3 effector domain. In addition, a type
of mutation known to produce dominant negative mutants of Rho proteins abolished
the interaction with both of these proteins. In contrast, Rho3 did not interact
with protein kinase C (Pkc1), an effector of another Rho family protein, Rho1,
nor did Rho1 interact with Exo70 or Myo2. Rho3 did interact with Bni1, another
effector of Rho1, but less efficiently than with Rho1. The interaction between
Rho3 and Exo70 and between Rho3 and Myo2 was also demonstrated with purified
proteins. The interaction between Exo70 and Rho3 in vitro was dependent on the
presence of GTP, since Rho3 complexed with guanosine 5'-O-(3-thiotriphosphate)
interacted more efficiently with Exo70 than Rho3 complexed with guanosine
5'-O-(3-thiodiphosphate). Overlapping subcellular localization of the Rho3 and
Exo70 proteins was demonstrated by indirect immunofluorescence. In addition,
patterns of localization of both Exo70 and Rho3 were altered when a dominant
active allele of RHO3, RHO3(E129,A131), which causes a morphological
abnormality, was expressed. These results provide a direct molecular basis for
the action of Rho3 on exocytosis and the actin cytoskeleton.
PMID: 10207081 [PubMed - indexed for MEDLINE]
768: EMBO J 1999 Apr 15;18(8):2229-40
Role of the essential yeast protein PSU1 in p6anscriptional enhancement by the
ligand-dependent activation function AF-2 of nuclear receptors.
Gaudon C, Chambon P, Losson R.
Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP,
College de France, BP 163, 67404 Illkirch Cedex, France.
Nuclear receptors (NRs) can function as ligandinducible transregulators in both
mammalian and yeast cells, indicating that important features of transcriptional
control have been conserved throughout evolution. We report here the isolation
and characterization of an essential yeast protein of unknown function, PSU1,
which exhibits properties expected for a co-activator/mediator of the
ligand-dependent activation function AF-2 present in the ligand-binding domain
(LBD, region E) of NRs. PSU1 interacts in a ligand-dependent manner with the LBD
of several NRs, including retinoic acid (RARalpha), retinoid X (RXRalpha),
thyroid hormone (TRalpha), vitamin D3 (VDR) and oestrogen (ERalpha) receptors.
Importantly, both in yeast and in vitro, these interactions require the
integrity of the AF-2 activating domain. When tethered to a heterologous
DNA-binding domain, PSU1 can activate transcription on its own. By using yeast
reporter cells that express PSU1 conditionally, we show that PSU1 is required
for transactivation by the AF-2 of ERalpha. Taken together these data suggest
that in yeast, PSU1 is involved in ligand-dependent transactivation by NRs.
Sequence analysis revealed that in addition to a highly conserved motif found in
a family of MutT-related proteins, PSU1 contains several alpha-helical
leucine-rich motifs sharing the consensus sequence LLxPhiL (x, any amino acid;
Phi, hydrophobic amino acid) in regions that elicit either transactivation or
NR-binding activity.
PMID: 10205176 [PubMed - indexed for MEDLINE]
769: J Immunol 1999 Apr 15;162(8):5019-24
Mutations that cause the Wiskott-Aldrich syndrome impair the interaction of
Wiskott-Aldrich syndrome protein (WASP) with WASP interacting protein.
Stewart DM, Tian L, Nelson DL.
Immunophysiology Section, Metabolism Branch, National Cancer Institute, National
Institutes of Health, Bethesda, MD 20892, USA.
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized
by thrombocytopenia, eczema, immune deficiency, and a proclivity toward lymphoid
malignancy. Lymphocytes of affected individuals show defects of activation,
motility, and cytoskeletal structure. The disease gene encodes a 502-amino acid
protein named the WAS protein (WASP). Studies have identified a number of
important interactions that place WASP in a role of integrating signaling
pathways with cytoskeletal function. We performed a two-hybrid screen to
identify proteins interacting with WASP and cloned a proline-rich protein as a
specific WASP interactor. Our clone of this protein, termed WASP interacting
protein (WIP) by others, shows a difference in seven amino acid residues,
compared with the previously published sequence revealing an additional profilin
binding motif. Deletion mutant analysis reveals that WASP residues 101-151 are
necessary for WASP-WIP interaction. Point mutant analyses in the two-hybrid
system and in vitro show impairment of WASP-WIP interaction with three WASP
missense mutants known to cause WAS. We conclude that impaired WASP-WIP
interaction may contribute to WAS.
PMID: 10202051 [PubMed - indexed for MEDLINE]
770: Nucleic Acids Res 1999 May 1;27(9):1978-84
The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G-G paired DNAs.
Sun H, Bennett RJ, Maizels N.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven,
CT 06520-8114, USA.
The Saccharomyces cerevisiae Sgs1p helicase localizes to the nucleolus and is
required to maintain the integrity of the rDNA repeats. Sgs1p is a member of the
RecQ DNA helicase family, which also includes Schizo-saccharomyces pombe Rqh1,
and the human BLM and WRN genes. These genes encode proteins which are essential
to maintenance of genomic integrity and which share a highly conserved helicase
domain. Here we show that recombinant Sgs1p helicase efficiently unwinds
guanine-guanine (G-G) paired DNA. Unwinding of G-G paired DNA is ATP- and
Mg2+-dependent and requires a short 3' single-stranded tail. Strikingly, Sgs1p
unwinds G-G paired substrates more efficiently than duplex DNAs, as measured
either in direct assays or by competition experiments. Sgs1p efficiently unwinds
G-G paired telomeric sequences, suggesting that one function of Sgs1p may be to
prevent telomere-telomere interactions which can lead to chromosome
non-disjunction. The rDNA is G-rich and has considerable potential for G-G
pairing. Diminished ability to unwind G-G paired regions may also explain the
deleterious effect of mutation of Sgs1 on rDNA stability, and the accelerated
aging characteristic of yeast strains that lack Sgs1 as well as humans deficient
in the related WRN helicase.
PMID: 10198430 [PubMed - indexed for MEDLINE]
771: Genetics 1999 Apr;151(4):1459-70
Dna2 mutants reveal interactions with Dna polymerase alpha and Ctf4, a Pol alpha
accessory factor, and show that full Dna2 helicase activity is not essential for
growth.
Formosa T, Nittis T.
Department of Biochemistry, University of Utah School of Medicine, Salt Lake
City, Utah 84132, USA. formosa@medschool.med.utah.edu
Mutations in the gene for the conserved, essential nuclease-helicase Dna2 from
the yeast Saccharomyces cerevisiae were found to interact genetically with POL1
and CTF4, which encode a DNA Polymerase alpha subunit and an associated protein,
suggesting that Dna2 acts in a process that involves Pol alpha. DNA2 alleles
were isolated that cause either temperature sensitivity, sensitivity to
alkylation damage, or both. The alkylation-sensitive alleles clustered in the
helicase domain, including changes in residues required for helicase activity in
related proteins. Additional mutations known or expected to destroy the ATPase
and helicase activities of Dna2 were constructed and found to support growth on
some media but to cause alkylation sensitivity. Only damage-sensitive alleles
were lethal in combination with a ctf4 deletion. Full activity of the Dna2
helicase function is therefore not needed for viability, but is required for
repairing damage and for tolerating loss of Ctf4. Arrest of dna2 mutants was
RAD9 dependent, but deleting this checkpoint resulted in either no effect or
suppression of defects, including the synthetic lethality with ctf4. Dna2
therefore appears to act in repair or lagging strand synthesis together with Pol
alpha and Ctf4, in a role that is optimal with, but does not require, full
helicase activity.
PMID: 10101169 [PubMed - indexed for MEDLINE]
772: Genetics 1999 Apr;151(4):1353-63
Suppression of a nuclear aep2 mutation in Saccharomyces cerevisiae by a base
substitution in the 5'-untranslated region of the mitochondrial oli1 gene
encoding subunit 9 of ATP synthase.
Ellis TP, Lukins HB, Nagley P, Corner BE.
Department of Biochemistry and Molecular Biology, Monash University, Clayton,
Victoria 3168, Australia.
Mutations in the nuclear AEP2 gene of Saccharomyces generate greatly reduced
levels of the mature form of mitochondrial oli1 mRNA, encoding subunit 9 of
mitochondrial ATP synthase. A series of mutants was isolated in which the
temperature-sensitive phenotype resulting from the aep2-ts1 mutation was
suppressed. Three strains were classified as containing a mitochondrial
suppressor: these lost the ability to suppress aep2-ts1 when their mitochondrial
genome was replaced with wild-type mitochondrial DNA (mtDNA). Many other
isolates were classified as containing dominant nuclear suppressors. The three
mitochondrion-encoded suppressors were localized to the oli1 region of mtDNA
using rho- genetic mapping techniques coupled with PCR analysis; DNA sequencing
revealed, in each case, a T-to-C nucleotide transition in mtDNA 16 nucleotides
upstream of the oli1 reading frame. It is inferred that the suppressing mutation
in the 5' untranslated region of oli1 mRNA restores subunit 9 biosynthesis by
accommodating the modified structure of Aep2p generated by the aep2-ts1 mutation
(shown here to cause the substitution of proline for leucine at residue 413 of
Aep2p). This mode of mitochondrial suppression is contrasted with that mediated
by heteroplasmic rearranged rho- mtDNA genomes bypassing the participation of a
nuclear gene product in expression of a particular mitochondrial gene. In the
present study, direct RNA-protein interactions are likely to form the basis of
suppression.
PMID: 10101162 [PubMed - indexed for MEDLINE]
773: Genetics 1999 Apr;151(4):1273-85
Analysis of mutations in the yeast mRNA decapping enzyme.
Tharun S, Parker R.
Departments of Molecular and Cellular Biology and Biochemistry and the Howard
Hughes Medical Institute, University of Arizona, Tucson, Arizona 85721-0106,
USA.
A major mechanism of mRNA decay in yeast is initiated by deadenylation, followed
by mRNA decapping, which exposes the transcript to 5' to 3' exonucleolytic
degradation. The decapping enzyme that removes the 5' cap structure is encoded
by the DCP1 gene. To understand the function of the decapping enzyme, we used
alanine scanning mutagenesis to create 31 mutant versions of the enzyme, and we
examined the effects of the mutations both in vivo and in vitro. Two types of
mutations that affected mRNA decapping in vivo were identified, including a
temperature-sensitive allele. First, two mutants produced decapping enzymes that
were defective for decapping in vitro, suggesting that these mutated residues
are required for enzymatic activity. In contrast, several mutants that
moderately affected mRNA decapping in vivo yielded decapping enzymes that had at
least the same specific activity as the wild-type enzyme in vitro. Combination
of alleles within this group yielded decapping enzymes that showed a strong loss
of function in vivo, but that still produced fully active enzymes in vitro. This
suggested that interactions of the decapping enzyme with other factors may be
required for efficient decapping in vivo, and that these particular mutations
may be disrupting such interactions. Interestingly, partial loss of decapping
activity in vivo led to a defect in normal deadenylation-dependent decapping,
but it did not affect the rapid deadenylation-independent decapping triggered by
early nonsense codons. This observation suggested that these two types of mRNA
decapping differ in their requirements for the decapping enzyme.
PMID: 10101156 [PubMed - indexed for MEDLINE]
774: Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3572-7
Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix
protein.
Jiang J, Zhang Y, Krainer AR, Xu RM.
W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY 11724, USA.
Human p32 (also known as SF2-associated p32, p32/TAP, and gC1qR) is a conserved
eukaryotic protein that localizes predominantly in the mitochondrial matrix. It
is thought to be involved in mitochondrial oxidative phosphorylation and in
nucleus-mitochondrion interactions. We report the crystal structure of p32
determined at 2.25 A resolution. The structure reveals that p32 adopts a novel
fold with seven consecutive antiparallel beta-strands flanked by one N-terminal
and two C-terminal alpha-helices. Three monomers form a doughnut-shaped
quaternary structure with an unusually asymmetric charge distribution on the
surface. The implications of the structure on previously proposed functions of
p32 are discussed and new specific functional properties are suggested.
PMID: 10097078 [PubMed - indexed for MEDLINE]
775: Gene 1999 Mar 18;229(1-2):183-91
Translation elongation factor 2 is encoded by a single essential gene in Candida
albicans.
Mendoza A, Serramia MJ, Capa L, Garcia-Bustos JF.
Research Department, Glaxo Wellcome, S.A., Severo Ochoa 2, E-28760, Tres Cantos,
Spain.
Translation elongation factor 2 (eEF2) is a large protein of more than 800 amino
acids which establishes complex interactions with the ribosome in order to
catalyze the conformational changes needed for translation elongation. Unlike
other yeasts, the pathogenic fungus Candida albicans was found to have a single
gene encoding this factor per haploid genome, located on chromosome 2.
Expression of this locus is essential for vegetative growth, as evidenced by
placing it under the control of a repressible promoter. This C. albicans gene,
named EFT2, was cloned and sequenced (EMBL accession number Y09664). Genomic and
cDNA sequence analysis identified common transcription initiation and
termination signals and an 842 amino acid open reading frame (ORF), which is
interrupted by a single intron. Despite some genetic differences, CaEFT2 was
capable of complementing a Saccharomyces cerevisiae Deltaeft1 Deltaeft2 null
mutant, which lacks endogenous eEF2, indicating that CaEFT2 can be expressed
from its own promoter and its intron can be correctly spliced in S. cerevisiae.
PMID: 10095118 [PubMed - indexed for MEDLINE]
776: Eur J Biochem 1999 Feb;259(3):939-45
Site-directed mutagenesis of proline 204 in the 'hinge' region of yeast
phosphoglycerate kinase.
McHarg J, Kelly SM, Price NC, Cooper A, Littlechild JA.
School of Chemistry, University of Exeter, UK.
Site-specific mutants have been produced in order to investigate the role of
proline 204 in the 'hinge' region of yeast phosphoglycerate kinase (PGK). This
totally conserved proline has been shown to be the only cis-proline in the high
resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T.
maritima PGK, and may therefore have a role in the independent folding of the
two domains or in the 'hinge' bending of the molecule during catalysis. The
residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe),
and the resulting proteins characterised by differential scanning calorimetry
(DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic
analysis. Although the secondary and tertiary structure of the Pro204His protein
is generally similar to that of the wild-type enzyme as assessed by CD, the
enzyme is less stable to heat and guanidinium chloride denaturation than the
wild-type. In the denaturation experiments two transitions were observed for
both the wild-type and the Pro204His mutant, as have been previously reported
for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990)
Biochemistry 29, 8683-8689]. The first transition is accompanied by an increase
in fluorescence intensity leading to a hyperfluorescent state, followed by the
second, corresponding to a decrease in fluorescence intensity. However, for the
Pro204His mutant, the first transition proceeded at lower concentrations of
guanidinium chloride and the second transition proceeded to the same extent as
for the wild-type protein, suggesting that sequence-distant interactions are
more rapidly disrupted in this mutant enzyme than in the wild-type enzyme, while
sequence-local interactions are disrupted in a similar way. The Michaelis
constants (K(m)) for both 3-phospho-D-glycerate and ATP are increased only by
three or fourfold, which confirms that, as expected, the substrate binding sites
are largely unaffected by the mutation. However, the turnover and efficiency of
the Pro204His mutant is severely impaired, indicating that the mechanism of
'hinge' bending is hindered. The Pro204Phe enzyme was shown to be significantly
less well folded than the wild-type and Pro204His enzymes, with considerable
loss of both secondary and tertiary structure. It is proposed that the proline
residue at 204 in the 'hinge' region of PGK plays a role in the stability and
catalytic mechanism of the enzyme.
PMID: 10092885 [PubMed - indexed for MEDLINE]
777: J Biol Chem 1999 Apr 2;274(14):9455-62
Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA
splicing.
Tsai WY, Chow YT, Chen HR, Huang KT, Hong RI, Jan SP, Kuo NY, Tsao TY, Chen CH,
Cheng SC.
Institute of Microbiology and Immunology, National Yang-Ming University
Shih-Pai, Taiwan, Republic of China.
The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential
splicing factor and is associated with the spliceosome during the splicing
reaction. We have previously shown that Prp19p is not tightly associated with
small nuclear ribonucleoprotein particles but is associated with a protein
complex consisting of at least eight protein components. By sequencing
components of the affinity-purified complex, we have identified Cef1p as a
component of the Prp19p-associated complex, Ntc85p. Cef1p could directly
interact with Prp19p and was required for pre-mRNA splicing both in vivo and in
vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required
for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p
self-interaction. We have identified a small region of 30 amino acid residues
near the carboxyl terminus required for both cell viability and protein-protein
interactions. Cef1p was associated with the spliceosome in the same manner as
Prp19p, i.e. concomitant with or immediately after dissociation of U4. The
anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and
at least three other components of the Prp19p-associated complex, suggesting
that the Prp19p-associated complex is likely associated with the spliceosome and
functions as an integral complex.
PMID: 10092627 [PubMed - indexed for MEDLINE]
778: J Gen Virol 1999 Mar;80 ( Pt 3):607-15
A cellular protein which binds hepatitis B virus but not hepatitis B surface
antigen.
Harvey TJ, Macnaughton TB, Park DS, Gowans EJ.
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston,
Queensland, Australia.
The envelope of hepatitis B virus (HBV) consists of three related proteins known
as the large (L), middle (M) and small (S) hepatitis B surface antigens (HBsAg).
L-HBsAg has a 108-119 amino acid extension at the N terminus compared with
M-HBsAg and contains the preS1 sequence of the HBV envelope. Previous research
has identified this region as the likely virus attachment protein which is
thought to interact with the cellular receptor for the virus. However, as the
receptor has still not been identified unequivocally, we used the preS1 region
of L-HBsAg to screen a human liver cDNA library by the yeast two-hybrid system.
Several positive clones were isolated which encoded cellular proteins that
interacted with the HBV preS1 protein. The specificity was examined in an
independent manner in experiments in which baculovirus-derived glutathione
S-transferase (GST)-preS1 was incubated with 35S-labelled protein expressed by
in vitro translation from the positive clones. The intensity of the interactions
using this alternative approach mirrored those observed in the yeast two-hybrid
system and two proteins (an unidentified protein and a mitochondrial protein)
were selected for further study. The specificity of the binding reaction between
the preS1 protein and these two proteins was further confirmed in a competition
assay; HBV purified from serum, but not purified HBsAg, was able to compete with
preS1 and thus block GST-preS1 binding to the unidentified protein but not to
the mitochondrial protein. The unidentified protein was then expressed as a
fusion protein with GST and this was able to bind HBV virions in a direct
manner.
PMID: 10091999 [PubMed - indexed for MEDLINE]
779: Genes Dev 1999 Mar 15;13(6):686-97
Characterization of the imitation switch subfamily of ATP-dependent
chromatin-remodeling factors in Saccharomyces cerevisiae.
Tsukiyama T, Palmer J, Landel CC, Shiloach J, Wu C.
Laboratory of Molecular Cell Biology, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland 20892, USA. ttsukiya@fhcrc.org
We have identified and characterized two Imitation Switch genes in Saccharomyces
cerevisiae, ISW1 and ISW2, which are highly related to Drosophila ISWI, encoding
the putative ATPase subunit of three ATP-dependent chromatin remodeling factors.
Purification of ISW1p reveals a four-subunit complex with nucleosome-stimulated
ATPase activity, as well as ATP-dependent nucleosome disruption and spacing
activities. Purification of ISW2p reveals a two-subunit complex also with
nucleosome-stimulated ATPase and ATP-dependent nucleosome spacing activities but
no detectable nucleosome disruption activity. Null mutations of ISW1, ISW2, and
CHD1 genes cause synthetic lethality in various stress conditions in yeast
cells, revealing the first in vivo functions of the ISWI subfamily of
chromatin-remodeling complexes and demonstrating their genetic interactions. A
single point mutation within the ATPase domain of both ISW1p and ISW2p
inactivated all ATP-dependent biochemical activities of the complexes, as well
as the ability of the genes to rescue the mutant phenotypes. This demonstrates
that the ATP-dependent chromatin-remodeling activities are essential for the in
vivo functions of both ISW1 and ISW2 complexes.
PMID: 10090725 [PubMed - indexed for MEDLINE]
780: J Cell Biol 1999 Mar 22;144(6):1187-202
A Cdc24p-Far1p-Gbetagamma protein complex required for yeast orientation during
mating.
Nern A, Arkowitz RA.
Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, CB2
2QH, United Kingdom.
Oriented cell growth requires the specification of a site for polarized growth
and subsequent orientation of the cytoskeleton towards this site. During mating,
haploid Saccharomyces cerevisiae cells orient their growth in response to a
pheromone gradient overriding an internal landmark for polarized growth, the bud
site. This response requires Cdc24p, Far1p, and a heterotrimeric G-protein. Here
we show that a two- hybrid interaction between Cdc24p and Gbeta requires Far1p
but not pheromone-dependent MAP-kinase signaling, indicating Far1p has a role in
regulating the association of Cdc24p and Gbeta. Binding experiments demonstrate
that Cdc24p, Far1p, and Gbeta form a complex in which pairwise interactions can
occur in the absence of the third protein. Cdc24p localizes to sites of
polarized growth suggesting that this complex is localized. In the absence of
CDC24-FAR1-mediated chemotropism, a bud site selection protein, Bud1p/Rsr1p, is
essential for morphological changes in response to pheromone. These results
suggest that formation of a Cdc24p-Far1p-Gbetagamma complex functions as a
landmark for orientation of the cytoskeleton during growth towards an external
signal.
PMID: 10087263 [PubMed - indexed for MEDLINE]
781: J Cell Biol 1999 Mar 8;144(5):963-75
The cortical localization of the microtubule orientation protein, Kar9p, is
dependent upon actin and proteins required for polarization.
Miller RK, Matheos D, Rose MD.
Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University,
Princeton, New Jersey 08544, USA.
In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle
requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical
protein that is required for the correct position of the mitotic spindle and the
orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)-
Kar9p localizes to a single spot at the tip of the growing bud and the mating
projection. However, the cortical localization of Kar9p does not require
microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377),
suggesting that Kar9p interacts with other proteins at the cortex. To
investigate Kar9p's cortical interactions, we treated cells with the
actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells,
Kar9p's cortical localization was completely dependent on polymerized actin.
Kar9p localization was also altered by mutations in four genes, spa2Delta,
pea2Delta, bud6Delta, and bni1Delta, required for normal polarization and actin
cytoskeleton functions and, of these, bni1Delta affected Kar9p localization most
severely. Like kar9Delta, bni1Delta mutants exhibited nuclear positioning
defects during mitosis and in shmoos. Furthermore, like kar9Delta, the bni1Delta
mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic
analysis placed BNI1 in the KAR9 pathway for nuclear migration. However,
analysis of kar9Delta bni1Delta double mutants suggested that Kar9p retained
some function in bni1Delta mitotic cells. Unlike the polarization mutants,
kar9Delta shmoos had a normal morphology and diploids budded in the correct
bipolar pattern. Furthermore, Bni1p localized normally in kar9Delta. We conclude
that Kar9p's function is specific for cytoplasmic microtubule orientation and
that Kar9p's role in nuclear positioning is to coordinate the interactions
between the actin and microtubule networks.
PMID: 10085294 [PubMed - indexed for MEDLINE]
782: J Cell Biol 1999 Mar 8;144(5):947-61
Control of mitotic spindle position by the Saccharomyces cerevisiae formin
Bni1p.
Lee L, Klee SK, Evangelista M, Boone C, Pellman D.
Department of Pediatric Oncology, The Dana-Farber Cancer Institute and
Department of Pediatric Hematology, The Children's Hospital, Harvard Medical
School, Boston, Massachusetts 02115, USA.
Alignment of the mitotic spindle with the axis of cell division is an essential
process in Saccharomyces cerevisiae that is mediated by interactions between
cytoplasmic microtubules and the cell cortex. We found that a cortical protein,
the yeast formin Bni1p, was required for spindle orientation. Two striking
abnormalities were observed in bni1Delta cells. First, the initial movement of
the spindle pole body (SPB) toward the emerging bud was defective. This
phenotype is similar to that previously observed in cells lacking the kinesin
Kip3p and, in fact, BNI1 and KIP3 were found to be in the same genetic pathway.
Second, abnormal pulling interactions between microtubules and the cortex
appeared to cause preanaphase spindles in bni1Delta cells to transit back and
forth between the mother and the bud. We therefore propose that Bni1p may
localize or alter the function of cortical microtubule-binding sites in the bud.
Additionally, we present evidence that other bipolar bud site determinants
together with cortical actin are also required for spindle orientation.
PMID: 10085293 [PubMed - indexed for MEDLINE]
783: Mol Cell Biol 1999 Apr;19(4):2967-76
An activator binding module of yeast RNA polymerase II holoenzyme.
Lee YC, Park JM, Min S, Han SJ, Kim YJ.
Center for Molecular Medicine, Samsung Biomedical Research Institute,
Sungkyunkwan University College of Medicine, Kangnam-ku, Seoul 135-230, Korea.
The Mediator complex of Saccharomyces cerevisiae is required for both general
and regulated transcription of RNA polymerase II (PolII) and is composed of two
stable subcomplexes (Srb4 and Rgr1 subcomplexes). To decipher the function of
each Mediator subcomplex and to delineate the functional relationship between
the subcomplexes, we characterized the compositions and biochemical activities
of PolII-Mediator complexes (holoenzymes) prepared from several Mediator mutant
strains of S. cerevisiae. We found that holoenzymes devoid of a functional Gal11
module were defective for activated but not basal transcription in a
reconstituted in vitro system. This activation-specific defect was correlated
with a crippled physical interaction to transcriptional activator proteins,
which could be bypassed by artificial recruitment of a mutant holoenzyme to a
promoter. Consistent with this observation, a direct interaction between Gal11
and gene-specific transcriptional activator proteins was detected by far-Western
analyses and column binding assays. In contrast, the srb5 deletion mutant
holoenzyme was defective for both basal and activated transcription, despite its
capacity for activator binding that is comparable to that of the wild-type
holoenzyme. These results demonstrate that the Gal11 module of the Rgr1
subcomplex is required for the efficient recruitment of PolII holoenzyme to a
promoter via activator-specific interactions, while the Srb4 subcomplex
functions in the modulation of general polymerase activity.
PMID: 10082564 [PubMed - indexed for MEDLINE]
784: Proc Natl Acad Sci U S A 1999 Mar 16;96(6):2656-61
The fission yeast homologue of Orc4p binds to replication origin DNA via
multiple AT-hooks.
Chuang RY, Kelly TJ.
Department of Molecular Biology and Genetics, Johns Hopkins University School of
Medicine, Baltimore, MD 21210, USA.
The origin recognition complex (ORC) was originally identified in the yeast
Saccharomyces cerevisiae as a protein that specifically binds to origins of DNA
replication. Although ORC appears to play an essential role in the initiation of
DNA replication in the cells of all eukaryotes, its interactions with DNA have
not been defined in species other than budding yeast. We have characterized a
Schizosaccharomyces pombe homologue of the ORC subunit, Orc4p. The homologue
(Orp4p) consists of two distinct functional domains. The C-terminal domain shows
strong sequence similarity to human, frog, and yeast Orc4 proteins, including
conserved ATP-binding motifs. The N-terminal domain contains nine copies of the
AT-hook motif found in a number of DNA-binding proteins, including the members
of the HMG-I(Y) family of chromatin proteins. AT-hook motifs are known from
biochemical and structural studies to mediate binding to the minor groove of
AT-tracts in DNA. Orp4p is essential for viability of Sc. pombe and is expressed
throughout the cell cycle. The Orp4 protein (and its isolated N-terminal domain)
binds to the Sc. pombe replication origin, ars1. The DNA binding properties of
Orp4p provide a plausible explanation for the characteristic features of Sc.
pombe origins of replication, which differ significantly from those of Sa.
cerevisiae.
PMID: 10077566 [PubMed - indexed for MEDLINE]
785: EMBO J 1999 Mar 15;18(6):1621-9
A trans-acting peptide activates the yeast a1 repressor by raising its
DNA-binding affinity.
Stark MR, Escher D, Johnson AD.
Department of Biochemistry and Biophysics, School of Medicine, University of
California, San Francisco, CA 94143-0414, USA.
The cooperative binding of gene regulatory proteins to DNA is a common feature
of transcriptional control in both prokaryotes and eukaryotes. It is generally
viewed as a simple energy coupling, through protein-protein interactions, of two
or more DNA-binding proteins. In this paper, we show that the simple view does
not account for the cooperative DNA binding of a1 and alpha2, two homeodomain
proteins from budding yeast. Rather, we show through the use of chimeric
proteins and synthetic peptides that, upon heterodimerization, alpha2 instructs
a1 to bind DNA. This change is induced by contact with a peptide contributed by
alpha2, and this contact converts a1 from a weak to a strong DNA-binding
protein. This explains, in part, how high DNA-binding specificity is achieved
only when the two gene regulatory proteins conjoin. We also provide evidence
that features of the a1-alpha2 interaction can serve as a model for other
examples of protein-protein interactions, including that between the herpes
virus transcriptional activator VP16 and the mammalian homeodomain-containing
protein Oct-l.
PMID: 10075932 [PubMed - indexed for MEDLINE]
786: Biotechnol Appl Biochem 1999 Apr;29 ( Pt 2):165-84
Mutational analysis of sickle haemoglobin (Hb) gelation.
Li X, Himanen JP, Martin de Llano JJ, Padovan JC, Chait BT, Manning JM.
Department of Biology, Mugar 414, Northeastern University, 360 Huntington
Avenue, Boston, MA 02115, USA.
The use of recombinant Hb has provided the advantage that any amino acid
substitution can be made at sites not represented by natural mutants or that
cannot be modified by chemical procedures. We have recently reported the
expression of human sickle Hb (HbS) in the yeast Saccharomyces cerevisiae that
carries a plasmid containing the human alpha- and beta-globin cDNA sequences;
N-terminal nascent protein processing is correct and a soluble correctly folded
Hb tetramer is produced. The yeast system produces a recombinant sickle Hb that
is identical by about a dozen biochemical and physiological criteria with the
natural sickle Hb purified from the red cells of sickle-cell anaemia patients.
Most importantly, the gelling concentration of this recombinant sickle Hb is the
same as that of the HbS purified from human sickle red cells. The misfolding of
Hb reported for the Escherichia coli-expressed protein is not apparent for Hb
expressed in yeast by any of the criteria that we have used for
characterization. These findings indicate that this system is well suited to the
production of HbS mutants to explore those areas of the HbS tetramer whose roles
in the gelation process are not yet defined and to measure quantitatively the
strength of such interactions at certain inter-tetrameric contact sites in the
deoxy-HbS aggregate. This article reviews our studies on a number of sickle Hb
mutants, including polymerization-enhancing HbS mutants and
polymerization-inhibiting HbS mutants.
PMID: 10075913 [PubMed - indexed for MEDLINE]
787: Methods 1999 Jan;17(1):28-37
Linking mRNA turnover and translation: assessing the polyribosomal association
of mRNA decay factors and degradative intermediates.
Mangus DA, Jacobson A.
Department of Molecular Genetics and Microbiology, University of Massachusetts
Medical School, Worcester, Massachusetts 01655-0122, USA.
mRNA decay is a multistep process, often dependent on the active translation of
an mRNA and on components of the translation apparatus. In Saccharomyces
cerevisiae, several trans-acting factors required for mRNA decay associate with
polyribosomes. We have explored the specificity of the interactions of these
factors with polyribosomes, using sucrose gradient sedimentation analysis of the
yeast UPF1 protein to test whether such interactions are altered when
polyribosomes are disrupted by treatment with EDTA, digestion with micrococcal
nuclease, or shifting of cells containing a temperature-sensitive eIF3 mutation
to the nonpermissive temperature. These experiments, as well as others assaying
the strength of factor association in high-salt sucrose gradients, lead us to
conclude that Upf1p is tightly bound to the smallest polyribosomes, but not to
the 40S or 60S ribosomal subunits. Similar experimental approaches were used to
determine whether mRNA decay initiates prior to mRNA release from polyribosomes.
Using sucrose gradient fractionation and Northern blotting, we can detect the
polysomal association of a PGK1 mRNA decay intermediate and conclude that mRNA
decay commences while an mRNA is still being translated. Copyright 1999 Academic
Press.
PMID: 10075880 [PubMed - indexed for MEDLINE]
788: J Virol 1999 Apr;73(4):2622-32
A brome mosaic virus intergenic RNA3 replication signal functions with viral
replication protein 1a to dramatically stabilize RNA in vivo.
Sullivan ML, Ahlquist P.
Institute for Molecular Virology, University of Wisconsin-Madison, Madison,
Wisconsin 53706, USA.
Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like
superfamily, encodes two RNA replication proteins. The 1a protein has putative
helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain.
Saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a BMV
RNA3 template produced by in vivo transcription of a DNA copy of RNA3. Although
insufficient for RNA3 replication, the expression of 1a protein alone results in
a dramatic and specific stabilization of the RNA3 template in yeast. As one step
toward understanding 1a-induced stabilization of RNA3, the interactions
involved, and its possible relation to RNA replication, we have identified the
cis-acting sequences required for this effect. We find that 1a-induced
stabilization is mediated by a 150- to 190-base segment of the RNA3 intergenic
region corresponding to a previously identified enhancer of RNA3 replication.
Moreover, this segment is sufficient to confer 1a-induced stability on a
heterologous beta-globin RNA. Within this intergenic segment, partial deletions
that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in
parallel decreases in the levels of negative- and positive-strand RNA3
replication products in yeast expressing 1a and 2a. In particular, a small
deletion encompassing a motif corresponding to the box B element of RNA
polymerase III promoters dramatically reduced the ability of RNAs to respond to
1a or 1a and 2a. These and other findings suggest that 1a-induced stabilization
likely reflects an early template selection step in BMV RNA replication.
PMID: 10074107 [PubMed - indexed for MEDLINE]
789: Genes Dev 1999 Mar 1;13(5):569-80
Interaction of the U1 snRNP with nonconserved intronic sequences affects 5'
splice site selection.
Puig O, Gottschalk A, Fabrizio P, Seraphin B.
Gene Expression Program, European Molecular Biology Laboratory, D-69117
Heidelberg, Germany.
Intron definition and splice site selection occur at an early stage during
assembly of the spliceosome, the complex mediating pre-mRNA splicing.
Association of U1 snRNP with the pre-mRNA is required for these early steps. We
report here that the yeast U1 snRNP-specific protein Nam8p is a component of the
commitment complexes, the first stable complexes assembled on pre-mRNA. In vitro
and in vivo, Nam8p becomes indispensable for efficient 5' splice site
recognition when this process is impaired as a result of the presence of
noncanonical 5' splice sites or the absence of a cap structure. Nam8p stabilizes
commitment complexes in the latter conditions. Consistent with this, Nam8p
interacts with the pre-mRNA downstream of the 5' splice site, in a region of
nonconserved sequence. Substitutions in this region affect splicing efficiency
and alternative splice site choice in a Nam8p-dependent manner. Therefore, Nam8p
is involved in a novel mechanism by which a snRNP component can affect splice
site choice and regulate intron removal through its interaction with a
nonconserved sequence. This supports a model where early 5' splice recognition
results from a network of interactions established by the splicing machinery
with various regions of the pre-mRNA.
PMID: 10072385 [PubMed - indexed for MEDLINE]
790: Genes Dev 1999 Mar 1;13(5):545-55
Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase
Glc7p.
Sassoon I, Severin FF, Andrews PD, Taba MR, Kaplan KB, Ashford AJ, Stark MJ,
Sorger PK, Hyman AA.
Cell Biology Program, European Molecular Biology Laboratory, 69117 Heidelberg,
Germany.
We have investigated the role of protein phosphorylation in regulation of
Saccharomyces cerevisiae kinetochores. By use of phosphatase inhibitors and a
type 1 protein phosphatase mutant (glc7-10), we show that the microtubule
binding activity, but not the centromeric DNA-binding activity, of the
kinetochore complex is regulated by a balance between a protein kinase and the
type 1 protein phosphatase (PP1) encoded by the GLC7 gene. glc7-10 mutant cells
exhibit low kinetochore-microtubule binding activity in vitro and a high
frequency of chromosome loss in vivo. Specifically, the Ndc10p component of the
centromere DNA-binding CBF3 complex is altered by the glc7-10 mutation; Ndc10p
is hyperphosphorylated in glc7-10 extracts. Furthermore, addition of recombinant
Ndc10p reconstitutes the microtubule-binding activity of a glc7-10 extract to
wild-type levels. Finally, the glc7-10-induced mitotic arrest is abolished in
spindle checkpoint mutants, suggesting that defects in kinetochore-microtubule
interactions caused by hyperphosphorylation of kinetochore proteins activate the
spindle checkpoint.
PMID: 10072383 [PubMed - indexed for MEDLINE]
791: Mol Gen Genet 1999 Feb;261(1):80-91
Functional implications of genetic interactions between genes encoding small
GTPases involved in vesicular transport in yeast.
Yoo JS, Grabowski R, Xing L, Trepte HH, Schmitt HD, Gallwitz D.
Department of Molecular Genetics, Max Planck Institute for Biophysical
Chemistry, Gottingen, Germany.
Ras-related, guanine nucleotide-binding proteins of the Ypt/Rab family play a
key role at defined steps in vesicular transport, both in yeast and in mammalian
cells. In yeast, Ypt1p has an essential function late in endoplasmic reticulum
(ER) to Golgi transport, and the redundant Ypt31/Ypt32 GTPases have been
proposed to act in transport through and/or from the Golgi. Here we report that
mutant alleles of YPT31 and YPT32, whose gene products have a reduced affinity
for GTP, are able to suppress the dominant lethal phenotype of YPT1(N121I).
Co-expression of YPT1(N121I) and the suppressor YPT31(N126I) allow essentially
undisturbed secretory transport in the absence of the respective wild-type
GTPases. Such mutant cells massively overaccumulate 60-100 nm vesicles and are
heat sensitive. It appears likely that the mutant GTPases, which are defective
in nucleotide binding, compete for the binding of common interacting protein(s).
These and other genetic interactions between YPT1, YPT31/32, ARF1 and SEC4
described here strongly support the view that Ypt31p and Ypt32p have a central,
Golgi-associated function in anterograde or retrograde transport.
PMID: 10071213 [PubMed - indexed for MEDLINE]
792: Mol Biol Cell 1999 Mar;10(3):609-26
Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during
nuclear fusion in Saccharomyces cerevisiae.
Brizzio V, Khalfan W, Huddler D, Beh CT, Andersen SS, Latterich M, Rose MD.
Department of Molecular Biology, Princeton University, Princeton, New Jersey
08544-1014, USA.
During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single
diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by
mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to
SEC71, a gene involved in protein translocation into the endoplasmic reticulum.
Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate
karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not
sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated
temperatures. Genetic interactions between kar7 and kar5 mutations were
suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p
was absent from the SPB and was not detected by Western blot or
immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding
an endoplasmic reticulum resident DnaJ protein required for nuclear fusion.
Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating
defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear
fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear
fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of
double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the
existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p,
and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.
PMID: 10069807 [PubMed - indexed for MEDLINE]
793: Microbiol Mol Biol Rev 1999 Mar;63(1):54-105
Cdc42: An essential Rho-type GTPase controlling eukaryotic cell polarity.
Johnson DI.
Department of Microbiology & Molecular Genetics and the Markey Center for
Molecular Genetics, University of Vermont, Burlington, Vermont 05405,
USA.dijohnso@zoo.uvm.edu
Cdc42p is an essential GTPase that belongs to the Rho/Rac subfamily of Ras-like
GTPases. These proteins act as molecular switches by responding to exogenous
and/or endogenous signals and relaying those signals to activate downstream
components of a biological pathway. The 11 current members of the Cdc42p family
display between 75 and 100% amino acid identity and are functional as well as
structural homologs. Cdc42p transduces signals to the actin cytoskeleton to
initiate and maintain polarized gorwth and to mitogen-activated protein
morphogenesis. In the budding yeast Saccharomyces cerevisiae, Cdc42p plays an
important role in multiple actin-dependent morphogenetic events such as bud
emergence, mating-projection formation, and pseudohyphal growth. In mammalian
cells, Cdc42p regulates a variety of actin-dependent events and induces the
JNK/SAPK protein kinase cascade, which leads to the activation of transcription
factors within the nucleus. Cdc42p mediates these processes through interactions
with a myriad of downstream effectors, whose number and regulation we are just
starting to understand. In addition, Cdc42p has been implicated in a number of
human diseases through interactions with its regulators and downstream
effectors. While much is known about Cdc42p structure and functional
interactions, little is known about the mechanism(s) by which it transduces
signals within the cell. Future research should focus on this question as well
as on the detailed analysis of the interactions of Cdc42p with its regulators
and downstream effectors.
Publication Types:
Review
Review, Tutorial
PMID: 10066831 [PubMed - indexed for MEDLINE]
794: J Biol Chem 1999 Mar 12;274(11):7576-82
Identification of determinants in E2 ubiquitin-conjugating enzymes required for
hect E3 ubiquitin-protein ligase interaction.
Nuber U, Scheffner M.
Deutsches Krebsforschungszentrum, Angewandte Tumorvirologie, Im Neuenheimer Feld
242, 69120 Heidelberg, Germany.
Members of the hect domain protein family are characterized by sequence
similarity of their C-terminal regions to the C terminus of E6-AP, an E3
ubiquitin-protein ligase. An essential intermediate step in E6-AP-dependent
ubiquitination is the formation of a thioester complex between E6-AP and
ubiquitin in the presence of distinct E2 ubiquitin-conjugating enzymes including
human UbcH5, a member of the UBC4/UBC5 subfamily of E2s. Similarly, several hect
domain proteins, including Saccharomyces cerevisiae RSP5, form ubiquitin
thioester complexes, indicating that hect domain proteins in general have E3
activity. We show here, by the use of chimeric E2s generated between UbcH5 and
other E2s, that a region of UbcH5 encompassing the catalytic site cysteine
residue is critical for its ability to interact with E6-AP and RSP5. Of
particular importance is a phenylalanine residue at position 62 of UbcH5 that is
conserved among the members of the UBC4/UBC5 subfamily but is not present in any
of the other known E2s, whereas the N-terminal 60 amino acids do not contribute
significantly to the specificity of these interactions. The conservation of this
phenylalanine residue throughout evolution underlines the importance of the
ability to interact with hect domain proteins for the cellular function of
UBC4/UBC5 subfamily members.
PMID: 10066826 [PubMed - indexed for MEDLINE]
795: Curr Opin Microbiol 1998 Apr;1(2):197-203
Protein chaperones and the heat shock response in Saccharomyces cerevisiae.
Morano KA, Liu PC, Thiele DJ.
Department of Biological Chemistry, University of Michigan Medical School, 1301
Catherine Road, Ann Arbor, MI 48109-0606, USA. kmorano@umich.edu
Recent studies have shed new light on the complexities of the heat shock
response in yeast. Multiple pathways for transcriptional induction of both
classic and novel heat shock proteins are emerging together with a more detailed
understanding of the interactions between protein chaperones and their
physiological targets. New roles for heat shock proteins in defense and recovery
from the impacts of thermal stress on critical cellular processes have expanded
our understanding of these elaborate and ubiquitous proteins.
Publication Types:
Review
Review, Tutorial
PMID: 10066474 [PubMed - indexed for MEDLINE]
796: EMBO J 1999 Mar 1;18(5):1137-45
Physical interactions among circadian clock proteins KaiA, KaiB and KaiC in
cyanobacteria.
Iwasaki H, Taniguchi Y, Ishiura M, Kondo T.
Division of Biological Science, Graduate School of Science, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
The kai gene cluster, which is composed of three genes, kaiA, kaiB and kaiC, is
essential for the generation of circadian rhythms in the unicellular
cyanobacterium Synechococcus sp. strain PCC 7942. Here we demonstrate the direct
association of KaiA, KaiB and KaiC in yeast cells using the two-hybrid system,
in vitro and in cyanobacterial cells. KaiC enhanced KaiA-KaiB interaction in
vitro and in yeast cells, suggesting that the three Kai proteins were able to
form a heteromultimeric complex. We also found that a long period mutation kaiA1
dramatically enhanced KaiA-KaiB interaction in vitro. Thus, direct
protein-protein association among the Kai proteins may be a critical process in
the generation of circadian rhythms in cyanobacteria.
PMID: 10064581 [PubMed - indexed for MEDLINE]
797: J Antimicrob Chemother 1998 Dec;42(6):747-53
Synergic effects of tactolimus and azole antifungal agents against
azole-resistant Candida albican strains.
Maesaki S, Marichal P, Hossain MA, Sanglard D, Vanden Bossche H, Kohno S.
The Second Department of Internal Medicine, Nagasaki University School of
Medicine, Japan.
We investigated the effects of combining tacrolimus and azole antifungal agents
in azole-resistant strains of Candida albicans by comparing the accumulation of
[3H]itraconazole. The CDR1-expressing resistant strain C26 accumulated less
itraconazole than the CaMDR-expressing resistant strain C40 or the
azole-sensitive strain B2630. A CDR1-expressing Saccharomyces cerevisiae mutant,
DSY415, showed a marked reduction in the accumulation of both fluconazole and
itraconazole. A CaMDR-expressing S. cerevisiae mutant, DSY416, also showed lower
accumulation of fluconazole, but not of itraconazole. The addition of sodium
azide, an electron-transport chain inhibitor, increased the intracellular
accumulation of itraconazole only in the C26 strain, and not in the C40 or B2630
strains. Addition of tacrolimus, an inhibitor of multidrug resistance proteins,
resulted in the highest increase in itraconazole accumulation in the C26 strain.
The combination of itraconazole and tacrolimus was synergic in azole-resistant
C. albicans strains. In the C26 strain, the MIC of itraconazole decreased from
>8 to 0.5 mg/L when combined with tacrolimus. Our results showed that two
multidrug resistance phenotypes (encoded by the CDR1 and CaMDR genes) in C.
albicans have different substrate specificity for azole antifungal agents and
that a combination of tacrolimus and azole antifungal agents is effective
against azole-resistant strains of C. albicans.
PMID: 10052898 [PubMed - indexed for MEDLINE]
798: Nat Struct Biol 1999 Feb;6(2):157-65
X-ray structural analysis of the yeast cell cycle regulator Swi6 reveals
variations of the ankyrin fold and has implications for Swi6 function.
Foord R, Taylor IA, Sedgwick SG, Smerdon SJ.
Division of Protein Structure, National Institute for Medical Research, London,
UK.
Swi6 is a 92,000 Mr protein common to two distinct transcriptional activation
complexes (SBF and MBF) that coordinate gene expression at the G1-S boundary of
the yeast cell cycle. The X-ray structure of a central 36,000 Mr fragment has
been determined and refined at 2.1 A resolution. The structure reveals a basic
framework of five ankyrin repeat modules that is elaborated through a series of
helical insertions distinguishing it from structures of other ankyrin repeat
proteins. A second domain contains an approximately 30-residue region of
extended structure that interacts with the ankyrin repeat core over a
substantial proportion of its surface. Conservation of residues buried by these
interactions indicates that all members of the Swi6/Cdc10 family share a similar
architecture. Several temperature-sensitive mutations within Swi6 and Cdc10
appear to disrupt these interdomain contacts rather than destabilize the ankyrin
repeat core. The unusual domain arrangement may be crucial for the modulation of
interactions with other co-regulatory molecules such as cyclin-CDK complexes,
and has implications for the quaternary interactions within the multisubunit SBF
and MBF transcription complexes.
PMID: 10048928 [PubMed - indexed for MEDLINE]
799: Biochem Soc Trans 1998 Nov;26(4):601-6
Interactions of the human, plant and yeast ornithine decarboxylase subunits and
human antizyme.
Illingworth C, Michael AJ.
Department of Genetics and Microbiology, Institute of Food Research, Norwich
Research Park, Colney, U.K.
PMID: 10047790 [PubMed - indexed for MEDLINE]
800: Exp Cell Res 1999 Feb 25;247(1):1-8
SNAREs and the secretory pathway-lessons from yeast.
Pelham HR.
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, United
Kingdom. hp@mrc-lmb.cam.ac.uk
SNARE proteins lie at the heart of the membrane fusion events in the secretory
and endocytic pathways. Physical interactions between them are thought not only
to provide the driving force for bringing membranes together, but also to
contribute to the specificity of vesicle targeting. Completion of the yeast
genome sequence has allowed the full set of SNAREs to be identified.
Characterization of these helps to define the number of distinct compartments
and the nature of the transport steps between them, but also shows that SNAREs
are by no means the sole determinants of fusion specificity. Evolutionary
conservation of SNAREs suggests that despite the differences in scale and
morphology, many features of membrane organization are similar in yeast and
animal cells. This review summarizes current knowledge of the yeast SNAREs and
the picture of the secretory pathway that emerges from such studies. Copyright
1999 Academic Press.
Publication Types:
Review
Review, Tutorial
PMID: 10047442 [PubMed - indexed for MEDLINE]
801: J Biol Chem 1999 Mar 5;274(10):6579-85
The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi
mannosyltransferase complex.
Jungmann J, Rayner JC, Munro S.
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge,
CB2 2QH, United Kingdom.
In the yeast Saccharomyces cerevisiae many of the N-linked glycans on cell wall
and periplasmic proteins are modified by the addition of mannan, a large
mannose-containing polysaccharide. Mannan comprises a backbone of approximately
50 alpha-1,6-linked mannoses to which are attached many branches consisting of
alpha-1,2-linked and alpha-1,3-linked mannoses. The initiation and subsequent
elongation of the mannan backbone is performed by two complexes of proteins in
the cis Golgi. In this study we show that the product of the MNN10/BED1 gene is
a component of one of these complexes, that which elongates the backbone.
Analysis of interactions between the proteins in this complex shows that Mnn10p,
and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are
indeed all components of the same large structure. Deletion of either Mnn10p, or
its homologue Mnn11p, results in defects in mannan synthesis in vivo, and
analysis of the enzymatic activity of the complexes isolated from mutant strains
suggests that Mnn10p and Mnn11p are responsible for the majority of the alpha-1,
6-polymerizing activity of the complex.
PMID: 10037752 [PubMed - indexed for MEDLINE]
802: J Theor Biol 1999 Mar 7;197(1):63-76
Deviations from Chargaff's second parity rule correlate with direction of
transcription.
Bell SJ, Forsdyke DR.
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L
3N6.
The distribution of deviations from Chargaff's second parity rule was examined
for overlapping sequence windows of a length (1 kb) predicted to be suitable for
detecting correlations with functional features of DNA. For long genomic
segments from E. coli, Saccharomyces cerevisiae, and Vaccinia virus, Chargaff
differences for the W bases and/or for the S bases correlate with transcription
direction and gene location. For W-rich genomes, the mRNA-synonymous strand
contains regions which, if extruded from negatively supercoiled DNA, would fold
to generate stem-loop structures with A-rich loops. Similarly, for S-rich
genomes the loops would be G-rich. We suggest that the disposition of genes in
nucleic acid sequences arises from their having to adapt to a preexisting mosaic
of genomic regions, each distinguished by its potential to extrude single-strand
loops enriched for a particular base (or two non-Watson-Crick pairing bases).
The mosaic would have facilitated the intrastrand and interstrand accounting
required for correction of mutations, and would have evolved in the early RNA
world before the emergence of protein-encoding capacity. The preexisting mosaic
would have determined transcription direction since there is pressure for all
mRNAs of a cell to have purine-rich loops, thus decreasing loop-loop
interactions which might lead to formation of "self" sense-antisense RNA
duplexes. Copyright 1999 Academic Press.
PMID: 10036208 [PubMed - indexed for MEDLINE]
803: Genomics 1999 Feb 15;56(1):59-69
A gene upregulated in the acoustically damaged chick basilar papilla encodes a
novel WD40 repeat protein.
Adler HJ, Winnicki RS, Gong TW, Lomax MI.
Department of Otolaryngology/Head-Neck Surgery, University of Michigan, Ann
Arbor, Michigan, 48109, USA.
The chick WDR1 gene is expressed at higher levels in the chick basilar papilla
after acoustic overstimulation. The 3.3-kb WDR1 cDNA encodes a novel 67-kDa
protein containing nine WD40 repeats, motifs that mediate protein-protein
interactions. The predicted WDR1 protein has high sequence identity to
WD40-repeat proteins in budding yeast (Saccharomyces cerevisiae), two slime
molds (Dictyostelium discoideum and Physarum polycephalum), and the roundworm
(Caenorhabditis elegans). The yeast and P. polycephalum proteins bind actin,
suggesting that the novel chick protein may be an actin-binding protein.
Sequence database comparisons identified mouse and human cDNAs with high
sequence identity to the chick WDR1 cDNA. The mouse Wdr1 and human WDR1 proteins
showed 95% sequence identity to each other and 86% identity to the chick WDR1
protein. Northern blot analysis of total RNA from the chick basilar papilla
after noise trauma revealed increased levels of a 3.1-kb transcript in the
lesioned area. The WDR1 gene was mapped to human chromosome 4, between 22 and 24
cM from the telomere of 4p. Copyright 1999 Academic Press.
PMID: 10036186 [PubMed - indexed for MEDLINE]
804: Yeast 1999 Jan 15;15(1):35-41
Identification and characterization of the genes for two topoisomerase
I-interacting proteins from Saccharomyces cerevisiae.
Park H, Sternglanz R.
Department of Biochemistry and Cell Biology, SUNY at Stony Brook 11794-5215,
USA.
The two-hybrid system was used to identify yeast genes encoding proteins that
interact with DNA topoisomerase I. Two new genes were found and named TOF1 and
TOF2. The Tof1 protein has 1238 amino acids and no obvious homologues in
databases. The Tof2 protein has 771 amino acids; it is quite closely related to
another yeast protein, the product of an uncharacterized ORF. Both tof1 and tof2
null mutants, and tof1-tof2 double mutants, grow normally and have normal levels
of topoisomerase I activity. TOF2 shows various genetic interactions with TOP1
and HPR1. The implications of these interactions for TOF2 function are
discussed.
PMID: 10028183 [PubMed - indexed for MEDLINE]
805: Yeast 1999 Jan 15;15(1):23-33
Saccharomyces cerevisiae IRR1 protein is indirectly involved in colony
formation.
Kurlandzka A, Rytka J, Rozalska B, Wysocka M.
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa,
Poland.
The ability of a microorganism to adhere to a solid support and to initiate a
colony is often the first stage of microbial infections. To date, studies on S.
cerevisiae cell-cell and cell-solid support interactions concerned only cell
agglutination during mating and flocculation. Colony formation has not been
studied before probably because this species is not pathogenic. However, S.
cerevisiae can be a convenient model to study this process, thanks to
well-developed genetics and the full knowledge of its nucleotide sequence. A
preliminary characterization of the recently cloned essential IRR1 gene
indicated that it may participate in cell-cell/substrate interactions. Here we
show that lowering the level of expression of IRR1 (after fusion with a
regulatory catalase A gene promoter) affects colony formation and disturbs
zygote formation and spore germination. All these processes involve cell-cell or
cell-solid support contacts. The IRR1 protein is localized in the cytosol as
verified by immunofluorescence microscopy, and confirmed by cell fractionation
and Western blotting. This indicates that Irr1p is not directly involved in the
cell-solid support adhesion, but may be an element of a communication pathway
between the cell and its surroundings.
PMID: 10028182 [PubMed - indexed for MEDLINE]
806: Cancer Res 1999 Feb 15;59(4):816-22
hMSH5: a human MutS homologue that forms a novel heterodimer with hMSH4 and is
expressed during spermatogenesis.
Bocker T, Barusevicius A, Snowden T, Rasio D, Guerrette S, Robbins D, Schmidt C,
Burczak J, Croce CM, Copeland T, Kovatich AJ, Fishel R.
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas
Jefferson University and Medical College, Philadelphia, Pennsylvania 19107, USA.
MutS homologues have been identified in nearly all organisms examined to date.
They play essential roles in maintaining mitotic genetic fidelity and meiotic
segregation fidelity. MutS homologues appear to function as a molecular switch
that signals genomic manipulation events. Here we describe the identification of
the human homologue of the Saccharomyces cerevisiae MSH5, which is known to
participate in meiotic segregation fidelity and crossing-over. The human MSH5
(MSH5) was localized to chromosome 6p22-21 and appears to play a role in
meiosis because expression is induced during spermatogenesis between the late
primary spermatocytes and the elongated spermatid phase. hMSH5 interacts
specifically with hMSH4, confirming the generality of functional heterodimeric
interactions in the eukaryotic MutS homologue, which also includes hMSH2-hMSH3
and hMSH2-hMSH6.
PMID: 10029069 [PubMed - indexed for MEDLINE]
807: Sci Total Environ 1999 Jan 12;225(1-2):69-79
Monitoring of estrogen mimics by a recombinant yeast assay: synergy between
natural and synthetic compounds?
Graumann K, Breithofer A, Jungbauer A.
Institute for Applied Microbiology, University of Agriculture, Forestry and
Biotechnology, Vienna, Austria.
Properties of mixtures of compounds exhibiting estrogenic potential have been
questioned in the past. Synergistic effects of endocrine disrupters have been
proposed, but could never be confirmed. In this study, the transactivational
potential of xenoestrogens and phytoestrogens has been evaluated in a yeast test
system. Pesticides such as endosulfan, dieldrin, atrazine, and the main
metabolites, desethylatrazine and desisopropylatrazine, have been tested and
their behavior as mixtures is compared to the behavior of the single compounds.
Our results are in contrast to a report (Tran et al., 1996) on the inhibitive
effects of xenoestrogens on 17 beta-estradiol-dependent transactivation.
Phytoestrogens have been investigated in a similar manner. A synergistic effect
could not be confirmed for both, xenoestrogens and phytoestrogens. These
compounds are either weak estrogens or completely lack estrogenic potential.
Their endocrine disrupting potential in more complex systems must be therefore
attributed to other molecular mechanisms such as to metabolic modification or
interference with steroidogenesis. This study shows that yeast systems are
useful tools for monitoring pure estrogenic properties.
PMID: 10028704 [PubMed - indexed for MEDLINE]
808: Mol Cell 1999 Jan;3(1):97-108
Erratum in:
Mol Cell 1999 Apr;3(4):following 541
A novel human SRB/MED-containing cofactor complex, SMCC, involved in
transcription regulation.
Gu W, Malik S, Ito M, Yuan CX, Fondell JD, Zhang X, Martinez E, Qin J, Roeder
RG.
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New
York, New York 10021, USA.
A novel human complex that can either repress activator-dependent transcription
mediated by PC4, or, at limiting TFIIH, act synergistically with PC4 to enhance
activator-dependent transcription has been purified. This complex contains
homologs of a subset of yeast mediator/holoenzyme components (including SRB7,
SRB10, SRB11, MED6, and RGR1), homologs of other yeast transcriptional
regulatory factors (SOH1 and NUT2), and, significantly, some components
(TRAP220, TRAP170/hRGR1, and TRAP100) of a human thyroid hormone
receptor-associated coactivator complex. The complex shows direct activator
interactions but, unlike yeast mediator, can act independently of the RNA
polymerase II CTD. These findings demonstrate both positive and negative
functional capabilities for the human complex, emphasize novel (CTD-independent)
regulatory mechanisms, and link the complex to other human coactivator
complexes.
PMID: 10024883 [PubMed - indexed for MEDLINE]
809: Infect Immun 1999 Mar;67(3):1063-71
Non-serum-dependent chemotactic factors produced by Candida albicans stimulate
chemotaxis by binding to the formyl peptide receptor on neutrophils and to an
unknown receptor on macrophages.
Edens HA, Parkos CA, Liang TW, Jesaitis AJ, Cutler JE, Miettinen HM.
Department of Microbiology, Montana State University-Bozeman, Bozeman, Montana
59717, USA. hedens@tex2.oscs.montana.edu
Serum-free culture filtrates of six Candida species and Saccharomyces cerevisiae
were found to contain chemoattractants for human polymorphonuclear leukocytes
(PMNs) and a mouse macrophage-like cell line, J774. The chemotactic factors
differed for the PMN and J774 cells, however, in terms of heat stability,
kinetics of liberation by the yeast cells, and divalent cation requirements for
production. The chemoattractant in Candida albicans culture filtrates appeared
to act through the formyl peptide receptor (FPR) of PMNs, since it was found to
induce chemotaxis of Chinese hamster ovary (CHO) cells that were expressing the
human FPR but did not induce chemotaxis of wild-type CHO cells. The C. albicans
culture filtrates also induced migration of PMNs across confluent monolayers of
a human gastrointestinal epithelial cell line, T84; migration occurred in the
basolateral-to-apical direction but not the reverse direction, unless the
epithelial tight junctions were disrupted. J774 cells did not migrate toward the
formylated peptide (fMet-Leu-Phe; fMLF), and chemotaxis toward the C. albicans
culture filtrate was not inhibited by an FPR antagonist
(t-butoxycarbonyl-Met-Leu-Phe), suggesting that a different receptor mediated
J774 cell chemotaxis. In conclusion, we have identified a receptor by which a
non-serum-dependent chemotactic factor (NSCF) produced by C. albicans induced
chemotaxis of PMNs. Additionally, we have shown that NSCF was active across
epithelial monolayers. These findings suggest that NSCFs produced by C. albicans
and other yeast species may influence host-pathogen interactions at the
gastrointestinal tract mucosal surface by inducing phagocytic-cell infiltration.
PMID: 10024544 [PubMed - indexed for MEDLINE]
810: Biochem J 1999 Mar 1;338 ( Pt 2):403-7
Characterization of the interaction domains of Ure2p, a prion-like protein of
yeast.
Fernandez-Bellot E, Guillemet E, Baudin-Baillieu A, Gaumer S, Komar AA, Cullin
C.
Centre de Genetique Moleculaire du C.N.R.S., Laboratoire Propre Associe a
l'Universite Pierre-et-Marie-Curie, 91190 Gif-sur-Yvette, France.
In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic
element [URE3] behaves as a prion. A hypothesis has been put forward which
states that [URE3] arises spontaneously from its cellular isoform Ure2p (the
product of the URE2 gene), and propagates through interactions of the N-terminal
domain of the protein, thus leading to its aggregation and loss of function. In
the present study, various N- and C-terminal deletion mutants of Ure2p were
constructed and their cross-interactions were tested in vitro and in vivo using
affinity binding and a two-hybrid analysis. We show that the self-interaction of
the protein is mediated by at least two domains, corresponding to the first
third of the protein (the so-called prion-forming domain) and the C-terminal
catalytic domain.
PMID: 10024516 [PubMed - indexed for MEDLINE]
811: Biochem J 1999 Mar 1;338 ( Pt 2):375-86
Comparison of the fibrin-binding activities in the N- and C-termini of
fibronectin.
Rostagno AA, Schwarzbauer JE, Gold LI.
Department of Pathology, New York University Medical Center, 400 East 34th
Street, New York, NY 10016, USA.
Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent
interactions. The N- and C-termini of Fn each contain one non-covalent
fibrin-binding site, which are composed of type 1 (F1) structural repeats. We
have previously localized the N-terminal site to the fourth and fifth F1 repeats
(4F1.5F1). In the current studies, using proteolytic and recombinant proteins
representing both the N- and C-terminal fibrin-binding regions, we localized and
characterized the C-terminal fibrin-binding site, compared the relative
fibrin-binding activities of both sites and determined the contribution of each
site to the fibrin-binding activity of intact Fn. By fibrin-affinity
chromatography, a protein composed of the 10F1 repeat through to the C-terminus
of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in
Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1
and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin
binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to
10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa
fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable
binding to fibrin-coated wells that was both competitively inhibited and
reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities
of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4
kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM,
respectively. The higher fibrin-binding affinity of the N-terminus was
substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal
antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to
fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal
anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the
N-terminal site appears to contribute to most of the binding activity of native
Fn to fibrin, the specific binding of the C-terminal site may strengthen this
interaction.
PMID: 10024513 [PubMed - indexed for MEDLINE]
812: Protein Expr Purif 1999 Feb;15(1):127-45
A kinetic locking-on strategy for bioaffinity purification: further studies with
alcohol dehydrogenase.
O'flaherty M, McMahon M, Mulcahy P.
Department of Applied Biology and Chemistry, Institute of Technology Carlow,
Ireland.
The kinetic locking-on strategy improves the selectivity of protein purification
procedures based on immobilized cofactor derivatives through use of
enzyme-specific substrate analogues in irrigants to promote biospecific
adsorption. This paper describes the development and application of this
strategy to the one-chromatographic step affinity purification of
NAD(P)+-dependent alcohol dehydrogenases using 8'-azo-linked immobilized
NAD(P)+, S6-linked and N6-linked immobilized NAD+, and N6-linked immobilized
NADP+ derivatives. These studies were carried out using alcohol dehydrogenases
from Saccharomyces cerevisiae (YADH, EC 1.1.1.1), equine liver (HLADH, EC
1.1.1.1), and Thermoanaerobium brockii (TBADH, EC 1.1.1.2). The results reveal
that the factors which require careful consideration before development of a
truly biospecific system based on the locking-on strategy include: (i) the
stability of the immobilized cofactor derivative; (ii) the spacer-arm
composition of the affinity derivative; (iii) the accessible immobilized
cofactor concentration; (iv) the soluble locking-on ligand concentration; (v)
the dissociation constant of locking-on ligand, and (vi) the identification and
elimination of nonbiospecific interference. The S6-linked immobilized NAD+
derivative (synthesized with a hydrophilic spacer arm) proved to be the most
suitable of the affinity adsorbents investigated in the present study for use
with the locking-on strategy. This conclusion was based primarily on the
observations that this affinity adsorbent was stable, retained cofactor activity
with the "test" enzymes under study, and was not prone to nonbiospecific
interactions. Using this immobilized derivative in conjunction with the
locking-on strategy, alcohol dehydrogenase from Saccharomyces cerevisiae was
purified to electrophoretic homogeneity in a single affinity chromatographic
step. Copyright 1999 Academic Press.
PMID: 10024480 [PubMed - indexed for MEDLINE]
813: J Cell Biochem 1999 Mar 1;72(3):356-67
180-kD bullous pemphigoid antigen/type XVII collagen: tissue-specific expression
and molecular interactions with keratin 18.
Aho S, Uitto J.
Department of Dermatology and Cutaneous Biology, Jefferson Medical College,
Philadelphia, Pennsylvania 19107, USA. sirpaaho@hotmail.com
The 180-kD bullous pemphigoid antigen (BPAG2) is a hemidesmosomal transmembrane
protein, also known as type XVII collagen. In this study, potential interactions
of BPAG2 with other proteins expressed in epidermal keratinocytes were explored
by yeast two-hybrid system using the amino-terminal intracellular domain of
BPAG2 as a bait. Several independent interacting clones encoding keratin 18
(K18) were identified when the keratinocyte cDNA library, cloned into the yeast
two-hybrid activation domain vector, was screened. The peptide sequence
responsible for the interaction of BPAG2 was restricted to amino acids 15-25,
and substitution of a valine residue in the middle of this sequence by a proline
(V23P) by site-directed mutagenesis abolished the interaction. Further
examination of the K18 sequences by restricted cDNA constructs in yeast
two-hybrid system identified a carboxyl-terminal segment corresponding to helix
2B domain as critical for BPAG2 binding. The interaction of BPAG2/K18 was
confirmed by an in vitro protein-protein interaction assay, which also confirmed
that normal human keratinocytes express K18 in culture. The tissue specific
expression of BPAG2 was first examined using a multi-tissue RNA blot. Human
multiple tissue cDNA panels representing a variety of adult and fetal tissues as
well as tumor cells were used as PCR-templates to study the expression patterns
of both BPAG2 and K18. The results demonstrated significant level of expression
of BPAG2, besides in epidermal keratinocytes, also in a variety of tissues with
predominant epithelial component, such as mammary, salivary and thyroid glands,
colon, prostate, testis, placenta, and adult and fetal thymus, as well as in
colon, pancreatic and prostatic adenocarcinoma cell lines, and an ovarian
carcinoma. As expected, K18 transcript is present in liver, pancreas, colon,
placenta, and in fetal kidney. Collectively, the results suggest that BPAG2 has
a relatively broad tissue distribution including specialized and simple
epithelia, and that within the tissues such as colon and placenta, BPAG2 may
have direct interactions with K18, a keratin characteristically expressed in a
simple epithelia.
PMID: 10022517 [PubMed - indexed for MEDLINE]
814: Mol Cell Biol 1999 Mar;19(3):2142-54
Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small
nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual
substrate specificity for U2 snRNA and tRNA.
Massenet S, Motorin Y, Lafontaine DL, Hurt EC, Grosjean H, Branlant C.
Laboratoire de Maturation des ARN et Enzymologie Moleculaire, UMR7567 CNRS-UHP,
Faculte des Sciences, 54506 Vandoeuvre-les-Nancy Cedex, France.
Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae
spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping
method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a
few Psi residues, which are located in segments involved in intermolecular
RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally
modified. When yeast mutants disrupted for one of the several pseudouridine
synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine
synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p
activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed,
Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in
vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the
S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase
characterized so far which exhibits a dual substrate specificity, acting on both
tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no
effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs
is not dependent on the Cbf5p-snoRNA guided mechanism.
PMID: 10022901 [PubMed - indexed for MEDLINE]
815: Mol Cell Biol 1999 Mar;19(3):2000-7
The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3'
exonuclease Exo1 have major roles in postreplication mutation avoidance in
Saccharomyces cerevisiae.
Tran HT, Gordenin DA, Resnick MA.
Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute
of Environmental Health Sciences, Research Triangle Park, North Carolina 27709,
USA.
Replication fidelity is controlled by DNA polymerase proofreading and
postreplication mismatch repair. We have genetically characterized the roles of
the 5'-->3' Exo1 and the 3'-->5' DNA polymerase exonucleases in mismatch repair
in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and
highly sensitive mutation detection systems that are based on long and short
homonucleotide runs. Genetic interactions were examined among DNA polymerase
epsilon (pol2-4) and delta (pol3-01) mutants defective in 3'-->5' proofreading
exonuclease, mutants defective in the 5'-->3' exonuclease Exo1, and mismatch
repair mutants (msh2, msh3, or msh6). These three exonucleases play an important
role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01
mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they
participate directly in postreplication mismatch repair as well as in other DNA
metabolic processes.
PMID: 10022887 [PubMed - indexed for MEDLINE]
816: Mol Cell Biol 1999 Mar;19(3):1627-39
Erratum in:
Mol Cell Biol 1999 May;19(5):3929
Cooperative binding of heat shock factor to the yeast HSP82 promoter in vivo and
in vitro.
Erkine AM, Magrogan SF, Sekinger EA, Gross DS.
Department of Biochemistry and Molecular Biology, Louisiana State University
Medical Center, Shreveport, Louisiana 71130, USA.
Previous work has shown that heat shock factor (HSF) plays a central role in
remodeling the chromatin structure of the yeast HSP82 promoter via constitutive
interactions with its high-affinity binding site, heat shock element 1 (HSE1).
The HSF-HSE1 interaction is also critical for stimulating both basal
(noninduced) and induced transcription. By contrast, the function of the
adjacent, inducibly occupied HSE2 and -3 is unknown. In this study, we examined
the consequences of mutations in HSE1, HSE2, and HSE3 on HSF binding and
transactivation. We provide evidence that in vivo, HSF binds to these three
sites cooperatively. This cooperativity is seen both before and after heat
shock, is required for full inducibility, and can be recapitulated in vitro on
both linear and supercoiled templates. Quantitative in vitro footprinting
reveals that occupancy of HSE2 and -3 by Saccharomyces cerevisiae HSF (ScHSF) is
enhanced approximately 100-fold through cooperative interactions with the
HSF-HSE1 complex. HSE1 point mutants, whose basal transcription is virtually
abolished, are functionally compensated by cooperative interactions with HSE2
and -3 following heat shock, resulting in robust inducibility. Using a
competition binding assay, we show that the affinity of recombinant HSF for the
full-length HSP82 promoter is reduced nearly an order of magnitude by a
single-point mutation within HSE1, paralleling the effect of these mutations on
noninduced transcript levels. We propose that the remodeled chromatin phenotype
previously shown for HSE1 point mutants (and lost in HSE1 deletion mutants)
stems from the retention of productive, cooperative interactions between HSF and
its target binding sites.
PMID: 10022851 [PubMed - indexed for MEDLINE]
817: EMBO J 1999 Feb 15;18(4):1071-80
The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of
exocytosis.
Guo W, Roth D, Walch-Solimena C, Novick P.
Department of Cell Biology, Yale University School of Medicine, PO Box 208002,
New Haven, CT 06520-8002, USA.
Polarized secretion requires proper targeting of secretory vesicles to specific
sites on the plasma membrane. Here we report that the exocyst complex plays a
key role in vesicle targeting. Sec15p, an exocyst component, can associate with
secretory vesicles and interact specifically with the rab GTPase, Sec4p, in its
GTP-bound form. A chain of protein-protein interactions leads from Sec4p and
Sec15p on the vesicle, through various subunits of the exocyst, to Sec3p, which
marks the sites of exocytosis on the plasma membrane. Sec4p may control the
assembly of the exocyst. The exocyst may therefore function as a rab effector
system for targeted secretion.
PMID: 10022848 [PubMed - indexed for MEDLINE]
818: Curr Opin Chem Biol 1999 Feb;3(1):64-70
Progress and variations in two-hybrid and three-hybrid technologies.
Drees BL.
Department of Genetics, Box 357360, University of Washington, Seattle WA,98195
USA. drees@u.washington.edu
The original yeast two-hybrid system and its variants have proven to be
effective tools for identification and analysis of protein-protein, protein-DNA
and protein-RNA interactions. The two-hybrid assay is being applied to the
entire complement of proteins of the yeast Saccharomyces cerevisiae to
characterize the network of protein-protein interactions in the eukaryotic cell.
The development of nontranscriptional cytosolic and membrane-associated
two-hybrid methods has made it possible to detect and examine a number of
protein-protein interactions in their normal cellular locations. Small-molecule
hybrid systems have been developed which can be used to study protein-ligand
interactions and to activate cellular processes by forcing protein associations.
Publication Types:
Review
Review, Tutorial
PMID: 10021404 [PubMed - indexed for MEDLINE]
819: Proc Natl Acad Sci U S A 1999 Feb 16;96(4):1738-43
A protein phosphatase 2C gene, LjNPP2C1, from Lotus japonicus induced during
root nodule development.
Kapranov P, Jensen TJ, Poulsen C, de Bruijn FJ, Szczyglowski K.
Michigan State University-Department of Energy Plant Research Laboratory,
Michigan State University, East Lansing, MI 48824, USA.
Symbiotic interactions between legumes and compatible strains of rhizobia result
in root nodule formation. This new plant organ provides the unique physiological
environment required for symbiotic nitrogen fixation by the bacterial
endosymbiont and assimilation of this nitrogen by the plant partner. We have
isolated two related genes (LjNPP2C1 and LjPP2C2) from the model legume Lotus
japonicus that encode protein phosphatase type 2C (PP2C). Expression of the
LjNPP2C1 gene was found to be enhanced specifically in L. japonicus nodules,
whereas the LjPP2C2 gene was expressed at a similar level in nodules and roots.
A glutathione S-transferase-LjNPP2C1 fusion protein was shown to have Mg2+- or
Mn2+-dependent and okadaic acid-insensitive PP2C activity in vitro. A chimeric
construct containing the full-length LjNPP2C1 cDNA, under the control of the
Saccharomyces cerevisiae alcohol dehydrogenase promoter, was found to be able to
complement a yeast PP2C-deficient mutant (pct1Delta). The transcript level of
the LjNPP2C1 gene was found to increase significantly in mature nodules, and its
highest expression level occurred after leghemoglobin (lb) gene induction, a
molecular marker for late developmental events in nodule organogenesis.
Expression of the LjNPP2C1 gene was found to be drastically altered in specific
L. japonicus lines carrying monogenic-recessive mutations in symbiosis-related
loci, suggesting that the product of the LjNPP2C1 gene may function at both
early and late stages of nodule development.
PMID: 9990094 [PubMed - indexed for MEDLINE]
820: Nat Genet 1999 Feb;21(2):204-8
Comment in:
Nat Genet. 1999 Feb;21(2):151-2
Interaction between Set1p and checkpoint protein Mec3p in DNA repair and
telomere functions.
Corda Y, Schramke V, Longhese MP, Smokvina T, Paciotti V, Brevet V, Gilson E,
Geli V.
Laboratoire d'Ingenierie et de Dynamique des Systemes Macromoleculaires, CNRS,
Marseille, France.
The yeast protein Set1p, inactivation of which alleviates telomeric position
effect (TPE), contains a conserved SET domain present in chromosomal proteins
involved in epigenetic control of transcription. Mec3p is required for efficient
DNA-damage-dependent checkpoints at G1/S, intra-S and G2/M (refs 3-7). We show
here that the SET domain of Set1p interacts with Mec3p. Deletion of SET1
increases the viability of mec3delta mutants after DNA damage (in a process that
is mostly independent of Rad53p kinase, which has a central role in checkpoint
control) but does not significantly affect cell-cycle progression. Deletion of
MEC3 enhances TPE and attenuates the Set1delta-induced silencing defect.
Furthermore, restoration of TPE in a Set1delta mutant by overexpression of the
isolated SET domain requires Mec3p. Finally, deletion of MEC3 results in
telomere elongation, whereas cells with deletions of both SET1 and MEC3 do not
have elongated telomeres. Our findings indicate that interactions between SET1
and MEC3 have a role in DNA repair and telomere function.
PMID: 9988274 [PubMed - indexed for MEDLINE]
821: J Biol Chem 1999 Feb 19;274(8):5252-8
Topology and functional domains of the yeast pore membrane protein Pom152p.
Tcheperegine SE, Marelli M, Wozniak RW.
Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2A7,
Canada.
Integral membrane proteins associated with the nuclear pore complex (NPC) are
likely to play an important role in the biogenesis of this structure. Here we
have examined the functional roles of domains of the yeast pore membrane protein
Pom152p in establishing its topology and its interactions with other NPC
proteins. The topology of Pom152p was evaluated by alkaline extraction, protease
protection, and endoglycosidase H sensitivity assays. The results of these
experiments suggest that Pom152p contains a single transmembrane segment with
its N terminus (amino acid residues 1-175) extending into the nuclear pore and
its C terminus (amino acid residues 196-1337) positioned in the lumen of the
nuclear envelope. The functional role of these different domains was
investigated in mutants that are dependent on Pom152p for viability. The
requirement for Pom152p in strains containing mutations allelic to the NPC
protein genes NIC96 and NUP59 could be alleviated by Pom152p's N terminus,
independent of its integration into the membrane. However, complementation of a
mutation in NUP170 required both the N terminus and the transmembrane segment.
Furthermore, mutations in NUP188 were rescued only by full-length Pom152p,
suggesting that the lumenal structures play an important role in the function of
pore-side NPC structures.
PMID: 9988776 [PubMed - indexed for MEDLINE]
822: Mol Endocrinol 1999 Feb;13(2):286-96
Estrogen receptor domains E and F: role in dimerization and interaction with
coactivator RIP-140.
Peters GA, Khan SA.
Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati
College of Medicine, Ohio 45627, USA.
We have used the yeast two-hybrid system to localize the ligand-dependent
dimerization domain of the estrogen receptor-alpha (ER) to region E in vivo. In
this system, the cDNAs corresponding to the A-D, E, E/F, A-E (deltaF), and
full-length (wtER) domains of the human ER were each cloned into the yeast
two-hybrid vectors GAL4 DB and GAL4 TA and expressed in different combinations
in yeast harboring a GAL1-lacZ reporter. The reporter was used as a relative
measure of the interaction between the ER domains, through reconstitution of
GAL4 activity. We found that the interaction of E or E/F domains of the ER with
full-length ER is estradiol dependent and estrogen responsive element
independent, as measured by the reconstitution of GAL4 activity from GAL4-E
domain-containing fusion protein interactions. In the presence of F domain, this
activity is reduced 10-fold. The results suggest that sequences in the F domain
are inhibitory to the dimerization signal that is present in the E region. We
propose that the full-length ER contains intrinsic dimerization restraints
contributed by regions outside domain E that are released upon binding hormone
agonist. In addition, we have demonstrated that coactivator RIP140 is able to
interact with the ER in vivo at the E domain of the receptor in the presence of
estrogen. Yeast two-hybrid analysis shows that RIP140 does not homodimerize in
the presence or absence of estrogens. We present evidence showing that the ER
has the inherent ability to interact with RIP140 in the presence of
antiestrogens, but sequences inherent in the ER itself that are present outside
of the E domain compromise this ability.
PMID: 9973258 [PubMed - indexed for MEDLINE]
823: Mol Biol Cell 1999 Feb;10(2):329-44
Detection of transient in vivo interactions between substrate and transporter
during protein translocation into the endoplasmic reticulum.
Dunnwald M, Varshavsky A, Johnsson N.
Max-Delbruck-Laboratorium, D-50829 Koln, Germany.
The split-ubiquitin technique was used to detect transient protein interactions
in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to
Sec62p, a component of the protein translocation machinery in the endoplasmic
reticulum of Saccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused
to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub
structure from the two halves of Ub, and the resulting cleavage by Ub-specific
proteases at the C terminus of Cub, serve as a gauge of proximity between the
two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p
is spatially close to the signal sequence of the prepro-alpha-factor in vivo.
This proximity is confined to the nascent polypeptide chain immediately
following the signal sequence. In addition, the extent of proximity depends on
the nature of the signal sequence. Cub fusions that bore the signal sequence of
invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than
otherwise identical test proteins bearing the signal sequence of
prepro-alpha-factor. An inactive derivative of Sec62p failed to interact with
signal sequences in this assay. These in vivo findings are consistent with
Sec62p being part of a signal sequence-binding complex.
PMID: 9950680 [PubMed - indexed for MEDLINE]
824: Mol Biol Cell 1999 Feb;10(2):283-96
The multiple roles of Cyk1p in the assembly and function of the actomyosin ring
in budding yeast.
Shannon KB, Li R.
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115,
USA.
The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud
junction during anaphase and has been shown to be required for the completion of
cytokinesis. In this study, video microscopy analysis of cells expressing green
fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a
dynamic component of the contractile ring during cytokinesis. Furthermore, in
the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size
change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p
in actomyosin ring assembly and dynamics, we have investigated the role of the
structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino terminal
portion containing the calponin homology domain binds to actin filaments and is
required for the assembly of actin filaments to the ring. This result supports
the hypothesis that Cyk1p/Iqg1p plays a direct role in F-actin recruitment.
Deletion of the domain harboring the eight IQ motifs abolishes the localization
of Cyk1p/Iqg1p to the bud neck, suggesting that Cyk1p/Iqg1p may be localized
through interactions with a calmodulin-like protein. Interestingly, deletion of
the COOH-terminal GTPase-activating protein-related domain does not affect
Cyk1p/Iqg1p localization or actin recruitment to the ring but prevents
actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p
binds to calmodulin, Cmd1p, in a calcium-dependent manner, and to Tem1p, a small
GTP-binding protein previously found to be required for the completion of
anaphase. These results demonstrate the critical function of Cyk1p/Iqg1p in
regulating various steps of actomyosin ring assembly and cytokinesis.
PMID: 9950677 [PubMed - indexed for MEDLINE]
825: J Biol Chem 1999 Feb 12;274(7):4027-35
Regulation of exocytosis by cyclin-dependent kinase 5 via phosphorylation of
Munc18.
Fletcher AI, Shuang R, Giovannucci DR, Zhang L, Bittner MA, Stuenkel EL.
Departments of Physiology, University of Michigan, Ann Arbor, Michigan 48109,
USA.
Munc18a, a mammalian neuronal homologue of Saccharomyces cerevisiae Sec1p
protein, is essential for secretion, likely as a result of its high affinity
interaction with the target SNARE protein syntaxin 1a (where SNARE is derived
from SNAP receptor (the soluble N-ethylmaleimide-sensitive fusion protein)).
However, this interaction inhibits vesicle SNARE interactions with syntaxin that
are required for secretory vesicles to achieve competency for membrane fusion.
As such, regulation of the interaction between Munc18a and syntaxin 1a may
provide an important mechanism controlling secretory responsiveness.
Cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division
kinases, co-purifies with Munc18a from rat brain, interacts directly with
Munc18a in vitro, and utilizes Munc18a as a substrate for phosphorylation. We
have now demonstrated that Cdk5 is capable of phosphorylating Munc18a in vitro
within a preformed Munc18a.syntaxin 1a heterodimer complex and that this results
in the disassembly of the complex. Using site-directed mutagenesis, the Cdk5
phosphorylation site on Munc18a was identified as Thr574. Stimulation of
secretion from neuroendocrine cells produced a corresponding rapid translocation
of cytosolic Cdk5 to a particulate fraction and an increase of Cdk5 kinase
activity. Inhibition of Cdk5 with olomoucine decreased evoked norepinephrine
secretion from chromaffin cells, an effect not observed with the inactive
analogue iso-olomoucine. The effects of olomoucine were independent of calcium
influx as evidenced by secretory inhibition in permeabilized chromaffin cells
and in cells under whole-cell voltage clamp. Furthermore, transfection and
expression in chromaffin cells of a neural specific Cdk5 activator, p25, led to
a strong increase in nicotinic agonist-induced secretory responses. Our data
suggest a model whereby Cdk5 acts to regulate Munc18a interaction with syntaxin
1a and thereby modulates the level of vesicle SNARE interaction with syntaxin 1a
and secretory responsiveness.
PMID: 9933594 [PubMed - indexed for MEDLINE]
826: Gene 1998 Dec 28;225(1-2):107-16
Isolation of hMRE11B: failure to complement yeast mre11 defects due to
species-specific protein interactions.
Chamankhah M, Wei YF, Xiao W.
Department of Microbiology, University of Saskatchewan, 107 Wiggins Road,
Saskatoon SK S7N 5E5, Canada.
The Saccharomyces cerevisiae MRE11 gene plays an important role in meiotic
recombination, mitotic DNA repair and telomere maintenance. We present the
isolation of hMRE11B cDNA from a human HeLa cell cDNA library as an MRE11
homolog. Compared to the previously identified hMRE11, hMRE11B contains an
additional 84bp sequence that results in a 28 amino-acid insertion close to the
C-terminus. The expression pattern of hMRE11B in different tissues shows the
presence of two mRNA species of approx. 2.6 and 7.5kb. Overexpression of hMRE11B
does not complement the alkylation sensitivity of the mre11 null and
temperature-sensitive mutant strains. In this study, we examine factors that may
explain this lack of complementation. First, both Northern and Western analyses
rule out the lack of hMRE11B transcription and/or translation in yeast. Second,
we demonstrate that hMre11B, like the yeast Mre11 protein, dimerizes in vivo in
a yeast two-hybrid system. This dimerization requires the C-terminal one-third
of hMre11B protein, which includes the 28 amino acids absent in hMre11. However,
hMre11B does not interact with Mre11, Rad50 and Xrs2. Hence, the lack of
protein-protein interaction between hMre11B and the yeast Mre11, Rad50, and Xrs2
may explain the inability of hMRE11B to complement the yeast mre11 mutants. We
rule out the hypothesis that the lack of interaction and, in turn of
complementation, is due to the absence of sequence homology at the C-terminal
domain of hMre11B compared to the yeast Mre11. Instead, we propose that the
C-terminus of hMre11B participates in protein-protein interaction and functions
in a species-specific manner.
PMID: 9931460 [PubMed - indexed for MEDLINE]
827: Biochemistry 1999 Jan 26;38(4):1365-70
Nonspecific weak actomyosin interactions: relocation of charged residues in
subdomain 1 of actin does not alter actomyosin function.
Wong WW, Doyle TC, Reisler E.
Department of Chemistry and Biochemistry and Molecular Biology Institute,
University of California, Los Angeles, California 90095, USA.
Yeast actin mutants with relocated charged residues within subdomain 1 were
constructed so we could investigate the functional importance of individual
clusters of acidic residues in mediating actomyosin weak-binding states in the
cross-bridge cycle. Past studies have established a functional role for three
distinct pairs of charged residues within this region of yeast actin (D2/E4,
D24/D25, and E99/E100); the loss of any one of these pairs resulted in the same
impairment in weak actomyosin interaction and in its function. However, the
specificity of myosin interaction with these sites has not yet been addressed.
To investigate this, we made and analyzed two new actin mutants, 4Ac/D24A/D25A
and 4Ac/E99A/E100A. In these mutants, the acidic residues of the D24/D25 or
E99/E100 sites were replaced with uncharged residues (alanines) and a pair of
acidic residues was inserted at the N-terminus, maintaining the overall charge
density of subdomain 1. Using the in vitro motility assays, we found that the
sliding and force generation properties of these mutant actins were identical to
those of wild-type actin. Similarly, actin-activated ATPase activities of the
mutant and wild-type actins were also indistinguishable. Additionally, the
binding of S1 to these mutant actins in the presence of ATP was similar to that
of wild-type actin. These results show that relocation of charged residues in
subdomain 1 of actin does not affect the weak actomyosin interactions and
actomyosin function.
PMID: 9930999 [PubMed - indexed for MEDLINE]
828: FEBS Lett 1999 Jan 22;443(1):41-7
C-terminal domains of human translation termination factors eRF1 and eRF3
mediate their in vivo interaction.
Merkulova TI, Frolova LY, Lazar M, Camonis J, Kisselev LL.
INSERM U248, Institut Curie, Paris, France.
At the termination step of protein synthesis, hydrolysis of the peptidyl-tRNA is
jointly catalysed at the ribosome by the termination codon and the polypeptide
release factor (eRF1 in eukaryotes). eRF1 forms in vivo and in vitro a stable
complex with release factor eRF3, an eRF1-dependent and ribosome-dependent
GTPase. The role of the eRF1-eRF3 complex in translation remains unclear. We
have undertaken a systematic analysis of the interactions between the human eRF1
and eRF3 employing a yeast two-hybrid assay. We show that the N-terminal parts
of eRF1 (positions 1-280) and of eRF3 (positions 1477) are either not involved
or non-essential for binding. Two regions in each factor are critical for mutual
binding: positions 478-530 and 628-637 of eRF3 and positions 281-305 and 411-415
of eRF1. The GTP binding domain of eRF3 is not involved in complex formation
with eRF1. The GILRY pentamer (positions 411-415) conserved in eukaryotes and
archaebacteria is critical for eRF1's ability to stimulate eRF3 GTPase. The
human eRF1 lacking 22 C-terminal amino acids remains active as a release factor
and promotes an eRF3 GTPase activity whereas C-terminally truncated eRF3 is
inactive as a GTPase.
PMID: 9928949 [PubMed - indexed for MEDLINE]
829: EMBO J 1999 Feb 1;18(3):754-62
Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain
co-chaperones.
Prodromou C, Siligardi G, O'Brien R, Woolfson DN, Regan L, Panaretou B, Ladbury
JE, Piper PW, Pearl LH.
Department of Biochemistry and Molecular Biology, University College London,
Gower Street, London WC1E 6BT, UK.
The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is
dependent on the binding and hydrolysis of ATP, and on interactions with a
variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We
have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and
Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism
spectroscopy and analytical ultracentrifugation, and determined the effect of
their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind
with sub-micromolar affinity, with Sti1 binding accompanied by a large
conformational change. Two co-chaperone molecules bind per Hsp90 dimer, and Sti1
itself is found to be a dimer in free solution. The inherent ATPase activity of
Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6,
although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90.
Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site
in the N-terminal domain of Hsp90. These results reveal an important role for
TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing
that the ATP-dependent step in Hsp90-mediated protein folding occurs after the
binding of the folding client protein, and suggesting that ATP hydrolysis
triggers client-protein release.
PMID: 9927435 [PubMed - indexed for MEDLINE]
830: EMBO J 1999 Feb 1;18(3):717-26
Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and
transforming mutations stabilize Myc.
Salghetti SE, Kim SY, Tansey WP.
Cold Spring Harbor Laboratory, 1 Bungtown Road, PO Box 100, Cold Spring Harbor,
NY 11724, USA.
The human proto-oncogene c-myc encodes a highly unstable transcription factor
that promotes cell proliferation. Although the extreme instability of Myc plays
an important role in preventing its accumulation in normal cells, little is
known about how Myc is targeted for rapid destruction. Here, we have
investigated mechanisms regulating the stability of Myc. We show that Myc is
destroyed by ubiquitin-mediated proteolysis, and define two elements in Myc that
oppositely regulate its stability: a transcriptional activation domain that
promotes Myc destruction, and a region required for association with the POZ
domain protein Miz-1 that stabilizes Myc. We also show that Myc is stabilized by
cancer-associated and transforming mutations within its transcriptional
activation domain. Our data reveal a complex network of interactions regulating
Myc destruction, and imply that enhanced protein stability contributes to
oncogenic transformation by mutant Myc proteins.
PMID: 9927431 [PubMed - indexed for MEDLINE]
831: EMBO J 1999 Feb 1;18(3):632-43
Bcl-xL regulates apoptosis by heterodimerization-dependent and -independent
mechanisms.
Minn AJ, Kettlun CS, Liang H, Kelekar A, Vander Heiden MG, Chang BS, Fesik SW,
Fill M, Thompson CB.
Gwen Knapp Center for Lupus and Immunology Research, Chicago, IL, USA.
A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is
responsible for interactions between Bcl-xL and BH3-containing death agonists.
Mutants were constructed which did not bind to Bax but retained anti-apoptotic
activity. Since Bcl-xL can form an ion channel in synthetic lipid membranes, the
possibility that this property has a role in heterodimerization-independent cell
survival was tested by replacing amino acids within the predicted
channel-forming domain with the corresponding amino acids from Bax. The
resulting chimera showed a reduced ability to adopt an open conductance state
over a wide range of membrane potentials. Although this construct retained the
ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation was
introduced that rendered the chimera incapable of heterodimerization, the
resulting protein failed to prevent both apoptosis in mammalian cells and
Bax-mediated growth defect in yeast. Similar to mammalian cells undergoing
apoptosis, yeast cells expressing Bax exhibited changes in mitochondrial
properties that were inhibited by Bcl-xL through heterodimerization-dependent
and -independent mechanisms. These data suggest that Bcl-xL regulates cell
survival by at least two distinct mechanisms; one is associated with
heterodimerization and the other with the ability to form a sustained ion
channel.
PMID: 9927423 [PubMed - indexed for MEDLINE]
832: EMBO J 1999 Feb 1;18(3):555-64
Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins
with overlapping function.
Poon PP, Cassel D, Spang A, Rotman M, Pick E, Singer RA, Johnston GC.
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova
Scotia, Canada B3H 4H7.
ARF proteins, which mediate vesicular transport, have little or no intrinsic
GTPase activity. They rely on the actions of GTPase-activating proteins (GAPs)
for their function. The in vitro GTPase activity of the Saccharomyces cerevisiae
ARF proteins Arf1 and Arf2 is stimulated by the yeast Gcs1 protein, and in vivo
genetic interactions between arf and gcs1 mutations implicate Gcs1 in vesicular
transport. However, the Gcs1 protein is dispensable, indicating that additional
ARF GAP proteins exist. We show that the structurally related protein Glo3,
which is also dispensable, also exhibits ARF GAP activity. Genetic and in vitro
approaches reveal that Glo3 and Gcs1 have an overlapping essential function at
the endoplasmic reticulum (ER)-Golgi stage of vesicular transport. Mutant cells
deficient for both ARF GAPs cannot proliferate, undergo a dramatic accumulation
of ER and are defective for protein transport between ER and Golgi. The
glo3Delta and gcs1Delta single mutations each interact with a sec21 mutation
that affects a component of COPI, which mediates vesicular transport within the
ER-Golgi shuttle, while increased dosage of the BET1, BOS1 and SEC22 genes
encoding members of a v-SNARE family that functions within the ER-Golgi
alleviates the effects of a glo3Delta mutation. An in vitro assay indicates that
efficient retrieval from the Golgi to the ER requires these two proteins. These
findings suggest that Glo3 and Gcs1 ARF GAPs mediate retrograde vesicular
transport from the Golgi to the ER.
PMID: 9927415 [PubMed - indexed for MEDLINE]
833: Biochemistry 1998 Dec 22;37(51):17673-9
Dual function C-terminal domain of dynamin-1: modulation of self-assembly by
interaction of the assembly site with SH3 domains.
Scaife R, Venien-Bryan C, Margolis RL.
Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS), Grenoble, France.
Impairment of endocytosis by mutational targeting of dynamin-1 GTPases can
result in paralysis and embryonic lethality. Dynamin-1 assembles at coated pits
where it functions to cleave vesicles from donor membranes. Receptor endocytosis
is modulated by SH3 (src homology 3) domain proteins, which directly bind to
dynamin C-terminal proline motif sequences, affecting both the dynamin GTPase
activity and its recruitment to coated pits. We have determined that
dynamin-dynamin interactions, which are required for dynamin helix formation,
involve these same SH3 domain-binding C-terminal proline motif sequences.
Consequently, SH3 domain proteins induce the in vitro disassembly of dynamin
helices. Our results therefore suggest the the dual function of the dynamin
C-terminus (involving amino acids 800-840) permits direct regulation of dynamin
assembly and function through interaction with SH3 domain proteins.
Additionally, the N-terminal GTPase domain plays an important role in assembly.
Finally, we show that the central PH (pleckstrin homology) domain exerts a
strong inhibitory effect on the capacity for dynamin-1 self-assembly.
PMID: 9922133 [PubMed - indexed for MEDLINE]
834: Biochemistry 1998 Dec 22;37(51):17637-41
Structure and function of the core histone N-termini: more than meets the eye.
Hansen JC, Tse C, Wolffe AP.
Department of Biochemistry, The University of Texas Health Science Center at San
Antonio 78284-7760, USA. hansen@bioc02.uthscsa.edu
For two decades, the core histone N-termini generally have been thought of as
unstructured domains whose function is to bind to DNA and screen negative
charge. New data indicates that both the molecular mechanisms of action and
biological functions of the core histone N-termini in chromatin are considerably
more complex. At the level of the chromatin fiber, multiple distinct functions
of the N-termini are required to achieve higher order chromatin condensation,
two of which apparently involve protein-protein rather than protein-DNA
interactions. In addition, the N-termini have been documented to participate in
specific interactions with many chromatin-associated regulatory proteins. Here,
we discuss evidence supporting the new concepts that when functioning in their
natural chromatin context, (1) the N-termini are engaged primarily in
protein-protein interactions, (2) as a consequence of these interactions the
N-termini adopt specific secondary structure, (3) posttranslational
modifications such as acetylation disrupt the ability of the N-termini to form
secondary structure, and (4) because the N-termini perform essential roles in
both chromatin condensation and also bind specific chromatin-associated
proteins, the global structure and function of any given region of the genome
will be determined predominantly by the core histone N-termini and their
specific interaction partners.
Publication Types:
Review
Review, Tutorial
PMID: 9922128 [PubMed - indexed for MEDLINE]
835: J Biol Chem 1999 Jan 29;274(5):2609-12
A built-in arginine finger triggers the self-stimulatory GTPase-activating
activity of rho family GTPases.
Zhang B, Zhang Y, Collins CC, Johnson DI, Zheng Y.
Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163,
USA.
Signal transduction through the Rho family GTPases requires regulated cycling of
the GTPases between the active GTP-bound state and the inactive GDP-bound state.
Rho family members containing an arginine residue at position 186 in the
C-terminal polybasic region were found to possess a self-stimulatory
GTPase-activating protein (GAP) activity through homophilic interaction,
resulting in significantly enhanced intrinsic GTPase activities. This arginine
residue functions effectively as an "arginine finger" in the GTPase activating
reaction to confer the catalytic GAP activity but is not essential for the
homophilic binding interactions of Rho family proteins. The arginine
186-mediated negative regulation seems to be absent from Cdc42, a Rho family
member important for cell-division cycle regulation, of lower eukaryotes, yet
appears to be a part of the turn-off machinery of Cdc42 from higher eukaryotes.
Introduction of the arginine 186 mutation into S. cerevisiae CDC42 led to
phenotypes consistent with down-regulated CDC42 function. Thus, specific Rho
family GTPases may utilize a built-in arginine finger, in addition to RhoGAPs,
for negative regulation.
PMID: 9915787 [PubMed - indexed for MEDLINE]
836: Eur J Biochem 1999 Jan;259(1-2):112-9
Protein interactions of Gts1p of Saccharomyces cerevisiae throughout a region
similar to a cytoplasmic portion of some ATP-binding cassette transporters.
Kawabata K, Mitsui K, Uno T, Tamura K, Tsurugi K.
Department of Internal Medicine 2, Yamanaashi Medical University, Yamanashi,
Japan.
The GTS1 gene product, Gts1p, has pleiotropic effects on the timing of budding,
cell size, heat tolerance, sporulation and the lifespan of the yeast
Saccharomyces cerevisiae. In this study, we found (using the yeast two-hybrid
system) that Gts1p forms homodimers throughout the 18-amino acid region 296-313
which has considerable similarity to a region downstream of the Walker
nucleotide-binding motif A of some ATP-binding cassette (ABC) transporters. The
region contains two aspartic acid residues at 301 and 310 preceded by
hydrophobic amino acid residues, and Gts1p with an Asp310 to Ala substitution
showed considerably reduced homodimerization, as shown by the two-hybrid assay.
Overexpression of the point-mutated Gts1p did not efficiently induce the
Gts1p-related phenotypes described above, suggesting that the homodimerization
of Gts1p is required for it to function in vivo. The C-terminal cytoplasmic
domain of the yeast ABC transporters Mdl1p (multidrug resistance-like
transporter) and Ycf1p (yeast cadmium factor or glutathione S-conjugate pump)
bound to Gts1p in the two-hybrid system, and the heterodimerization activity of
the Gts1p with the Asp301 to Ala substitution was more affected than the Gts1p
with the Asp310 to Ala substitution. Overexpression of GTS1 considerably
reduced, and disruption of GTS1 slightly decreased, cellular resistance to
cycloheximide, cadmium, cisplatin and 1-chloro-2,4-dinitrophenol, which (except
for cycloheximide) are all substrates of Ycf1p. These results suggest that Gts1p
interacts with some ABC transporters through the binding site overlapping that
of homodimerization and modulates their activity.
PMID: 9914482 [PubMed - indexed for MEDLINE]
837: Mol Gen Genet 1998 Dec;260(5):492-502
GRISEA, a copper-modulated transcription factor from Podospora anserina involved
in senescence and morphogenesis, is an ortholog of MAC1 in Saccharomyces
cerevisiae.
Borghouts C, Osiewacz HD.
Abteilung Molekulare Entwicklungsbiologie und Biotechnologie, Botanisches
Institut Johann Wolfgang Goethe Universitat, Frankfurt am Main, Germany.
The initial characterization of Grisea suggested that this gene codes for a
transcription factor involved in the genetic control of cellular copper
homeostasis in Podospora anserina. Here we demonstrate that GRISEA activates in
vivo gene expression in Saccharomyces cerevisiae and is characterized by a
modular organization. The DNA-binding domain was mapped to the first 168
N-terminal amino acids and the transactivation domain to the C-terminal half of
the protein. Increased levels of copper in the growth medium lead to repression
of the transactivation function possibly via intramolecular interactions between
parts of the DNA-binding domain and the transactivation domain. The wild-type
copy of Grisea was found to complement the phenotype of the mac1-1 mutant of S.
cerevisiae. GRISEA is able to bind to the promoter of CTR1, a MAC1 target gene
that encodes a high-affinity copper transporter. Taken together, the data
reported here and in earlier investigations indicate that GRISEA is an ortholog
of the yeast transcription factor MAC1 and suggest at least a partial
conservation of the molecular machinery involved in the control of cellular
copper homeostasis in eukaryotes. Remarkably, in P. anserina, the spectrum of
phenotypes affected by this regulatory protein is much broader than that known
in yeast and includes morphogenetic traits as well as lifespan and senescence.
PMID: 9894921 [PubMed - indexed for MEDLINE]
838: Nature 1999 Jan 7;397(6714):69-72
GABA(A)-receptor-associated protein links GABA(A) receptors and the
cytoskeleton.
Wang H, Bedford FK, Brandon NJ, Moss SJ, Olsen RW.
Molecular Biology Institute, University of California, Los Angeles 90095, USA.
Type-A receptors for the neurotransmitter GABA (gamma-aminobutyric acid) are
ligand-gated chloride channels that mediate inhibitory neurotransmission. Each
subunit of the pentameric receptor protein has ligand-binding sites in the
amino-terminal extracellular domain and four membrane-spanning regions, one of
which forms a wall of the ion channel. Each subunit also has a large
intracellular loop that may be a target for protein kinases and be required for
subcellular targeting and membrane clustering of the receptor, perhaps by
anchoring the receptor to the cytoskeleton. Neurotransmitter receptors need to
be positioned in high density in the cell membrane at sites postsynaptic to
nerve terminals releasing that neurotransmitter. Other members of the
superfamily of ligand-gated ion-channel receptors associate in
postsynaptic-membrane clusters by binding to the proteins rapsyn or gephyrin.
Here we identify a new cellular protein, GABA(A)-receptor-associated protein
(GABARAP), which can interact with the gamma2 subunit of GABA(A) receptors.
GABARAP binds to GABA(A) receptors both in vitro and in vivo, and co-localizes
with the punctate staining of GABA(A) receptors on cultured cortical neurons.
Sequence analysis shows similarity between GABARAP and light chain-3 of
microtubule-associated proteins 1A and 1B. Moreover, the N terminus of GABARAP
is highly positively charged and features a putative tubulin-binding motif. The
interactions among GABA(A) receptors, GABARAP and tubulin suggest a mechanism
for the targeting and clustering of GABA(A) receptors.
PMID: 9892355 [PubMed - indexed for MEDLINE]
839: Mol Cell Biol 1999 Feb;19(2):1547-57
Interactions between a nuclear transporter and a subset of nuclear pore complex
proteins depend on Ran GTPase.
Seedorf M, Damelin M, Kahana J, Taura T, Silver PA.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, and The Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Proteins to be transported into the nucleus are recognized by members of the
importin-karyopherin nuclear transport receptor family. After docking at the
nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous
pore channel. Once cargo is released, the importin then moves back through the
channel for new rounds of transport. Thus, importin and exportin, another member
of this family involved in export, are thought to continuously shuttle between
the nuclear interior and the cytoplasm. In order to understand how nuclear
transporters traverse the NPC, we constructed functional protein fusions between
several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and
Kap95p, and the green fluorescent protein (GFP). Complexes containing nuclear
transporters were isolated by using highly specific anti-GFP antibodies.
Pse1-GFP was studied in the most detail. Pse1-GFP is in a complex with
importin-alpha and -beta (Srp1p and Kap95p in yeast cells) that is sensitive to
the nucleotide-bound state of the Ran GTPase. In addition, Pse1p associates with
the nucleoporins Nsp1p, Nup159p, and Nup116p, while Sxm1p, Xpo1p, and Kap95p
show different patterns of interaction with nucleoporins. Association of Pse1p
with nucleoporins also depends on the nucleotide-bound state of Ran; when Ran is
in the GTP-bound state, the nucleoporin association is lost. A mutant form of
Pse1p that does not bind Ran also fails to interact with nucleoporins. These
data indicate that transport receptors such as Pse1p interact in a Ran-dependent
manner with certain nucleoporins. These nucleoporins may represent major docking
sites for Pse1p as it moves in or out of the nucleus via the NPC.
PMID: 9891088 [PubMed - indexed for MEDLINE]
840: Mol Cell Biol 1999 Feb;19(2):1325-33
Antagonistic interactions between yeast chaperones Hsp104 and Hsp70 in prion
curing.
Newnam GP, Wegrzyn RD, Lindquist SL, Chernoff YO.
School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230,
USA.
The maintenance of [PSI], a prion-like form of the yeast release factor Sup35,
requires a specific concentration of the chaperone protein Hsp104: either
deletion or overexpression of Hsp104 will cure cells of [PSI]. A major puzzle of
these studies was that overexpression of Hsp104 alone, from a heterologous
promoter, cures cells of [PSI] very efficiently, yet the natural induction of
Hsp104 with heat shock, stationary-phase growth, or sporulation does not. These
observations pointed to a mechanism for protecting the genetic information
carried by the [PSI] element from vicissitudes of the environment. Here, we show
that simultaneous overexpression of Ssa1, a protein of the Hsp70 family,
protects [PSI] from curing by overexpression of Hsp104. Ssa1 protein belongs to
the Ssa subfamily, members of which are normally induced with Hsp104 during heat
shock, stationary-phase growth, and sporulation. At the molecular level, excess
Ssa1 prevents a shift of Sup35 protein from the insoluble (prion) to the soluble
(cellular) state in the presence of excess Hsp104. Overexpression of Ssa1 also
increases nonsense suppression by [PSI] when Hsp104 is expressed at its normal
level. In contrast, hsp104 deletion strains lose [PSI] even in the presence of
overproduced Ssa1. Overproduction of the unrelated chaperone protein Hsp82
(Hsp90) neither cured [PSI] nor antagonized the [PSI]-curing effect of
overproduced Hsp104. Our results suggest it is the interplay between Hsp104 and
Hsp70 that allows the maintenance of [PSI] under natural growth conditions.
PMID: 9891066 [PubMed - indexed for MEDLINE]
841: Mol Cell Biol 1999 Feb;19(2):1056-67
A complex containing RNA polymerase II, Paf1p, Cdc73p, Hpr1p, and Ccr4p plays a
role in protein kinase C signaling.
Chang M, French-Cornay D, Fan HY, Klein H, Denis CL, Jaehning JA.
Department of Biochemistry and Molecular Genetics and Program in Molecular
Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262,
USA.
Yeast contains at least two complex forms of RNA polymerase II (Pol II), one
including the Srbps and a second biochemically distinct form defined by the
presence of Paf1p and Cdc73p (X. Shi et al., Mol. Cell. Biol. 17:1160-1169,
1997). In this work we demonstrate that Ccr4p and Hpr1p are components of the
Paf1p-Cdc73p-Pol II complex. We have found many synthetic genetic interactions
between factors within the Paf1p-Cdc73p complex, including the lethality of
paf1Delta ccr4Delta, paf1Delta hpr1Delta, ccr4Delta hpr1Delta, and ccr4Delta
gal11Delta double mutants. In addition, paf1Delta and ccr4Delta are lethal in
combination with srb5Delta, indicating that the factors within and between the
two RNA polymerase II complexes have overlapping essential functions. We have
used differential display to identify several genes whose expression is affected
by mutations in components of the Paf1p-Cdc73p-Pol II complex. Additionally, as
previously observed for hpr1Delta, deleting PAF1 or CDC73 leads to elevated
recombination between direct repeats. The paf1Delta and ccr4Delta mutations, as
well as gal11Delta, demonstrate sensitivity to cell wall-damaging agents, rescue
of the temperature-sensitive phenotype by sorbitol, and reduced expression of
genes involved in cell wall biosynthesis. This unusual combination of effects on
recombination and cell wall integrity has also been observed for mutations in
genes in the Pkc1p-Mpk1p kinase cascade. Consistent with a role for this novel
form of RNA polymerase II in the Pkc1p-Mpk1p signaling pathway, we find that
paf1Delta mpk1Delta and paf1Delta pkc1Delta double mutants do not demonstrate an
enhanced phenotype relative to the single mutants. Our observation that the
Mpk1p kinase is fully active in a paf1Delta strain indicates that the
Paf1p-Cdc73p complex may function downstream of the Pkc1p-Mpk1p cascade to
regulate the expression of a subset of yeast genes.
PMID: 9891041 [PubMed - indexed for MEDLINE]
842: Mol Cell Biol 1999 Feb;19(2):1049-55
Selective interaction of vitamin D receptor with transcriptional coactivators by
a vitamin D analog.
Takeyama K, Masuhiro Y, Fuse H, Endoh H, Murayama A, Kitanaka S, Suzawa M,
Yanagisawa J, Kato S.
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo
113, Japan.
The nuclear vitamin D receptor (VDR) is a member of a nuclear receptor
superfamily and acts as a ligand-dependent transcription factor. A family of
cotranscriptional activators (SRC-1, TIF2, and AIB-1) interacts with and
activates the transactivation function of nuclear receptors in a
ligand-dependent way. We examined interaction of VDR with these coactivators
that was induced by several vitamin D analogs, since they exert differential
subsets of the biological action of vitamin D through unknown mechanisms. Unlike
other vitamin D analogs tested, OCT (22-oxa-1alpha,25-dihydroxyvitamin D3)
induced interaction of VDR with TIF2 but not with SRC-1 or AIB-1. Consistent
with these interactions, only TIF2 was able to potentiate the transactivation
function of VDR bound to OCT. Thus, the present findings suggest that the
structure of VDR is altered in a vitamin D analog-specific way, resulting in
selective interactions of VDR with coactivators. Such selective interaction of
coactivators with VDR may specify the array of biological actions of a vitamin D
analog like OCT, possibly through activating a particular set of target gene
promoters.
PMID: 9891040 [PubMed - indexed for MEDLINE]
843: J Biol Chem 1999 Jan 22;274(4):1928-33
Heterochromatin organization of a natural yeast telomere. Recruitment of Sir3p
through interaction with histone H4 N terminus is required for the establishment
of repressive structures.
Venditti S, Vega-Palas MA, Di Mauro E.
Fondazione "Istituto Pasteur-Fondazione Cenci-Bolognetti", c/o Dipartimento di
Genetica e Biologia Molecolare Universita "La Sapienza", P. le A. Moro 5,
00185-Roma Italy.
The chromatin organization of eukaryotic telomeres is essential for telomeric
function and is currently receiving great attention. In yeast, the structural
organization of telomeres involves a complex interplay of telomeric proteins
that results in the formation of heterochromatin. This telomeric heterochromatin
involves homotypic and heterotypic protein interactions that have been
summarized in a general model. Recent analyses have focused on the study of the
structural complexity at yeast telomeres to the level of specific nucleosomes
and of the distribution of protein complexes in a natural telomeric region
(LIII). In this report, we further analyze the structural complexity of LIII and
the implication of this structure on telomeric silencing. It is shown that the
establishment of repressive heterochromatin structures at LIII requires the
recruitment of Sir3p through interaction with the N terminus of histone H4. The
establishment of such structures does not require acetylation of any of four
lysines located in the H4 N terminus (lysines 5, 8, 12, and 16).
PMID: 9890947 [PubMed - indexed for MEDLINE]
844: Nat Struct Biol 1999 Jan;6(1):22-7
Structure of HAP1-18-DNA implicates direct allosteric effect of protein-DNA
interactions on transcriptional activation.
King DA, Zhang L, Guarente L, Marmorstein R.
The Wistar Institute and The Department of Chemistry, University of
Pennsylvania, Philadelphia 19104, USA.
HAP1 is a yeast transcriptional activator that binds with equal affinity to the
dissimilar upstream activation sequences UAS1 and UAS(CYC7), but activates
transcription differentially when bound to each site. HAP1-18 harbors an amino
acid change in the DNA binding domain. While binding UAS1 poorly, HAP1-18 binds
UAS(CYC7) with wild-type properties and activates transcription at elevated
levels relative to HAP1. We have determined the structure of HAP1-18-UAS(CYC7)
and have compared it to HAP1-UAS(CYC7). Unexpectedly, the single amino acid
substitution in HAP1-18 nucleates a significantly altered hydrogen bond
interface between the protein and DNA resulting in DNA conformational changes
and an ordering of one N-terminal arm of the protein dimer along the DNA minor
groove. These observations, together with a large subset of transcriptionally
defective mutations in the HAP1 DNA-binding domain that map to the HAP1-DNA
interface, suggest that protein-DNA interactions may have direct allosteric
effects on transcriptional activation.
PMID: 9886287 [PubMed - indexed for MEDLINE]
845: Toxicol Appl Pharmacol 1999 Jan 1;154(1):76-83
New screening methods for chemicals with hormonal activities using interaction
of nuclear hormone receptor with coactivator.
Nishikawa J, Saito K, Goto J, Dakeyama F, Matsuo M, Nishihara T.
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka,
Suita, Osaka, 565-0871, Japan. nishihara@phs.osaka-u.ac.jp
The endocrine system exerts important functions in a multitude of physiological
processes including embryogenesis, differentiation, and homeostasis. Xenobiotics
may modify natural endocrine function and so affect human health and wildlife.
It is necessary, therefore, to understand the degree to which xenobiotics can
disrupt endocrine systems. The key targets of endocrine disruptors are nuclear
hormone receptors, which bind to steroid hormones and regulate their gene
transcription. We have developed relevant assay systems based on the
ligand-dependent interaction between nuclear hormone receptor and coactivator.
The coactivators used in this study contained CBP, p300, RIP140, SRC1, TIF1, and
TIF2. By two hybrid assay in yeast, the interactions of estrogen receptor with
RIP140, SRC1, TIF1, and TIF2 were detected and they were completely dependent on
the presence of estrogen. Specificity of this assay was assessed by determining
the effect of steroids, known estrogen receptor agonists, and phytoestrogens.
The pattern of response to chemicals were consistent with estrogenic activity
measured by other assay systems, indicating that this assay system is reliable
for measuring estrogenic activity. In addition, we carried out in vitro binding
studies: GST pull-down assay and surface plasmon resonance analysis. The
estrogen receptor also bound to coactivator in response to chemicals depending
on their estrogenic activity in vitro. These data demonstrate that the
measurement of interaction between steroid hormone receptor and coactivator
serves as a useful tool for identifying chemicals that interact with steroid
receptors. Copyright 1999 Academic Press.
PMID: 9882594 [PubMed - indexed for MEDLINE]
846: J Biochem (Tokyo) 1999 Jan;125(1):130-7
Identification of SEC12, SED4, truncated SEC16, and EKS1/HRD3 as multicopy
suppressors of ts mutants of Sar1 GTPase.
Saito Y, Yamanushi T, Oka T, Nakano A.
Molecular Membrane Biology Laboratory, RIKEN, Wako, Saitama, 351-0198, Japan.
The yeast SAR1 gene encodes a low-molecular-weight GTPase which is essential for
the formation of transport vesicles from the endoplasmic reticulum (ER). To
understand how the Sar1p function is regulated in its GTPase cycle, we searched
for multicopy suppressors of sar1 temperature-sensitive mutants and identified
SEC12, SED4, truncated SEC16, and EKS1. EKS1 turns out to be identical to HRD3,
which was independently isolated as a gene implicated in the degradation of an
HMG-CoA reductase isozyme, Hmg2p. In this paper, we show that the product of
EKS1/HRD3 is a type-I transmembrane glycoprotein and resides in the ER. The
eks1/hrd3 disrupted cells are normal in growth and transport of cargo proteins,
but missecrete BiP (Kar2p). The overexpression of EKS1/HRD3, which stabilizes
Hmg2p, did not affect the stability of wild-type or mutant Sar1p or any early
Sec proteins we examined. These results suggest that the role of Eks1p/Hrd3p is
not involved in the ER protein degradation in general but rather required for
the maintenance of the ER membrane functions. The novel genetic interactions
unveiled between SAR1, SEC12, SEC16, and SED4 will provide useful information as
to how the complex machinery of vesicle budding operates.
PMID: 9880808 [PubMed - indexed for MEDLINE]
847: Biochim Biophys Acta 1999 Jan 6;1426(2):323-34
The KTR and MNN1 mannosyltransferase families of Saccharomyces cerevisiae.
Lussier M, Sdicu AM, Bussey H.
Department of Biology, McGill University, 1205 Dr. Penfield Avenue, Montreal,
Que. H3A 1B1, Canada.
Glycosylation constitutes one of the most important of all the
post-translational modifications and may have numerous effects on the function,
structure, physical properties and targeting of particular proteins. Eukaryotic
glycan structures are progressively elaborated in the secretory pathway.
Following the addition of a core N-linked carbohydrate in the endoplasmic
reticulum, glycoproteins move to the Golgi complex where the elongation of
O-linked sugar chains and processing of complex N-linked oligosaccharide
structures take place. In order to better define how such post-translational
modifications occur, we have been studying the yeast KTR and MNN1
mannosyltransferase gene families. The KTR family contains nine members: KRE2,
YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6 and KTR7. The MNN1 family contains six
members: MNN1, TTP1, YGL257c, YNR059w, YIL014w and YJL86w. In this review, we
address protein structure, sequence similarities and enzymatic activity in the
context of each gene family. In addition, a description of the known function of
many family members in O- and N-linked glycosylation is included. Finally, the
genetic interactions and functional redundancies within a gene family are also
discussed.
Publication Types:
Review
Review, Tutorial
PMID: 9878809 [PubMed - indexed for MEDLINE]
848: Biochim Biophys Acta 1999 Jan 6;1426(2):309-22
Asparagine-linked glycosylation in the yeast Golgi.
Dean N.
Department of Biochemistry and Cell Biology, Institute for Cell and
Developmental Biology, State University of New York, Stony Brook, NY 11794-5215,
USA. ndean@mcbsgi.bio.sunnysb.edu
The Golgi complex is the site where the terminal carbohydrate modification of
proteins and lipids occurs. These carbohydrates play a variety of biological
roles, ranging from the stabilization of glycoprotein structure to the provision
of ligands for cell-cell interactions to the regulation of cell surface
properties. Progress in our understanding of the biosynthesis and regulation of
glycoconjugates has been accelerating at a rapid pace. Recent advances in the
field of yeast glycobiology have been particularly impressive. This review
focuses on glycosylation of proteins in the Golgi of the yeast Saccharomyces
cerevisiae, with emphasis on the candidate mannosyltransferases that participate
in the synthesis of N-linked oligosaccharides. Current views on how these
enzymes may be regulated and how glycosylation relates on other cellular
processes are also discussed.
Publication Types:
Review
Review, Tutorial
PMID: 9878803 [PubMed - indexed for MEDLINE]
849: J Mol Biol 1998 Dec 18;284(5):1341-51
Conserved core structure in the internal transcribed spacer 1 of the
Schizosaccharomyces pombe precursor ribosomal RNA.
Lalev AI, Nazar RN.
Department of Molecular Biology and Genetics, University of Guelph, Guelph,
Ontario, N1G 2W1, Canada.
The structure of the internal transcribed spacer 1 (ITS1) in Schizosaccharomyces
pombe was examined with respect to phylogenetically conserved features in yeasts
as well as the binding of transacting factors that potentially play a role in
ribosomal maturation. Computer analyses and probes for nuclease protection
indicate a compact, more highly organized structure than previously proposed in
Saccharomyces cerevisiae, with distinct structural features which can be
recognized in S. cerevisiae. These include a central extended hairpin structure
as well as smaller hairpins immediately adjacent to the maturing termini.
Comparisons with ITS sequences in more diverse organisms indicate that the same
features also can be recognized. This is especially clear in organisms which
contain very short sequences in which the putative structures are much less
ambiguous. Again nuclease protection analyses in one of these, Verticillium
albo-atrum, confirm a central hairpin with additional hairpins linked to the
maturing termini. Protein binding and gel retardation studies with the S. pombe
ITS1 further indicate that, as observed in the 3' external transcripted spacer
(ETS) region, the extended hairpin is not only the site of intermediate RNA
cleavage during rRNA processing, but also a site for specific interactions with
one or more soluble factors. Taken together with other analyses on transcribed
spacer regions, the present data provide evidence that the spacer regions act
not only to organize the maturing terminal sequences but also may serve to
organize specific soluble factors, possibly acting in a manner which is
analogous with that of the free small nucleolar ribonucleo protein particles
(snoRNPs). Copyright 1998 Academic Press
PMID: 9878354 [PubMed - indexed for MEDLINE]
850: EMBO J 1999 Jan 4;18(1):58-64
Epistatic interactions of deletion mutants in the genes encoding the F1-ATPase
in yeast Saccharomyces cerevisiae.
Lai-Zhang J, Xiao Y, Mueller DM.
Department of Biochemistry and Molecular Biology, The Chicago Medical School,
North Chicago, IL 60064, USA.
The F1-ATPase is a multimeric enzyme (alpha3 beta3 gamma delta epsilon)
primarily responsible for the synthesis of ATP under aerobic conditions. The
entire coding region of each of the genes was deleted separately in yeast,
providing five null mutant strains. Strains with a deletion in the genes
encoding alpha-, beta-, gamma- or delta-subunits were unable to grow, while the
strain with a null mutation in epsilon was able to grow slowly on medium
containing glycerol as the carbon source. In addition, strains with a null
mutation in gamma or delta became 100% rho0/rho- and the strain with the null
mutation in gamma grew much more slowly on medium containing glucose. These
additional phenotypes were not observed in strains with the double mutations:
Delta alpha Delta gamma, Delta beta Delta gamma, Deltaatp11 Delta gamma, Delta
alpha Delta delta, Delta beta Delta delta or Deltaatp11 Delta delta. These
results indicate that epsilon is not an essential component of the ATP synthase
and that mutations in the genes encoding the alpha- and beta-subunits and in
ATP11 are epistatic to null mutations in the genes encoding the gamma- and
delta-subunits. These data suggest that the propensity to form rho0/rho-
mutations in the gamma and delta null deletion mutant stains and the slow
growing phenotypes of the null gamma mutant strain are due to the assembly of F1
deficient in the corresponding subunit. These results have profound implications
for the physiology of normal cells.
PMID: 9878050 [PubMed - indexed for MEDLINE]
851: Genetics 1999 Jan;151(1):31-44
A region of the Sir1 protein dedicated to recognition of a silencer and required
for interaction with the Orc1 protein in saccharomyces cerevisiae.
Gardner KA, Rine J, Fox CA.
Department of Biomolecular Chemistry, University of Wisconsin, Madison,
Wisconsin 53706, USA.
Silencing of the cryptic mating-type loci HMR and HML requires the recognition
of DNA sequence elements called silencers by the Sir1p, one of four proteins
dedicated to the assembly of silenced chromatin in Saccharomyces cerevisiae. The
Sir1p is thought to recognize silencers indirectly through interactions with
proteins that bind the silencer DNA directly, such as the origin recognition
complex (ORC). Eight recessive alleles of SIR1 were discovered that encode
mutant Sir1 proteins specifically defective in their ability to recognize the
HMR-E silencer. The eight missense mutations all map within a 17-amino-acid
segment of Sir1p, and this segment was also required for Sir1p's interaction
with Orc1p. The mutant Sir1 proteins could function in silencing if tethered to
a silencer directly through a heterologous DNA-binding domain. Thus the amino
acids identified are required for Sir1 protein's recognition of the HMR-E
silencer and interaction with Orc1p, but not for its ability to function in
silencing per se. The approach used to find these mutations may be applicable to
defining interaction surfaces on proteins involved in other processes that
require the assembly of macromolecular complexes.
PMID: 9872946 [PubMed - indexed for MEDLINE]
852: Genes Dev 1998 Dec 15;12(24):3843-56
Maintenance of sister-chromatid cohesion at the centromere by the Drosophila
MEI-S332 protein.
Tang TT, Bickel SE, Young LM, Orr-Weaver TL.
Whitehead Institute and Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts USA 02142, USA.
Sister-chromatid cohesion is essential for the faithful segregation of
chromosomes during cell division. Recently biochemical analysis with Xenopus
extracts suggests that cohesion is established during S phase by a cohesion
complex but that other proteins must maintain it in mitosis. The Drosophila
melanogaster MEI-S332 protein is present on centromeres in mitosis and meiosis
and is essential for cohesion at the centromeres in meiosis II. Here, we analyze
the timing of MEI-S332 assembly onto centromeres and the functional domains of
the MEI-S332 protein. We find that MEI-S332 is first detectable on chromosomes
during prometaphase, and this localization is independent of microtubules.
MEI-S332 contains two separable functional domains, as mutations within these
domains show intragenic complementation. The carboxy-terminal basic region is
required for chromosomal localization. The amino-terminal coiled-coil domain may
facilitate protein-protein interactions between MEI-S332 and male meiotic
proteins. MEI-S332 interacts with itself in the yeast two-hybrid assay and in
immunoprecipitates from Drosophila oocyte and embryo extracts. Thus it appears
that MEI-S332 assembles into a multimeric protein complex that localizes to
centromeric regions during prometaphase and is required for the maintenance of
sister-chromatid cohesion until anaphase, rather than its establishment in S
phase.
PMID: 9869638 [PubMed - indexed for MEDLINE]
853: Nature 1998 Dec 10;396(6711):587-90
Decoupling of nucleotide- and microtubule-binding sites in a kinesin mutant.
Song H, Endow SA.
Department of Microbiology, Duke University Medical Center, Durham, North
Carolina 27710, USA.
Molecular motors require ATP to move along microtubules or actin filaments. To
understand how molecular motors function, it is crucial to know how binding of
the motor to its filamentous track stimulates the hydrolysis of ATP by the
motor, enabling it to move along the filament. A mechanism for the enhanced ATP
hydrolysis has not been elucidated, but it is generally accepted that
conformational changes in the motor proteins occur when they bind to
microtubules or actin filaments, facilitating the release of ADP. Here we report
that a mutation in the motor domain of the microtubule motor proteins Kar3 and
Ncd uncouples nucleotide- and microtubule-binding by the proteins, preventing
activation of the motor ATPase by microtubules. Unlike the wild-type motors, the
mutants bind tightly to both ADP and microtubules, indicating that interactions
between the nucleotide- and microtubule-binding sites are blocked. The region of
the motor that includes the mutated amino acid could transmit or undergo a
conformational change required to convert the motor ATPase into a
microtubule-stimulated state.
PMID: 9859995 [PubMed - indexed for MEDLINE]
854: Mol Cell Biol 1999 Jan;19(1):826-34
Ribosomal protein S14 of Saccharomyces cerevisiae regulates its expression by
binding to RPS14B pre-mRNA and to 18S rRNA.
Fewell SW, Woolford JL Jr.
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh,
Pennsylvania 15213, USA.
Production of ribosomal protein S14 in Saccharomyces cerevisiae is coordinated
with the rate of ribosome assembly by a feedback mechanism that represses
expression of RPS14B. Three-hybrid assays in vivo and filter binding assays in
vitro demonstrate that rpS14 directly binds to an RNA stem-loop structure in
RPS14B pre-mRNA that is necessary for RPS14B regulation. Moreover, rpS14 binds
to a conserved helix in 18S rRNA with approximately five- to sixfold-greater
affinity. These results support the model that RPS14B regulation is mediated by
direct binding of rpS14 either to its pre-mRNA or to rRNA. Investigation of
these interactions with the three-hybrid system reveals two regions of rpS14
that are involved in RNA recognition. D52G and E55G mutations in rpS14 alter the
specificity of rpS14 for RNA, as indicated by increased affinity for RPS14B RNA
but reduced affinity for the rRNA target. Deletion of the C terminus of rpS14,
where multiple antibiotic resistance mutations map, prevents binding of rpS14 to
RNA and production of functional 40S subunits. The emetine-resistant protein,
rpS14-EmRR, which contains two mutations near the C terminus of rpS14, does not
bind either RNA target in the three-hybrid or in vitro assays. This is the first
direct demonstration that an antibiotic resistance mutation alters binding of an
r protein to rRNA and is consistent with the hypothesis that antibiotic
resistance mutations can result from local alterations in rRNA structure.
PMID: 9858605 [PubMed - indexed for MEDLINE]
855: Mol Cell Biol 1999 Jan;19(1):602-11
Genetic evidence for Pak1 autoinhibition and its release by Cdc42.
Tu H, Wigler M.
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Pak1 protein kinase of Schizosaccharomyces pombe, a member of the
p21-GTPase-activated protein kinase (PAK) family, participates in signaling
pathways including sexual differentiation and morphogenesis. The regulatory
domain of PAK proteins is thought to inhibit the kinase catalytic domain, as
truncation of this region renders kinases more active. Here we report the
detection in the two-hybrid system of the interaction between Pak1 regulatory
domain and the kinase catalytic domain. Pak1 catalytic domain binds to the same
highly conserved region on the regulatory domain that binds Cdc42, a GTPase
protein capable of activating Pak1. Two-hybrid, mutant, and genetic analyses
indicated that this intramolecular interaction rendered the kinase in a closed
and inactive configuration. We show that Cdc42 can induce an open configuration
of Pak1. We propose that Cdc42 interaction disrupts the intramolecular
interactions of Pak1, thereby releasing the kinase from autoinhibition.
PMID: 9858584 [PubMed - indexed for MEDLINE]
856: Mol Cells 1998 Oct 31;8(5):606-13
Isoform-specific interaction of the cytoplasmic domains of Na,K-ATPase.
Yoon T, Lee K.
College of Pharmacy, Ewha Woman's University, Seoul, Korea.
The Na,K-ATPase is a heterodimer consisting of an alpha and a beta subunit,
which exchanges intracellular Na+ for extracellular K+ using the energy of ATP
hydrolysis. Several studies have demonstrated that the enzyme exists as an
(alphabeta)2 heterotetramer, an oligomer of alphabeta dimers within the cell
membrane, at least during some portion of the transport cycle although its
functional significance is unknown. In our study, we employed the yeast
two-hybrid system to identify the cytoplasmic domains of the Na,K-ATPase which
might be involved in intersubunit and/or intrasubunit interactions to form
higher order oligmers. Our data demonstrate that the N-terminus and the
cytoplasmic loop 1 of the alpha2 subunit interact with each other, while those
of the alpha1 subunit do not, suggesting that the interaction is
isoform-specific. Therefore, the N-terminal and the cytoplasmic loop 1 might be
the regions where the alpha2 subunit, which are involved in alpha alpha
interactions, stabilize Na,K-ATPase as alphabeta protomer, diprotomer, or higher
order oligomer because the interaction can be intrasubunit as well as
intersubunit interactions. Our study suggests that there may be an
isoform-specific difference in the alpha-alpha interaction and that the
isoform-specific interaction may contribute significantly to the differences of
the physiological function and regulation among the alpha isoforms.
PMID: 9856349 [PubMed - indexed for MEDLINE]
857: Biochem J 1999 Jan 1;337 ( Pt 1):89-95
The role of the C-terminal region in phosphoglycerate mutase.
Walter RA, Nairn J, Duncan D, Price NC, Kelly SM, Rigden DJ, Fothergill-Gilmore
LA.
Department of Biochemistry, University of Edinburgh, George Square, Edinburgh
EH8 9XD, Scotland, U.K.
Removal of the C-terminal seven residues from phosphoglycerate mutase from
Saccharomyces cerevisiae by limited proteolysis is associated with loss of
mutase activity, but no change in phosphatase activity. The presence of the
cofactor 2, 3-bisphosphoglycerate, or of the cofactor and substrate
3-phosphoglycerate together, confers protection against proteolysis. The
substrate alone offers no protection. Replacement of either or both of the two
lysines at the C-terminus by glycines has only limited effects on the kinetic
properties of phosphoglycerate mutase, indicating that these residues are
unlikely to be involved in crucial electrostatic interactions with the
substrate, intermediate or product in the reaction. However, the double-mutant
form of the enzyme is more sensitive to proteolysis and is no longer protected
against proteolysis by the presence of cofactor. The proteolysed wild-type and
two of the mutated forms of the enzyme show a reduced response to
2-phosphoglycollate, which enhances the instability of the phospho form of the
native enzyme. The phosphoglycerate mutase from Schizosaccharomyces pombe, which
lacks the analogous C-terminal tail, has an inherently lower mutase activity and
is also less responsive to stimulation by 2-phosphoglycollate. It is proposed
that the C-terminal region of phosphoglycerate mutase helps to maintain the
enzyme in its active phosphorylated form and assists in the retention of the
bisphosphoglycerate intermediate at the active site. However, its role seems not
to be to contribute directly to ligand binding, but rather to exert indirect
effects on the transfer of the phospho group between substrate, enzyme,
intermediate and product.
PMID: 9854029 [PubMed - indexed for MEDLINE]
858: Biol Pharm Bull 1998 Nov;21(11):1215-7
Evaluation of fluorescence polarization method for binding study in
carbohydrate-lectin interaction.
Oda Y, Kinoshita M, Nakayama K, Kakehi K.
Faculty of Pharmaceutical Sciences, Kinki University, Higashi-osaka, Japan.
The fluorescence polarization (FP) technique was evaluated to determine
molecular interaction between plant lectins and polysaccharides, yeast cells and
glycopeptide after labeling the lectins with fluorescein isothiocyanate. Use of
Lycoris radiata agglutinin allowed determination of the molecular interactions
with large biomolecules containing mannose oligomers and polymers. Another
example using a fluorescein-labeled glycopeptide also indicated that use of the
FP method would allow easy observation of the molecular interactions on the
quantitative base. The present technique is highly sensitive and facile because
it does not require any washing procedures before measurement.
PMID: 9853416 [PubMed - indexed for MEDLINE]
859: J Biol Chem 1998 Dec 18;273(51):34653-60
Promoter structure and transcriptional activation with chromatin templates
assembled in vitro. A single Gal4-VP16 dimer binds to chromatin or to DNA with
comparable affinity.
Pazin MJ, Hermann JW, Kadonaga JT.
Department of Biology and Center for Molecular Genetics, University of
California, San Diego, La Jolla, California 92093-0347, USA.
To gain a better understanding of the role of chromatin in the regulation of
transcription by RNA polymerase II, we examined the relation between promoter
structure and the ability of Gal4-VP16 to function with chromatin templates
assembled in vitro. First, to investigate whether there are synergistic
interactions among multiple bound factors, we studied promoter constructions
containing one or five Gal4 sites and found that a single recognition site is
sufficient for Gal4-VP16 to bind to chromatin, to induce nucleosome
rearrangement, and to activate transcription. Notably, we observed that
Gal4-VP16 binds to a single site in chromatin with affinity comparable with that
which it binds to naked DNA, even in the absence of ATP-dependent nucleosome
remodeling activity. Second, to explore the relation between translational
nucleosome positioning and transcriptional activation, we analyzed a series of
promoter constructions in which nucleosomes were positioned by Gal4-VP16 at
different locations relative to the RNA start site. These experiments revealed
that the positioning of a nucleosome over the RNA start site is not an absolute
barrier to transcriptional activation. Third, to determine the contribution of
core promoter elements to transcriptional activation with chromatin templates,
we tested the ability of Gal4-VP16 to activate transcription with TATA box-
versus DPE-driven core promoters and found that the TATA box is not required to
achieve transcriptional activation by Gal4-VP16 with chromatin templates. These
results suggest that a single protomer of a strong activator is able to bind to
chromatin, to induce nucleosome remodeling, and to activate transcription in
conjunction with a broad range of chromatin structures and core promoter
elements.
PMID: 9852139 [PubMed - indexed for MEDLINE]
860: J Biol Chem 1998 Dec 18;273(51):34328-34
Evidence for a salt bridge between transmembrane segments 5 and 6 of the yeast
plasma-membrane H+-ATPase.
Gupta SS, DeWitt ND, Allen KE, Slayman CW.
Departments of Genetics and Cellular & Molecular Physiology, Yale University
School of Medicine, New Haven, Connecticut 06520, USA.
The plasma-membrane H+-ATPase of Saccharomyces cerevisiae, which belongs to the
P2 subgroup of cation-transporting ATPases, is encoded by the PMA1 gene and
functions physiologically to pump protons out of the cell. This study has
focused on hydrophobic transmembrane segments M5 and M6 of the H+-ATPase. In
particular, a conserved aspartate residue near the middle of M6 has been found
to play a critical role in the structure and biogenesis of the ATPase.
Site-directed mutants in which Asp-730 was replaced by an uncharged residue (Asn
or Val) were abnormally sensitive to trypsin, consistent with the idea that the
proteins were poorly folded, and immunofluorescence confocal microscopy showed
them to be arrested in the endoplasmic reticulum. Similar defects are known to
occur when either Arg-695 or His-701 in M5 is replaced by a neutral residue
(Dutra, M. B., Ambesi, A., and Slayman, C. W. (1998) J. Biol. Chem. 273,
17411-17417). To search for possible charge-charge interactions between Asp-730
and Arg-695 or His-701, double mutants were constructed in which positively and
negatively charged residues were swapped or eliminated. Strikingly, two of the
double mutants (R695D/D730R and R695A/D730A) regained the capacity for normal
biogenesis and displayed near-normal rates of ATP hydrolysis and ATP-dependent
H+ pumping. These results demonstrate that neither Arg-695 nor Asp-730 is
required for enzymatic activity or proton transport, but suggest that there is a
salt bridge between the two residues, linking M5 and M6 of the 100-kDa
polypeptide.
PMID: 9852098 [PubMed - indexed for MEDLINE]
861: RNA 1998 Dec;4(12):1675-86
Corrected and republished from:
RNA 1998 Oct; 4(10):1239-50
Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae.
Dix I, Russell CS, O'Keefe RT, Newman AJ, Beggs JD.
Institute of Cell and Molecular Biology, University of Edinburgh, United
Kingdom.
We present here the first insights into the organization of proteins on the RNA
in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly
labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting
proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller
proteins requiring an intact Sm site. Site-specific crosslinking showed that
Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with
five different regions of the 5' stem-loop, but not with the Sm site or 3'
stem-loop. Both internal loops in the 5' domain are essential for Prp8p to
associate with the snRNP, but the conserved loop 1 is not, although this is the
region to which Prp8p crosslinks most strongly. The extensive contacts between
Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in
spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing
that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p
may be the principal anchoring factor for exons in the spliceosome. This and the
close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p
support and extend the proposal that Snu114p mimics U5 loop 1 during a
translocation event in the spliceosome.
Publication Types:
Corrected and Republished Article
PMID: 9848662 [PubMed - indexed for MEDLINE]
862: Proc Natl Acad Sci U S A 1998 Dec 8;95(25):14897-902
Identification of a proline-binding motif regulating CD2-triggered T lymphocyte
activation.
Nishizawa K, Freund C, Li J, Wagner G, Reinherz EL.
Laboratory of Immunobiology, Dana-Farber Cancer Institute, and Department of
Medicine, Harvard Medical School, Boston, MA 02115, USA.
An intracellular protein termed CD2 binding protein 2 (CD2BP2), which binds to a
site containing two PPPGHR segments within the cytoplasmic region of CD2, was
identified. Mutagenesis and NMR analysis demonstrated that the CD2 binding
region of CD2BP2 includes a 17-aa motif (GPY[orF]xxxxM[orV]xxWxxx GYF), also
found in several yeast and Caenorhabditis elegans proteins of unknown function.
In Jurkat T cells, over-expression of the isolated CD2BP2 domain binding to CD2
enhances the production of interleukin 2 on crosslinking of CD2 but not the T
cell receptor. Hence, a proline-binding module distinct from SH3 and WW domains
regulates protein-protein interactions.
PMID: 9843987 [PubMed - indexed for MEDLINE]
863: Proc Natl Acad Sci U S A 1998 Dec 8;95(25):14799-804
The terminal tail region of a yeast myosin-V mediates its attachment to vacuole
membranes and sites of polarized growth.
Catlett NL, Weisman LS.
Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA.
The Saccharomyces cerevisiae myosin-V, Myo2p, has been implicated in the
polarized movement of several organelles and is essential for yeast viability.
We have shown previously that Myo2p is required for the movement of a portion of
the lysosome (vacuole) into the bud and consequently for proper inheritance of
this organelle during cell division. Class V myosins have a globular carboxyl
terminal tail domain that is proposed to mediate localization of the myosin,
possibly through interaction with organelle-specific receptors. Here we describe
a myo2 allele whose phenotypes support this hypothesis. vac15-1/myo2-2 has a
single mutation in this globular tail domain, causing defects in vacuole
movement and inheritance. Although a portion of wild-type Myo2p fractionates
with the vacuole, the myo2-2 gene product does not. In addition, the mutant
protein does not concentrate at sites of active growth, the predominant location
of wild-type Myo2p. Although deletion of the tail domain is lethal, the myo2-2
gene product retains the essential functions of Myo2p. Moreover, myo2-2 does not
cause the growth defects and lethal genetic interactions seen in myo2-66, a
mutant defective in the actin-binding domain. These observations suggest that
the myo2-2 mutation specifically disrupts interactions with selected myosin
receptors, namely those on the vacuole membrane and those at sites of polarized
growth.
PMID: 9843969 [PubMed - indexed for MEDLINE]
864: Mol Biol Cell 1998 Dec;9(12):3533-45
Specific molecular chaperone interactions and an ATP-dependent conformational
change are required during posttranslational protein translocation into the
yeast ER.
McClellan AJ, Endres JB, Vogel JP, Palazzi D, Rose MD, Brodsky JL.
Department of Biological Sciences, University of Pittsburgh, Pittsburgh,
Pennsylvania 15260, USA.
The posttranslational translocation of proteins across the endoplasmic reticulum
(ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK
homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the
cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they
cannot substitute for one another, possibly because they interact with specific
DnaJ homologues on each side of the ER membrane. To investigate this
possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a
GST-63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We
observed that BiP, but not Ssa1p, is able to associate with GST-63Jp and that
Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the
ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the
release of an unfolded polypeptide from Ssa1p but not from BiP. To understand
further how BiP drives protein translocation, we purified four dominant lethal
mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis,
fails to undergo an ATP-dependent conformational change, and cannot interact
with GST-63Jp. Measurements of protein translocation into reconstituted
proteoliposomes indicate that the mutants inhibit translocation even in the
presence of wild-type BiP. We conclude that a conformation- and ATP-dependent
interaction of BiP with the J domain of Sec63p is essential for protein
translocation and that the specificity of hsc70 action is dictated by their DnaJ
partners.
PMID: 9843586 [PubMed - indexed for MEDLINE]
865: Mol Biol Cell 1998 Dec;9(12):3475-92
Functional characterization of a Nup159p-containing nuclear pore subcomplex.
Belgareh N, Snay-Hodge C, Pasteau F, Dagher S, Cole CN, Doye V.
Centre National de la Recherche Scientifique, UMR144, Institut Curie, 75 248
Paris cedex 05, France.
Nup159p/Rat7p is an essential FG repeat-containing nucleoporin localized at the
cytoplasmic face of the nuclear pore complex (NPC) and involved in poly(A)+ RNA
export and NPC distribution. A detailed structural-functional analysis of this
nucleoporin previously demonstrated that Nup159p is anchored within the NPC
through its essential carboxyl-terminal domain. In this study, we demonstrate
that Nup159p specifically interacts through this domain with both Nsp1p and
Nup82p. Further analysis of the interactions within the Nup159p/Nsp1p/Nup82p
subcomplex using the nup82Delta108 mutant strain revealed that a deletion within
the carboxyl-terminal domain of Nup82p prevents its interaction with Nsp1p but
does not affect the interaction between Nup159p and Nsp1p. Moreover,
immunofluorescence analysis demonstrated that Nup159p is delocalized from the
NPC in nup82Delta108 cells grown at 37 degrees C, a temperature at which the
Nup82Delta108p mutant protein becomes degraded. This suggests that Nup82p may
act as a docking site for a core complex composed of the repeat-containing
nucleoporins Nup159p and Nsp1p. In vivo transport assays further revealed that
nup82Delta108 and nup159-1/rat7-1 mutant strains have little if any defect in
nuclear protein import and protein export. Together our data suggest that the
poly(A)+ RNA export defect previously observed in nup82 mutant cells might be
due to the loss from the NPCs of the repeat-containing nucleoporin Nup159p.
PMID: 9843582 [PubMed - indexed for MEDLINE]
866: EMBO J 1998 Dec 1;17(23):7105-17
A map of the binding site for catalytic domain 5 in the core of a group II
intron ribozyme.
Konforti BB, Liu Q, Pyle AM.
Department of Biochemistry and Molecular Biophysics, Columbia University, 701 W.
168th Street, Room 616, Hammer Health Sciences Center, New York, NY 10032, USA.
Group II introns are ribozymes with a complex tertiary architecture that is of
great interest as a model for RNA folding. Domain 5 (D5) is a highly conserved
region of the intron that is considered one of the most critical structures in
the catalytic core. Despite its central importance, the means by which D5
interacts with other core elements is unclear. To obtain a map of potential
interaction sites, dimethyl sulfate was used to footprint regions of the intron
that are involved in D5 binding. These studies were complemented by measurements
of D5 binding to a series of truncated intron derivatives. In this way, the
minimal region of the intron required for strong D5 association was defined and
the sites most likely to represent thermodynamically significant positions of
tertiary contact were identified. These studies show that ground-state D5
binding is mediated by tertiary contacts to specific regions of D1, including a
tetraloop receptor and an adjacent three-way junction. In contrast, D2 and D3
are not found to stabilize D5 association. These data highlight the significance
of D1-D5 interactions and will facilitate the identification of specific
tertiary contacts between them.
PMID: 9843514 [PubMed - indexed for MEDLINE]
867: Biochemistry 1998 Nov 10;37(45):15842-9
DNA sequence-specific recognition by the Saccharomyces cerevisiae "TATA" binding
protein: promoter-dependent differences in the thermodynamics and kinetics of
binding.
Petri V, Hsieh M, Jamison E, Brenowitz M.
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York
10461, USA.
The equilibrium binding and association kinetics of the Saccharomyces cerevisiae
TATA Binding Protein (TBP) to the E4 and Major Late promoters of adenovirus
(TATATATA and TATAAAAG, respectively), have been directly compared by
quantitative DNase I titration and quench-flow "footprinting". The equilibrium
binding of TBP to both promoters is described by the equilibrium TBP + DNA"TATA"
left and right arrow TBP-DNA"TATA". The salt dependence of TBP binding to both
promoters is identical within experimental error while the temperature
dependence differs significantly. The observed rate of association follows
simple second-order kinetics over the TBP concentration ranges investigated. The
salt and temperature dependencies of the second-order association rate constants
for TBP binding the two promoters reflect the dependencies determined by
equilibrium binding. The TBP-E4 promoter interaction is entropically driven at
low temperature and enthalpically driven at high temperature while the TBP-Major
Late promoter reaction is entropically driven over virtually the entire
temperature range investigated. These data suggest that the reaction mechanisms
of TBP-promoter interactions are TATA sequence-specific and provide for
differential regulation of promoters as a function of environmental variables.
PMID: 9843390 [PubMed - indexed for MEDLINE]
868: Biochemistry 1998 Nov 10;37(45):15726-36
SH3 binding domains in the dopamine D4 receptor.
Oldenhof J, Vickery R, Anafi M, Oak J, Ray A, Schoots O, Pawson T, von Zastrow
M, Van Tol HH.
Department of Pharmacology, Institute of Medical Science, University of Toronto,
Ontario, Canada.
The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to
the dopamine D2-like receptor family. Functionally, the D2-like receptors are
characterized by their ability to inhibit adenylyl cyclase. The dopamine D4
receptor as well as many other catecholaminergic receptors contain several
putative SH3 binding domains. Most of these sites in the D4 receptor are located
in a polymorphic repeat sequence and flanking sequences in the third
intracellular loop. Here we demonstrate that this region of the D4 receptor can
interact with a large variety of SH3 domains of different origin. The strongest
interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The
repeat sequence itself is not essential in this interaction. The data presented
indicate that the different SH3 domains in the adapter proteins interact in a
cooperative fashion with two distinct sites immediately upstream and downstream
from the repeat sequence. Removal of all the putative SH3 binding domains in the
third intracellular loop of the dopamine D4 receptor resulted in a receptor that
could still bind spiperone and dopamine. Dopamine could not modulate the
coupling of these mutant receptors to adenylyl cyclase and MAPK, although
dopamine modulated receptor-G protein interaction appeared normal. The receptor
deletion mutants show strong constitutive internalization that may account for
the deficiency in functional activation of second messengers. The data indicates
that the D4 receptor contains SH3 binding sites and that these sites fall within
a region involved in the control of receptor internalization.
PMID: 9843378 [PubMed - indexed for MEDLINE]
869: Microbiol Mol Biol Rev 1998 Dec;62(4):1492-553
Posttranscriptional control of gene expression in yeast.
McCarthy JE.
Posttranscriptional Control Group, Department of Biomolecular Sciences,
University of Manchester Institute of Science and Technology (UMIST), Manchester
M60 1QD, United Kingdom. J.McCarthy@umist.ac.uk
Studies of the budding yeast Saccharomyces cerevisiae have greatly advanced our
understanding of the posttranscriptional steps of eukaryotic gene expression.
Given the wide range of experimental tools applicable to S. cerevisiae and the
recent determination of its complete genomic sequence, many of the key
challenges of the posttranscriptional control field can be tackled particularly
effectively by using this organism. This article reviews the current knowledge
of the cellular components and mechanisms related to translation and mRNA decay,
with the emphasis on the molecular basis for rate control and gene regulation.
Recent progress in characterizing translation factors and their protein-protein
and RNA-protein interactions has been rapid. Against the background of a growing
body of structural information, the review discusses the thermodynamic and
kinetic principles that govern the translation process. As in prokaryotic
systems, translational initiation is a key point of control. Modulation of the
activities of translational initiation factors imposes global regulation in the
cell, while structural features of particular 5' untranslated regions, such as
upstream open reading frames and effector binding sites, allow for gene-specific
regulation. Recent data have revealed many new details of the molecular
mechanisms involved while providing insight into the functional overlaps and
molecular networking that are apparently a key feature of evolving cellular
systems. An overall picture of the mechanisms governing mRNA decay has only very
recently begun to develop. The latest work has revealed new information about
the mRNA decay pathways, the components of the mRNA degradation machinery, and
the way in which these might relate to the translation apparatus. Overall, major
challenges still to be addressed include the task of relating principles of
posttranscriptional control to cellular compartmentalization and polysome
structure and the role of molecular channelling in these highly complex
expression systems.
Publication Types:
Review
Review, Academic
PMID: 9841679 [PubMed - indexed for MEDLINE]
870: J Immunol Methods 1998 Nov 1;220(1-2):179-88
A yeast surface display system for the discovery of ligands that trigger cell
activation.
Cho BK, Kieke MC, Boder ET, Wittrup KD, Kranz DM.
Department of Biochemistry, University of Illinois, Urbana 61801, USA.
Opposing cells often communicate signalling events using multivalent
interactions between receptors present on their cell surface. For example, T
cells are typically activated when the T cell receptor (TCR) and its associated
costimulatory molecules are multivalently engaged by the appropriate ligands
present on an antigen presenting cell. In this report, yeast expressing high
cell-surface levels of a TCR ligand (a recombinant antibody to the TCR Vbeta
domain) were shown to act as 'pseudo' antigen presenting cells and induce T cell
activation as monitored by increased levels of CD25 and CD69 and by
downregulation of cell surface TCR. Similar levels of T cell activation could
occur even when a 30-fold excess of irrelevant yeast was present, suggesting
that such a yeast display system, by virtue of its ability to present ligands
multivalently, may be used in highly sensitive procedures to identify novel
polypeptides that interact multivalently with cell surface receptors and thereby
trigger specific cellular responses.
PMID: 9839939 [PubMed - indexed for MEDLINE]
871: Nucleic Acids Res 1998 Dec 15;26(24):5707-18
Crystal structure of the MATa1/MATalpha2 homeodomain heterodimer in complex with
DNA containing an A-tract.
Li T, Jin Y, Vershon AK, Wolberger C.
Department of Biophysics and Biophysical Chemistry and The Howard Hughes Medical
Institute,Johns Hopkins University School of Medicine, 725 North Wolfe Street,
Baltimore, MD 21205-2185, USA.
The crystal structure of the heterodimer formed by the DNA binding domains of
the yeast mating type transcription factors, MATa1 and MATalpha2, bound to a 21
bp DNA fragment has been determined at 2.5 A resolution. The DNA fragment in the
present study differs at four central base pairs from the DNA sequence used in
the previously studied ternary complex. These base pair changes give rise to a
(dA5).(dT5) tract without changing the overall base composition of the DNA. The
resulting A-tract occurs near the center of the overall 60 degrees bend in the
DNA. Comparison of the two structures shows that the structural details of the
DNA bend are maintained despite the DNA sequence changes. Analysis of the
A5-tract DNA subfragment shows that it contains a bend toward the minor groove
centered at one end of the A-tract. The observed bend is larger than that
observed in the crystal structures of A-tracts embedded in uncomplexed DNA,
which are straight and have been presumed to be quite rigid. Variation of the
central DNA base sequence reverses the two AT base pairs contacted in the minor
groove by Arg7 of the alpha2 N-terminal arm without significantly altering the
DNA binding affinity of the a1/alpha2 heterodimer. The Arg7 side chain
accommodates the sequence change by forming alternate H bond interactions, in
agreement with the proposal that minor groove base pair recognition is
insensitive to base pair reversal. Furthermore, the minor groove spine of
hydration, which stabilizes the narrowed minor groove caused by DNA bending, is
conserved in both structures. We also find that many of the water-mediated
hydrogen bonds between the a1 and alpha2 homeodomains and the DNA are highly
conserved, indicating an important role for water in stabilization of the
a1/alpha2-DNA complex.
PMID: 9838003 [PubMed - indexed for MEDLINE]
872: Nucleic Acids Res 1998 Dec 15;26(24):5562-7
In vitro assembly of an archaeal D-L-N RNA polymerase subunit complex reveals a
eukaryote-like structural arrangement.
Eloranta JJ, Kato A, Teng MS, Weinzierl RO.
Department of Biochemistry, Imperial College of Science, Technology and
Medicine, Exhibition Road, London SW7 2AY, UK.
Archaeal RNA polymerases (RNAPs) resemble the eukaryotic nuclear RNAPs in
complexity, and many of their subunits display a high degree of sequence
similarity to their eukaryotic counterparts. Here we describe specific
protein-protein contacts present between individual recombinant RNAP subunits
from the archaeon Methanococcus jannaschii. Subunits D and L interact
specifically with each other in two-hybrid assays. D also interacts under the
same conditions with the RPB11 and AC19 subunits from the yeast Saccharomyces
cerevisiae, suggesting that essential elements of the binding surface between
these proteins have been conserved across the archaeal/eukaryotic evolutionary
domain boundary. Interactions between L and RPB3 or AC40 were, however, not
detectable. Recombinant D and L subunits associate under in vitro conditions and
copurify with each other during size-exclusion chromatography. Addition of an
another recombinant subunit (N) to the D-L complex results in the formation of a
triple complex. This D-L-N complex resembles the RPB3-RPB11-RPB10 or
AC40-AC19-RPB10 complexes in eukaryotic RNAPIIand RNAPI/RNAPIII, respectively.
Our data provide evidence for a close similarity in the quaternary arrangement
of a subset of archaeal and eukaryotic RNA polymerase subunits and the
conservation of the protein-protein contacts formed between them.
PMID: 9837983 [PubMed - indexed for MEDLINE]
873: Genetics 1998 Dec;150(4):1407-17
Mapping of a yeast G protein betagamma signaling interaction.
Dowell SJ, Bishop AL, Dyos SL, Brown AJ, Whiteway MS.
Glaxo Wellcome Research and Development, Stevenage, SG1 2NY, United Kingdom.
sd14041@glaxowellcome.co.uk
The mating pathway of Saccharomyces cerevisiae is widely used as a model system
for G protein-coupled receptor-mediated signal transduction. Following receptor
activation by the binding of mating pheromones, G protein betagamma subunits
transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta
(Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues
in Ste4p required for the interaction with Ste5p. These residues define a new
signaling interface close to the Ste20p binding site within the Gbetagamma
coiled-coil. Ste4p mutants defective in the Ste5p interaction interact
efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in
signal transduction because cells expressing these mutants are sterile. Ste4
L65S is temperature-sensitive for its interaction with Ste5p, and also for
signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic
interaction defect with Ste4 L65S, providing strong evidence that Ste4p and
Ste5p interact directly in vivo through an interface that involves hydrophobic
residues. The correlation between disruption of the Ste4p-Ste5p interaction and
sterility confirms the importance of this interaction in signal transduction.
Identification of the Gbetagamma coiled-coil in Ste5p binding may set a
precedent for Gbetagamma-effector interactions in more complex organisms.
PMID: 9832519 [PubMed - indexed for MEDLINE]
874: Genes Dev 1998 Nov 15;12(22):3482-7
Allosteric interactions between capping enzyme subunits and the RNA polymerase
II carboxy-terminal domain.
Cho EJ, Rodriguez CR, Takagi T, Buratowski S.
Harvard Medical School, Boston, Massachusetts 02115 USA.
mRNA capping is a cotranscriptional event mediated by the association of capping
enzyme with the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase
II. In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two
subunits, the mRNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase
(Ceg1). Here we map interactions between Ceg1, Cet1, and the CTD. Although the
guanylyltransferase subunit can bind alone to the CTD, it cannot be guanylylated
unless the triphosphatase subunit is also present. Therefore, the yeast mRNA
guanylyltransferase is regulated by allosteric interactions with both the
triphosphatase and CTD.
PMID: 9832501 [PubMed - indexed for MEDLINE]
875: Proc Natl Acad Sci U S A 1998 Nov 24;95(24):14278-83
Physical interaction between components of DNA mismatch repair and nucleotide
excision repair.
Bertrand P, Tishkoff DX, Filosi N, Dasgupta R, Kolodner RD.
Charles A. Dana Division of Human Cancer Genetics, Dana-Farber Cancer Institute,
44 Binney Street, Boston, MA 02115, USA.
Nucleotide excision repair (NER) and DNA mismatch repair are required for some
common processes although the biochemical basis for this requirement is unknown.
Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2
as "bait," and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10,
RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2
coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10,
RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1
pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in
MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2
mutations were epistatic to the mutator phenotype observed in NER-deficient
strains. These data suggest that MSH2 and possibly other components of DNA
mismatch repair exist in a complex with NER proteins, providing a biochemical
and genetical basis for these proteins to function in common processes.
PMID: 9826691 [PubMed - indexed for MEDLINE]
876: Proc Natl Acad Sci U S A 1998 Nov 24;95(24):14272-7
An artificial cell-cycle inhibitor isolated from a combinatorial library.
Cohen BA, Colas P, Brent R.
Department of Molecular Biology, Massachusetts General Hospital, 50 Blossom
Street, Boston, MA 02114, USA.
Understanding the genetic networks that operate inside cells will require the
dissection of interactions among network members. Here we describe a peptide
aptamer isolated from a combinatorial library that distinguishes among such
interactions. This aptamer binds to cyclin-dependent kinase 2 (Cdk2) and
inhibits its kinase activity. In contrast to naturally occurring inhibitors,
such as p21(Cip1), which inhibit the activity of Cdk2 on all its substrates,
inhibition by pep8 has distinct substrate specificity. We show that the aptamer
binds to Cdk2 at or near its active site and that its mode of inhibition is
competitive. Expression of pep8 in human cells retards their progression through
the G1 phase of the cell cycle. Our results suggest that the aptamer inhibits
cell-cycle progression by blocking the activity of Cdk2 on substrates needed for
the G1-to-S transition. This work demonstrates the feasibility of selection of
artificial proteins to perform functions not developed during evolution. The
ability to select proteins that block interactions between a gene product and
some partners but not others should make sophisticated genetic manipulations
possible in human cells and other currently intractable systems.
PMID: 9826690 [PubMed - indexed for MEDLINE]
877: J Mol Biol 1998 Dec 4;284(3):673-87
Functional and structural characterization of the prp3 binding domain of the
yeast prp4 splicing factor.
Ayadi L, Callebaut I, Saguez C, Villa T, Mornon JP, Banroques J.
Centre de Genetique Moleculaire du CNRS, Laboratoire Propre Associe a
l'Universite P. & M. Curie, Gif-sur-Yvette, 91198, France.
Nuclear pre-mRNA splicing occurs in a large RNA-protein complex containing four
small nuclear ribonucleoprotein particles (snRNPs) and additional protein
factors. The yeast Prp4 (yPrp4) protein is a specific component of the U4/U6 and
U4/U6-U5 snRNPs, which associates transiently with the spliceosome before the
first step of splicing. In this work, we used the in vivo yeast two-hybrid
system and in vitro immunoprecipitation assays to show that yPrp4 interacts with
yPrp3, another U4/U6 snRNP protein. To investigate the domain of yPrp4 that
directly contacts yPrp3, we introduced deletions in the N-terminal half of yPrp4
and point mutations in the C-terminal half of the molecule, and we tested the
resulting prp4 mutants for cell viability and for their ability to interact with
yPrp3. We could not define any particular sequence in the first 161 amino acid
residues that are specifically required for protein-protein interactions.
However, deletion of a small basic-rich region of 30 amino acid residues is
lethal to the cells. Analysis of the C terminus prp4 mutants obtained clearly
shows that this region of yPrp4 represents the primary domain of interaction
with yPrp3. Interestingly, yPrp4 shows significant similarity in its C-terminal
half to the beta-subunits of G proteins. We have generated a three-dimensional
computer model of this domain, consisting of a seven-bladed beta-propeller based
on the crystalline structure of beta-transducin. Several lines of evidence
suggested that yPrp4 is contacting yPrp3 through a large flat surface formed by
the long variable loops linking the beta-strands of the propeller. This surface
could be used as a scaffold for generating an RNA-protein complex. Copyright
1998 Academic Press
PMID: 9826507 [PubMed - indexed for MEDLINE]
878: J Biol Chem 1998 Nov 27;273(48):32360-8
Interactions of p62(dok) with p210(bcr-abl) and Bcr-Abl-associated proteins.
Bhat A, Johnson KJ, Oda T, Corbin AS, Druker BJ.
Division of Hematology and Medical Oncology, Oregon Health Sciences University,
Portland, Oregon 97201, USA.
A 62-kDa Ras GTPase-activating protein (RasGAP)-associated protein is
tyrosine-phosphorylated under a variety of circumstances including growth factor
stimulation and in cells transformed by activated tyrosine kinases. A cDNA for
p62(dok), reported to be the RasGAP-associated 62-kDa protein, was recently
cloned from Abl-transformed cells. In this study, the interactions of p62(dok)
with Bcr-Abl and associated proteins were examined. In 32D myeloid cells and
Rat-1 fibroblasts transformed by p210(bcr-abl), p62(dok) is
tyrosine-phosphorylated and co-immunoprecipitates with Bcr-Abl, RasGAP, and
CrkL, a Src homology 2 (SH2) and SH3 domain-containing adaptor protein.
Tyrosine-phosphorylated p62(dok) from cells expressing p210(bcr-abl) bound
directly to the SH2 domains of Abl and CrkL in a gel overlay assay. Previous
work has shown that an SH2 domain deletion mutant of Bcr-Abl is defective in
transforming fibroblasts but remains capable of inducing myeloid growth factor
independence. In both fibroblasts and myeloid cells expressing this mutant,
p62(dok) is underphosphorylated as compared with cells expressing full-length
p210(bcr-abl) but remains capable of associating with Bcr-Abl. However, in a gel
overlay assay, p62(dok) from cells expressing the SH2 domain deletion was
incapable of associating directly with SH2 domains of Abl and CrkL.
Interestingly, no direct binding between Bcr-Abl and p62(dok) could be
demonstrated in a yeast two-hybrid assay. These data suggest that indirect
interactions mediate the interaction between Bcr-Abl and p62(dok) and that the
SH2 domain of Bcr-Abl is required for hyperphosphorylation of p62(dok). Further,
hyperphosphorylation of p62(dok) correlates with the ability of Bcr-Abl to
transform fibroblasts but not with the induction of growth factor independence
in myeloid cells.
PMID: 9822717 [PubMed - indexed for MEDLINE]
879: J Biol Chem 1998 Nov 27;273(48):32254-64
Identification of highly conserved amino-terminal segments of dTAFII230 and
yTAFII145 that are functionally interchangeable for inhibiting TBP-DNA
interactions in vitro and in promoting yeast cell growth in vivo.
Kotani T, Miyake T, Tsukihashi Y, Hinnebusch AG, Nakatani Y, Kawaichi M, Kokubo
T.
Division of Gene Function in Animals, Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
TFIID is a multiprotein complex composed of TBP and several TAFIIs. Small
amino-terminal segments (TAF N-terminal domain (TAND)) of Drosophila TAFII230
(dTAFII230) and yeast TAFII145 (yTAFII145) bind strongly to TBP and inhibit
TBP-DNA interactions. yTAFII145 TAND (yTAND) was divided into two subdomains,
yTANDI10-37 and yTANDII46-71, that function cooperatively. Here, we identify
dTANDII within the amino terminus of dTAFII230 at 118-143 amino acids in
addition to dTANDI18-77, reported previously. dTANDII exhibits pronounced
sequence similarity to yTANDII, and the two were shown to be functionally
equivalent in binding to TBP and inhibiting TBP-DNA interactions in vitro.
Alanine scanning mutation analysis demonstrated that Phe-57 (yTANDII) and
Tyr-129 (dTANDII) are critically required for the interaction with TBP. Yeast
strains containing mutant yTAFII145 lacking yTANDI or yTANDII showed a
temperature-sensitive growth phenotype. The conserved core of dTANDII could
substitute for the yTANDII core, and Phe-57 or Tyr-129 described above was
critically required for the function of this segment in promoting normal cell
growth at 37 degreesC. In these respects, the impact of yTANDII mutations on
cell growth paralleled their effects on TBP binding in vitro, strongly
suggesting that the yTAFII145-TBP interaction and its negative effects on TFIID
binding to core promoters are physiologically important.
PMID: 9822704 [PubMed - indexed for MEDLINE]
880: Mol Cell Biol 1998 Dec;18(12):7466-77
The 2 micrometer plasmid stability system: analyses of the interactions among
plasmid- and host-encoded components.
Velmurugan S, Ahn YT, Yang XM, Wu XL, Jayaram M.
Department of Microbiology and Institute of Cell and Molecular Biology,
University of Texas at Austin, Austin, Texas 78712, USA.
The stable inheritance of the 2 micrometer plasmid in a growing population of
Saccharomyces cerevisiae is dependent on two plasmid-encoded proteins (Rep1p and
Rep2p), together with the cis-acting locus REP3 (STB). In this study we
demonstrate that short carboxy-terminal deletions of Rep1p and Rep2p severely
diminish their normal capacity to localize to the yeast nucleus. The nuclear
targeting, as well as their functional role in plasmid partitioning, can be
restored by the addition of a nuclear localization sequence to the amino or the
carboxy terminus of the shortened Rep proteins. Analyses of deletion derivatives
of the Rep proteins by using the in vivo dihybrid genetic test in yeast, as well
as by glutathione S-transferase fusion trapping assays in vitro demonstrate that
the amino-terminal portion of Rep1p (ca. 150 amino acids long) is responsible
for its interactions with Rep2p. In a monohybrid in vivo assay, we have
identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of
interacting with the STB locus. The Shf1 protein expressed in Escherichia coli
can bind with high specificity to the STB sequence in vitro. In a yeast strain
deleted for the SHF1 locus, a 2 micrometer circle-derived plasmid shows
relatively poor stability.
PMID: 9819432 [PubMed - indexed for MEDLINE]
881: Mol Cell Biol 1998 Dec;18(12):7344-52
CNS1 encodes an essential p60/Sti1 homolog in Saccharomyces cerevisiae that
suppresses cyclophilin 40 mutations and interacts with Hsp90.
Dolinski KJ, Cardenas ME, Heitman J.
Departments of Genetics, Pharmacology and Cancer Biology, and Medicine, Howard
Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina
27710, USA.
Cyclophilins are cis-trans-peptidyl-prolyl isomerases that bind to and are
inhibited by the immunosuppressant cyclosporin A (CsA). The toxic effects of CsA
are mediated by the 18-kDa cyclophilin A protein. A larger cyclophilin of 40
kDa, cyclophilin 40, is a component of Hsp90-steroid receptor complexes and
contains two domains, an amino-terminal prolyl isomerase domain and a
carboxy-terminal tetratricopeptide repeat (TPR) domain. There are two
cyclophilin 40 homologs in the yeast Saccharomyces cerevisiae, encoded by the
CPR6 and CPR7 genes. Yeast strains lacking the Cpr7 enzyme are viable but
exhibit a slow-growth phenotype. In addition, we show here that cpr7 mutant
strains are hypersensitive to the Hsp90 inhibitor geldanamycin. When
overexpressed, the TPR domain of Cpr7 alone complements both cpr7 mutant
phenotypes, while overexpression of the cyclophilin domain of Cpr7, full-length
Cpr6, or human cyclophilin 40 does not. The open reading frame YBR155w, which
has moderate identity to the yeast p60 homolog STI1, was isolated as a
high-copy-number suppressor of the cpr7 slow-growth phenotype. We show that this
Sti1 homolog Cns1 (cyclophilin seven suppressor) is constitutively expressed,
essential, and found in protein complexes with both yeast Hsp90 and Cpr7 but not
with Cpr6. Cyclosporin A inhibited Cpr7 interactions with Cns1 but not with
Hsp90. In summary, our findings identify a novel component of the Hsp90
chaperone complex that shares function with cyclophilin 40 and provide evidence
that there are functional differences between two conserved sets of Hsp90
binding proteins in yeast.
PMID: 9819421 [PubMed - indexed for MEDLINE]
882: Mol Cell Biol 1998 Dec;18(12):7304-16
Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus
E3: role of complex formation and the E3 N-terminal domain.
Romano PR, Zhang F, Tan SL, Garcia-Barrio MT, Katze MG, Dever TE, Hinnebusch AG.
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and
Human Development, Bethesda, Maryland 20892, USA.
The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits
protein synthesis by phosphorylating translation initiation factor 2alpha
(eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits
PKR in virus-infected cells, presumably by sequestering dsRNA activators.
Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by
phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs).
We found that expression of E3 in yeast overcomes the lethal effect of PKR in a
manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by
E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in
particular, also is required for anti-PKR function. Because the E3 N-terminal
region does not contribute to dsRNA binding in vitro, it appears that
sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR.
This conclusion was supported by the fact that E3 activity was antagonized, not
augmented, by overexpressing the catalytically defective PKR-K296R protein
containing functional DRBMs. Coimmunoprecipitation experiments showed that a
majority of PKR in yeast extracts was in a complex with E3, whose formation was
completely dependent on the dsRNA binding activity of E3 and enhanced by the
N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding
experiments, segments of E3 and PKR containing their respective DRBMs interacted
in a manner requiring E3 residues Lys-167 and Arg-168. We also detected
interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid
and lambda repressor dimerization assays. In the latter case, the N-terminal
half of E3 interacted with the kinase domain of PKR, dependent on E3 residue
Trp-66. We propose that effective inhibition of PKR in yeast requires formation
of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically
interacts with the protein kinase domain of PKR.
PMID: 9819417 [PubMed - indexed for MEDLINE]
883: Mol Cell Biol 1998 Dec;18(12):7205-15
Identification of a polar region in transmembrane domain 6 that regulates the
function of the G protein-coupled alpha-factor receptor.
Dube P, Konopka JB.
Program in Molecular and Cellular Biology, State University of New York, Stony
Brook, New York 11794-5222, USA.
The alpha-factor pheromone receptor (Ste2p) of the yeast Saccharomyces
cerevisiae belongs to the family of G protein-coupled receptors that contain
seven transmembrane domains (TMDs). Because polar residues can influence
receptor structure by forming intramolecular contacts between TMDs, we tested
the role of the five polar amino acids in TMD6 of the alpha-factor receptor by
mutating these residues to nonpolar leucine. Interestingly, a subset of these
mutants showed increased affinity for ligand and constitutive receptor activity.
The mutation of the most polar residue, Q253L, resulted in 25-fold increased
affinity and a 5-fold-higher basal level of signaling that was equal to about
19% of the alpha-factor induced maximum signal. Mutation of the adjacent
residue, S254L, caused weaker constitutive activity and a 5-fold increase in
affinity. Comparison of nine different mutations affecting Ser254 showed that an
S254F mutation caused higher constitutive activity, suggesting that a large
hydrophobic amino acid residue at position 254 alters transmembrane helix
packing. Thus, these studies indicate that Gln253 and Ser254 are likely to be
involved in intramolecular interactions with other TMDs. Furthermore, Gln253 and
Ser254 fall on one side of the transmembrane helix that is on the opposite side
from residues that do not cause constitutive activity when mutated. These
results suggest that Gln253 and Ser254 face inward toward the other TMDs and
thus provide the first experimental evidence to suggest the orientation of a TMD
in this receptor. Consistent with this, we identified two residues in TMD7
(Ser288 and Ser292) that are potential contact residues for Gln253 because
mutations affecting these residues also cause constitutive activity. Altogether,
these results identify a new domain of the alpha-factor receptor that regulates
its ability to enter the activated conformation.
PMID: 9819407 [PubMed - indexed for MEDLINE]
884: Curr Opin Chem Biol 1998 Oct;2(5):597-603
Technological advances in high-throughput screening.
Fernandes PB.
Small Molecule Therapeutics Inc., Monmouth Junction, NJ 08852, USA.
fernandes@smtherapeutics.com
A variety of assay technologies continue to be developed for high-throughput
screening. These include cell-based assays, surrogate systems using microbial
cells such as yeast and bacterial two-hybrid and three-hybrid systems, and
systems to measure nucleic acid-protein and receptor-ligand interactions.
Modifications have been developed for cell-free, homogeneous assay systems, such
as time-resolved fluorescence, fluorescence polarization and the scintillation
proximity assay. Innovations in engineering and chemistry have led to delivery
systems for nanoliter volumes and sensitive biosensors for ultra-high-throughout
screening conducted in nanoliter and picoliter volumes. Spectroscopic methods
have been extended to read single molecule fluorescence. Technologies are being
developed to identify new targets from genomic information in order to design
the next generation of screens.
Publication Types:
Review
Review, Tutorial
PMID: 9818185 [PubMed - indexed for MEDLINE]
885: J Cell Biol 1998 Nov 16;143(4):887-99
Nuclear import and the evolution of a multifunctional RNA-binding protein.
Rosenblum JS, Pemberton LF, Bonifaci N, Blobel G.
Laboratory of Cell Biology, Howard Hughes Medical Institute and Rockefeller
University, New York, New York 10021, USA.
La (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate
and other common RNA sequence/structural motifs. By virtue of these
interactions, La is present in a myriad of nuclear and cytoplasmic
ribonucleoprotein complexes in vivo where it may function as an RNA-folding
protein or RNA chaperone. We have recently characterized the nuclear import
pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or
karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now
determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by
Kap108p. Unexpectedly, this domain does not coincide with the previously
identified nuclear localization signal of human La. Furthermore, when expressed
in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces
pombe, Drosophila, and human La proteins are independent of Kap108p. We have
been able to reconstitute the nuclear import of human La into permeabilized HeLa
cells using the recombinant human factors karyopherin alpha2, karyopherin beta1,
Ran, and p10. As such, the yeast and human La proteins are imported using
different sequence motifs and dissimilar karyopherins. Our results are
consistent with an intermingling of the nuclear import and evolution of La.
PMID: 9817748 [PubMed - indexed for MEDLINE]
886: RNA 1998 Nov;4(11):1357-72
Synthetic lethal interactions with conditional poly(A) polymerase alleles
identify LCP5, a gene involved in 18S rRNA maturation.
Wiederkehr T, Pretot RF, Minvielle-Sebastia L.
Department of Cell Biology, Biozentrum, University of Basel, Switzerland.
To identify new genes involved in 3'-end formation of mRNAs in Saccharomyces
cerevisiae, we carried out a screen for synthetic lethal mutants with the
conditional poly(A) polymerase allele, pap1-7. Five independent
temperature-sensitive mutations called Icp1 to Icp5 (for lethal with conditional
pap1 allele) were isolated. Here, we describe the characterization of the
essential gene LCP5 which codes for a protein with a calculated molecular mass
of 40.8 kD. Unexpectedly, we found that mutations in LCP5 caused defects in
pre-ribosomal RNA (pre-rRNA) processing, whereas mRNA 3'-end formation in vitro
was comparable to wild-type. Early cleavage steps (denoted A0 to A2) that lead
to the production of mature 18S rRNA were impaired. In vivo depletion of Lcp5p
also inhibited pre-rRNA processing. As a consequence, mutant and depleted cells
showed decreased levels of polysomes compared to wild-type cells. Indirect
immunofluorescence indicated a predominant localization of Lcp5p in the
nucleolus. In addition, antibodies directed against Lcp5p specifically
immunoprecipitated the yeast U3 snoRNA snR17, suggesting that the protein is
directly involved in pre-rRNA processing.
PMID: 9814757 [PubMed - indexed for MEDLINE]
887: Proc Natl Acad Sci U S A 1998 Nov 10;95(23):13543-8
A transcriptional activating region with two contrasting modes of protein
interaction.
Ansari AZ, Reece RJ, Ptashne M.
Program in Molecular Biology, Memorial Sloan Kettering Cancer Center, Box 595,
1275 York Avenue, New York, NY 10021, USA.
A C-terminal segment of the yeast activator Gal4 manifests two functions: When
tethered to DNA, it elicits gene activation, and it binds the inhibitor Gal80.
Here we examine the effects on these two functions of cysteine and proline
substitutions. We find that, although certain cysteine substitutions diminish
interaction with Gal80, those substitutions have little effect on the activating
function in vivo and interaction with TATA box-binding protein (TBP) in vitro.
Proline substitutions introduced near residues critical for Gal80 binding
abolish that interaction but once again have no effect on the activating
function. Crosslinking experiments show that a defined position in the
activating peptide is in close proximity to TBP and Gal80 in the two separate
reactions and show that binding of the inhibitor blocks binding to TBP. Thus,
the same stretch of amino acids are involved in two quite different
protein-protein interactions: binding to Gal80, which depends on a precise
sequence and the formation of a defined secondary structure, or interactions
with the transcriptional machinery in vivo, which are not impaired by
perturbations of either sequence or structure.
PMID: 9811836 [PubMed - indexed for MEDLINE]
888: Mol Cell Endocrinol 1998 Aug 25;143(1-2):133-42
Studies of dehydroepiandrosterone (DHEA) with the human estrogen receptor in
yeast.
Nephew KP, Sheeler CQ, Dudley MD, Gordon S, Nayfield SG, Khan SA.
Medical Sciences Program, Indiana University School of Medicine, Bloomington
47405-4401, USA. knephew@indiana.edu
Dehydroepiandrosterone (DHEA) is a C19 adrenal steroid synthesized in the human
adrenal cortex and serving as a biosynthetic precursor to testosterone and
17beta-estradiol. Despite the fact that it is one of the most abundant steroid
hormones in circulation, the physiological role of DHEA in humans remains
unclear. The action of DHEA itself, such as its interactions with receptors and
nuclear transcription factors, is not well understood, and a specific DHEA
receptor has yet to be identified. Although the activity of DHEA can be due to
its metabolism into androgens and estrogens, DHEA has been shown to interact
with the androgen receptor and the estrogen receptor (ER) in vitro. We
demonstrate in this study that DHEA (3beta-Hydroxy-5alpha-androstan-17-one)
inhibits 17beta-estradiol (E2) binding to its receptor in vivo in yeast. DHEA
stimulates human ER dimerization in yeast, as determined by ER fusion protein
interactions, GAL4 reconstitution and subsequent measurement of increased
beta-galactosidase activity. DHEA causes an increase in estrogen response
element-dependent beta-galactosidase activity, demonstrating that the ER dimer
induced by DHEA is transcriptionally active, but at a concentration of DHEA
about 1000 times greater than E2. Inclusion of the nuclear receptor co-activator
RIP140 in the yeast enhances ER transactivation by DHEA or E2 in a
ligand-dependent manner; moreover, only in the presence of RIP140 is DHEA able
to stimulate beta-galactosidase activity to levels similar to those achieved by
E2. Ligand-receptor interaction for other C19-steroids was also examined. While
5-androstene-3beta, 17beta-diol (ADIOL) displayed estrogenic activity in this
system, 4-androstene-17-dione (androstenedione) and 4-androstene-17beta-ol,3-one
(testosterone) did not. We have investigated whether DHEA can interact with the
human ER in vivo. Our findings demonstrate a mechanism by which DHEA interacts
directly with estrogen signaling systems; however, because DHEA is several
orders of magnitude less potent than E2 in this system, we conclude that it
essentially is not an estrogen agonist.
PMID: 9806358 [PubMed - indexed for MEDLINE]
889: J Biol Chem 1998 Nov 13;273(46):30279-86
Lipid products of phosphoinositide 3-kinase interact with Rac1 GTPase and
stimulate GDP dissociation.
Missy K, Van Poucke V, Raynal P, Viala C, Mauco G, Plantavid M, Chap H,
Payrastre B.
Institut Federatif de Recherche en Immunologie Cellulaire et Moleculaire,
Universite Paul Sabatier and Centre Hospitalo-Universitaire de Toulouse, INSERM
Unite 326, France.
A number of reports suggest that under different conditions leading to
cytoskeleton reorganization the GTPase Rac1 and possibly RhoA are downstream
targets of phosphoinositide 3-kinase (PI 3-kinase). In order to gain more
insight into this particular signaling pathway, we have addressed the question
of a possible direct interaction of PI 3-kinase products with the Rho family
GTPases RhoA, Rac1, and Cdc42. Using recombinant proteins, we found that Rac1
and, to a lesser extent, RhoA but not Cdc42 were capable to selectively bind to
phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in a mixture of crude
brain phosphoinositides. Nucleotide-depleted Rac1 was the most efficient, but
the GDP- and GTP-bound forms retained significant PtdIns(3,4,5)P3 binding
activity. This protein-lipid association involved electrostatic as well as
hydrophobic interactions, since both phosphate groups located at specific
positions of the inositol ring and fatty-acyl chains were absolutely required.
Based on the sequence of Rac1, two potential binding sites were identified, one
at the C terminus and one in the extra alpha-helical domain. Deletion of these
two domains resulted in a complete loss of binding to PI 3-kinase products.
Finally, PtdIns(3, 4,5)P3 strongly stimulated GDP dissociation from Rac1 in a
dose-dependent manner. In agreement, data obtained in intact cells suggest that
PtdIns(3,4,5)P3 might target Rac1 to peculiar membrane domains, allowing
formation of specific clusters containing not only small GTPases but other
partners bearing pleckstrin homology domains such as specific exchange factors
required for Rac1 and RhoA activation.
PMID: 9804788 [PubMed - indexed for MEDLINE]
890: FEBS Lett 1998 Oct 16;437(1-2):56-60
Identification of a possible MAP kinase cascade in Arabidopsis thaliana based on
pairwise yeast two-hybrid analysis and functional complementation tests of yeast
mutants.
Mizoguchi T, Ichimura K, Irie K, Morris P, Giraudat J, Matsumoto K, Shinozaki K.
Laboratory of Plant Molecular Biology, Institute of Physical and Chemical
Research (RIKEN), Tsukuba Life Science Center, Ibaraki, Japan.
A possible MAP kinase (MAPK) cascade of Arabidopsis thaliana was identified on
the basis of both yeast 2-hybrid analysis and complementation analysis of yeast
mutants. Specific protein-protein interactions between ATMPK4 (a MAPK) and MEK1
(a MAPKK) and interactions between MEK1 and ATMEKK1 (a MAPKKK) were detected by
using the 2-hybrid system. A growth defect of the yeast mpk1delta mutant was
reversed by coexpression of ATMPK4 and MEK1. Coexpression of the N-terminal
deletion form of ATMEKK1 increased the ability of MEK1 to suppress a growth
defect of the yeast pbs2delta mutant. These results suggest that ATMPK4, MEK1,
and ATMEKK1 may interact with each other and constitute a specific MAPK cascade
in Arabidopsis. This is the first demonstration of a possible MAPK cascade in
plants.
PMID: 9804171 [PubMed - indexed for MEDLINE]
891: Mol Biol Cell 1998 Nov;9(11):3071-83
Hsp90 is required for pheromone signaling in yeast.
Louvion JF, Abbas-Terki T, Picard D.
Departement de Biologie Cellulaire, Universite de Geneve Sciences III, CH-1211
Geneve 4, Switzerland.
The heat-shock protein 90 (Hsp90) is a cytosolic molecular chaperone that is
highly abundant even at normal temperature. Specific functions for Hsp90 have
been proposed based on the characterization of its interactions with certain
transcription factors and kinases including Raf in vertebrates and flies. We
therefore decided to address the role of Hsp90 for MAP kinase pathways in the
budding yeast, an organism amenable to both genetic and biochemical analyses. We
found that both basal and induced activities of the pheromone-signaling pathway
depend on Hsp90. Signaling is defective in strains expressing low levels or
point mutants of yeast Hsp90 (Hsp82), or human Hsp90beta instead of the
wild-type protein. Ste11, a yeast equivalent of Raf, forms complexes with
wild-type Hsp90 and depends on Hsp90 function for accumulation. For budding
yeast, Ste11 represents the first identified endogenous "substrate" of Hsp90.
Moreover, Hsp90 functions in steroid receptor and pheromone signaling can be
genetically separated as the Hsp82 point mutant T525I and the human Hsp90beta
are specifically defective for the former and the latter, respectively. These
findings further corroborate the view that molecular chaperones must also be
considered as transient or stable components of signal transduction pathways.
PMID: 9802897 [PubMed - indexed for MEDLINE]
892: Dev Genes Evol 1998 Oct;208(8):440-6
Specific interactions between vestigial and scalloped are required to promote
wing tissue proliferation in Drosophila melanogaster.
Paumard-Rigal S, Zider A, Vaudin P, Silber J.
Institut Jacques Monod, L.G.Q.M., 2, Place Jussieu, Tour 43, F-75251 Paris cedex
05, France.
The two genes vestigial (vg) and scalloped (sd) are required for wing
development in Drosophila melanogaster. They present similar patterns of
expression in second and third instar wing discs and similar wing mutant
phenotypes. vg encodes a nuclear protein without any recognized nucleic
acid-binding motif. Sd is a transcription factor homologous to the human TEF-1
factor whose promoter activity depends on cell-specific cofactors. We postulate
that Vg could be a cofactor of Sd in the wing morphogenetic process and that,
together, they could constitute a functional transcription complex. We
investigated genetic interactions between the two genes. We show here that vg
and sd co-operate in vivo in a manner dependent on the structure of the Vg
protein. We ectopically expressed vg in the patch (ptc) domains. We show
evidence that wing-like outgrowths induced by ectopic expression of vg are
severely reduced in vg or sd mutant backgrounds. Accordingly, we demonstrate
that ptc-GAL4-driven expression of vg induces both expressions of the endogenous
vg and sd genes and that the two Vg and Sd proteins have to be produced together
to promote wing proliferation. Furthermore, we show an interaction between the
two proteins by double hybrid experiments in yeast. Our results therefore
support the hypothesis that Sd and Vg directly interact in vivo to form a
complex regulating the proliferation of wing tissue.
PMID: 9799424 [PubMed - indexed for MEDLINE]
893: Genetics 1998 Nov;150(3):987-1005
Sth1p, a Saccharomyces cerevisiae Snf2p/Swi2p homolog, is an essential ATPase in
RSC and differs from Snf/Swi in its interactions with histones and
chromatin-associated proteins.
Du J, Nasir I, Benton BK, Kladde MP, Laurent BC.
Department of Microbiology and Immunology and Morse Institute for Molecular
Genetics, State University of New York, Brooklyn, New York 11203, USA.
The essential Sth1p is the protein most closely related to the conserved
Snf2p/Swi2p in Saccharomyces cerevisiae. Sth1p purified from yeast has a
DNA-stimulated ATPase activity required for its function in vivo. The finding
that Sth1p is a component of a multiprotein complex capable of ATP-dependent
remodeling of the structure of chromatin (RSC) in vitro, suggests that it
provides RSC with ATP hydrolysis activity. Three sth1 temperature-sensitive
mutations map to the highly conserved ATPase/helicase domain and have cell cycle
and non-cell cycle phenotypes, suggesting multiple essential roles for Sth1p.
The Sth1p bromodomain is required for wild-type function; deletion mutants
lacking portions of this region are thermosensitive and arrest with highly
elongated buds and 2C DNA content, indicating perturbation of a unique function.
The pleiotropic growth defects of sth1-ts mutants imply a requirement for Sth1p
in a general cellular process that affects several metabolic pathways.
Significantly, an sth1-ts allele is synthetically sick or lethal with previously
identified mutations in histones and chromatin assembly genes that suppress
snf/swi, suggesting that RSC interacts differently with chromatin than Snf/Swi.
These results provide a framework for understanding the ATP-dependent RSC
function in modeling chromatin and its connection to the cell cycle.
PMID: 9799253 [PubMed - indexed for MEDLINE]
894: Virology 1998 Oct 25;250(2):302-15
The vaccinia virus E3L gene product interacts with both the regulatory and the
substrate binding regions of PKR: implications for PKR autoregulation.
Sharp TV, Moonan F, Romashko A, Joshi B, Barber GN, Jagus R.
Center of Marine Biotechnology, University of Maryland Biotechnology Institute,
Baltimore, Maryland, 21202, USA.
The vaccinia virus E3L gene product, pE3, is a dsRNA binding protein that
prevents activation of the interferon-induced, dsRNA-activated protein kinase,
PKR. Activation of PKR, which results in phosphorylation of the translation
initiation factor, eIF2alpha, leads to the inhibition of protein synthesis, a
process involved in defense against virus infection. The E3L gene product has a
conserved dsRNA binding domain (DRBD) in its carboxyl-terminal region and has
been shown to function in vitro by sequestration of dsRNA. We have utilized in
vitro binding assays and the yeast two-hybrid system to demonstrate direct
interactions of pE3 with PKR. By these methods, we demonstrate that pE3
interacts with two distinct regions in PKR, the amino-terminal (amino acids
1-99) located in the regulatory domain and the carboxyl-terminal (amino acids
367-523) located in the catalytic domain. The amino-terminal region of PKR that
interacts with pE3 contains a conserved DRBD, suggesting that PKR can form
nonfunctional heterodimers with pE3, analogous to those seen with other dsRNA
binding proteins. Interaction of pE3 with the amino-terminal region of PKR is
enhanced by dsRNA. In contrast, dsRNA reduces the interaction of pE3 with the
carboxyl-terminal region of PKR. Competition experiments demonstrate that the
carboxyl-terminal region of PKR, to which pE3 binds, overlaps the region with
which eIF2alpha and the pseudosubstrate pK3 interact, suggesting that pE3 may
also prevent PKR activation by masking the substrate binding domain. Like pE3,
the amino-terminal region of PKR also interacts with the carboxyl-terminal
domain of PKR. These interactions increase our understanding of the mechanisms
by which pE3 downregulates PKR. In addition, the PKR-PKR interactions observed
leads us to suggest a novel autoregulatory mechanism for activation of PKR in
which dsRNA binding to the DRBD(s) induces a conformational change that results
in release of the amino terminal region from the substrate binding domain,
allowing access to eIF2alpha and its subsequent phosphorylation. Copyright 1998
Academic Press
PMID: 9792841 [PubMed - indexed for MEDLINE]
895: Biochem Biophys Res Commun 1998 Oct 29;251(3):903-6
The use of the yeast two hybrid system to evaluate ErbB-3 interactions with SH2
domain containing proteins.
Yoo JY, Hamburger AW.
Molecular and Cellular Biology Program, University of Maryland, Baltimore,
Maryland, 21201, USA.
Several mutations in the tyrosine kinase domain of ErbB-3 have been postulated
to render this enzyme catalytically inactive. To test which amino acid mutations
in ErbB-3 might be critical for kinase inactivation, we used a yeast two hybrid
assay of protein-protein interaction. We monitored restoration of ErbB-3 kinase
activity by investigating the ability of wild type or mutant ErbB-3 to associate
with the SH2 containing proteins Syp and Phosphatidyl-inositol-3-kinase (PI3K).
Our results demonstrate that changing individual amino acids to tyrosine kinase
consensus sequences did not increase the interaction of ErbB-3 with Syp or PI3K.
Mutation of the consensus Asp832 of rat ErbB-3 to Asn observed in human and
bovine ErbB-3 significantly increased the interaction of ErbB-3 and Syp and PI3K
11 or 26 fold respectively. A double mutant (Asp832Asn, Asp757 His) exhibited a
96 or 350 fold increase in the ability to bind PI3K and Syp. Copyright 1998
Academic Press.
PMID: 9791008 [PubMed - indexed for MEDLINE]
896: Biochem Biophys Res Commun 1998 Oct 29;251(3):732-6
Shs1p: a novel member of septin that interacts with spa2p, involved in polarized
growth in saccharomyces cerevisiae.
Mino A, Tanaka K, Kamei T, Umikawa M, Fujiwara T, Takai Y.
Department of Molecular Biology and Biochemistry, Osaka University Medical
School, Suita, 565-0871, Japan.
The Rho family small G proteins regulate various cell functions including
cytokinesis. We have shown that Bni1p, a potential target of Rho1p, interacts
with Spa2p and that Spa2p is required for the localization of Bni1p at the
growth sites in Saccharomyces cerevisiae. We isolated here a novel member of the
septin family, implicated in cytokinesis, as a Spa2p-binding protein by the
yeast two-hybrid method. We named this gene SHS1 (Seventh Homolog of Septin).
The shs1 mutant cells showed cytokinesis deficiency and Shs1p was localized at
the bud neck in budded cells. The Spa2p-Shs1p interactions may play an important
role in cytokinesis. Copyright 1998 Academic Press.
PMID: 9790978 [PubMed - indexed for MEDLINE]
897: Nat Biotechnol 1998 Oct;16(10):946-50
Erratum in:
Nat Biotechnol 1998 Nov;16(11):1074
Comment in:
Nat Biotechnol. 1998 Oct;16(10):906.
Identification of a calcium channel modulator using a high throughput yeast
two-hybrid screen.
Young K, Lin S, Sun L, Lee E, Modi M, Hellings S, Husbands M, Ozenberger B,
Franco R.
Wyeth-Ayerst Research, CNS Disorders, Princeton, NJ 08543, USA.
The interaction of the N-type calcium channel beta3 subunit with the alpha1B
subunit alters the activation/inactivation kinetics and the maximal conductance
of the channel. The defined protein-protein interaction of the human alpha1B and
beta3 subunits provides a target for small-molecule modulation of N-type channel
activity. We describe a high throughput screen based on a counterselection yeast
two-hybrid assay, which was used to identify small molecules that disrupt
alpha1B-beta3 subunit interactions and inhibit N-type calcium channel activity.
These small molecules may be a new class of calcium channel antagonists with
therapeutic potential.
PMID: 9788351 [PubMed - indexed for MEDLINE]
898: Nucleic Acids Res 1998 Nov 1;26(21):4965-74
A specific RNA-protein interaction at yeast polyadenylation efficiency elements.
Chen S, Hyman LE.
Department of Biochemistry, Tulane University School of Medicine, 1430 Tulane
Avenue, New Orleans,LA 70112-2699, USA.
The specific RNA-protein interactions responsible for the production of mature
3' ends of eukaryotic mRNAs are not well understood. Sequence elements at the 3'
ends of yeast genes have been identified that specify the position of the
poly(A) site and the efficiency of polyadenylation. To provide additional
insights into the interaction between important sequences that direct 3'-end
formation in vivo and nuclear proteins, we utilized gel mobility shift assays
and UV-crosslinking studies. The data indicate that a protein, with an apparent
molecular weight of 80 kDa, interacts specifically with pre-mRNA at the
(UA)3efficiency element. Although the interaction is specific, it can be
competed by RNA sequences that do not contain the same type of efficiency
element; that is, a sequence lacking a (UA)3repeat. This result implies that the
protein binding site is flexible. Using immunoprecipitation techniques, the
protein has been identified as Hrp1, a heteronuclear RNA binding protein. The
role of Hrp1p in 3'-end formation including RNA processing and transcription
termination is addressed.
PMID: 9776761 [PubMed - indexed for MEDLINE]
899: Mol Cell Biol 1998 Nov;18(11):6365-73
Erratum in:
Mol Cell Biol 2000 Mar;20(5):1898
Cak1 is required for Kin28 phosphorylation and activation in vivo.
Espinoza FH, Farrell A, Nourse JL, Chamberlin HM, Gileadi O, Morgan DO.
Departments of Physiology and Biochemistry & Biophysics, University of
California, San Francisco, California 94143-0444, USA.
Complete activation of most cyclin-dependent protein kinases (CDKs) requires
phosphorylation by the CDK-activating kinase (CAK). In the budding yeast,
Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as
Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major
CDK involved in cell cycle control. We addressed the possibility that Cak1 is
also required for the activation of other yeast CDKs, such as Kin28, Pho85, and
Srb10. We generated three new temperature-sensitive cak1 mutant strains, which
arrested at the restrictive temperature with nonuniform budding morphology. All
three cak1 mutants displayed significant synthetic interactions with
loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the
phosphorylation and activity of both Cdc28 and Kin28 but did not affect the
activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits
Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1
in insect cells. We conclude that Cak1 is required for the activating
phosphorylation of Kin28 as well as that of Cdc28.
PMID: 9774652 [PubMed - indexed for MEDLINE]
900: J Biol Chem 1998 Oct 23;273(43):28341-5
Interaction of Bnr1p with a novel Src homology 3 domain-containing Hof1p.
Implication in cytokinesis in Saccharomyces cerevisiae.
Kamei T, Tanaka K, Hihara T, Umikawa M, Imamura H, Kikyo M, Ozaki K, Takai Y.
Department of Molecular Biology and Biochemistry, Osaka University Medical
School, Suita 565-0871, Osaka, Japan.
Proteins containing the formin homology (FH) domains FH1 and FH2 are involved in
cytokinesis or establishment of cell polarity in a variety of organisms. We have
shown that the FH proteins Bni1p and Bnr1p are potential targets of the Rho
family small GTP-binding proteins and bind to an actin-binding protein,
profilin, at their proline-rich FH1 domains to regulate reorganization of the
actin cytoskeleton in the yeast Saccharomyces cerevisiae. We found here that a
novel Src homology 3 (SH3) domain-containing protein, encoded by YMR032w,
interacted with Bnr1p in a GTP-Rho4p-dependent manner through the FH1 domain of
Bnr1p and the SH3 domain of Ymr032wp. Ymr032wp weakly bound to Bni1p. Ymr032wp
was homologous to cdc15p, which is involved in cytokinesis in
Schizosaccharomyces pombe, and we named this gene HOF1 (homolog of cdc 15). Both
Bnr1p and Hof1p were localized at the bud neck, and both the bnr1 and hof1
mutations showed synthetic lethal interactions with the bni1 mutation. The hof1
mutant cells showed phenotypes similar to those of the septin mutants,
indicating that HOF1 is involved in cytokinesis. These results indicate that
Bnr1p directly interacts with Hof1p as well as with profilin to regulate
cytoskeletal functions in S. cerevisiae.
PMID: 9774458 [PubMed - indexed for MEDLINE]
901: J Biol Chem 1998 Oct 23;273(43):28085-90
Negative charges in the C-terminal domain stabilize the alphaB-crystallin
complex.
Boelens WC, Croes Y, de Ruwe M, de Reu L, de Jong WW.
Department of Biochemistry, University of Nijmegen, P. O. Box 9101, 6500 HB
Nijmegen, The Netherlands. w.boelens@bioch.kun.nl
alphaB-Crystallin is one of the six known mammalian small heat-shock proteins
(sHsps). These are characterized by the presence of a conserved sequence of
80-100 residues, which constitutes the putative C-terminal domain. Like other
sHsps, alphaB-crystallin forms multimeric globular complexes, often in
combination with related sHsps. Here we show that in a yeast two-hybrid system,
alphaB-crystallin can specifically interact with itself as well as with
alphaA-crystallin and Hsp27. Analyses of the separate domains show that the
conserved C-terminal domain (CalphaB) is essential for this interaction between
subunits. To try and detect residues that are important in subunit interaction,
the CalphaB domain was used in a two-hybrid screen as bait to select randomly
mutated CalphaB mutants. In this way we obtained nine mutants that were still
able to interact with wild-type CalphaB despite the presence of up to 15
replacements. Similarly, we obtained 16 mutants that were unable to bind,
because of the presence of just three to nine replacements. In binding CalphaB
mutants, lysine residues were most often replaced by glutamic acid residues, and
in non-binding CalphaB mutants, acidic residues were often found to be replaced
by non-charged residues. This indicates that negative charges are important for
subunit interaction and we propose a model to explain this role of acidic
residues. Furthermore, we observed that two homologs of alphaB-crystallin,
alphaA-crystallin and Hsp27, generally interact similarly with the binding and
non-binding CalphaB mutants as does alphaB-crystallin. This suggests that
interactions involved in the complex formation of these three sHsps are largely
comparable.
PMID: 9774426 [PubMed - indexed for MEDLINE]
902: J Biol Chem 1998 Oct 23;273(43):28073-7
Inhibition of the interaction between tyrosine-based motifs and the medium chain
subunit of the AP-2 adaptor complex by specific tyrphostins.
Crump CM, Williams JL, Stephens DJ, Banting G.
Department of Biochemistry, School of Medical Sciences, University of Bristol,
Bristol BS8 1TD, United Kingdom.
Several intracellular membrane trafficking events are mediated by
tyrosine-containing motifs found within the cytosolic domains of certain
integral membrane proteins. Many of these tyrosine motifs conform to the
consensus YXXPhi (where Phi represents a bulky hydrophobic residue). This YXXPhi
motif has been shown to interact with the medium chain subunits of adaptor
complexes that generally link relevant integral membrane protein cytosolic
domains to the clathrin coat involved in vesicle formation. The motif YXXPhi is
also very similar to motifs that are targets for phosphorylation by tyrosine
kinases. Tyrosine kinase inhibitors known as tyrphostins are structural
analogues of tyrosine, and so it is possible that tyrphostins could also inhibit
interactions between medium chains and YXXPhi motifs. TGN38 is a type I integral
membrane protein containing a tyrosine motif, YQRL, within the cytosolic domain.
We have previously shown that this motif interacts directly with the medium
chain subunit of the plasma membrane localized AP-2 adaptor complex (mu2). We
have investigated a range of tyrphostins and demonstrated a specific inhibition
of the interaction between mu2 and the TGN38 cytosolic domain by tyrphostin A23
through in vitro analysis and the yeast two-hybrid system. These data raise the
exciting possibility that different membrane traffic events could be inhibited
by specific tyrphostins.
PMID: 9774424 [PubMed - indexed for MEDLINE]
903: J Biol Chem 1998 Oct 23;273(43):27761-4
Oncoprotein TLS interacts with serine-arginine proteins involved in RNA
splicing.
Yang L, Embree LJ, Tsai S, Hickstein DD.
Medical Research Service, Veterans Affairs Puget Sound Health Care System,
Seattle, Washington 98108, USA.
The gene encoding the human TLS protein, also termed FUS, is located at the site
of chromosomal translocations in human leukemias and sarcomas where it forms a
chimeric fusion gene with one of several different genes. To identify
interacting partners of TLS, we screened a yeast two-hybrid cDNA library
constructed from mouse hematopoietic cells using the C-terminal region of TLS in
the bait plasmid. Two cDNAs encoding members of the serine-arginine (SR) family
of proteins were isolated. The first SR protein is the mouse homolog of human
splicing factor SC35, and the second SR member is a novel 183-amino acid protein
that we term TASR (TLS-associated serine-arginine protein). cDNA cloning of
human TASR indicated that mouse and human TASR have identical amino acid
sequences. The interactions between TLS and these two SR proteins were confirmed
by co-transfection and immunoprecipitation studies. In vivo splicing assays
indicated that SC35 and TASR influence splice site selection of adenovirus E1A
pre-mRNA. TLS may recruit SR splicing factors to specific target genes through
interaction with its C-terminal region, and chromosomal translocations that
truncate the C-terminal region of TLS may prevent this interaction. Thus TLS
translocations may alter RNA processing and play a role in malignant
transformation.
PMID: 9774382 [PubMed - indexed for MEDLINE]
904: EMBO J 1998 Oct 15;17(20):6028-38
Nucleosome structure of the yeast CHA1 promoter: analysis of
activation-dependent chromatin remodeling of an RNA-polymerase-II-transcribed
gene in TBP and RNA pol II mutants defective in vivo in response to acidic
activators.
Moreira JM, Holmberg S.
Department of Genetics, Institute of Molecular Biology, University of
Copenhagen, Oster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark.
The Saccharomyces cerevisiae CHA1 gene encodes the catabolic L-serine
(L-threonine) dehydratase. We have previously shown that the transcriptional
activator protein Cha4p mediates serine/threonine induction of CHA1 expression.
We used accessibility to micrococcal nuclease and DNase I to determine the in
vivo chromatin structure of the CHA1 chromosomal locus, both in the non-induced
state and upon induction. Upon activation, a precisely positioned nucleosome
(nuc-1) occluding the TATA box and the transcription start site is removed. A
strain devoid of Cha4p showed no chromatin alteration under inducing conditions.
Five yeast TBP mutants defective in different steps in activated transcription
abolished CHA1 expression, but failed to affect induction-dependent chromatin
rearrangement of the promoter region. Progressive truncations of the RNA
polymerase II C-terminal domain caused a progressive reduction in CHA1
transcription, but no difference in chromatin remodeling. Analysis of swi1,
swi3, snf5 and snf6, as well as gcn5, ada2 and ada3 mutants, suggested that
neither the SWI/SNF complex nor the ADA/GCN5 complex is involved in efficient
activation and/or remodeling of the CHA1 promoter. Interestingly, in a sir4
deletion strain, repression of CHA1 is partly lost and activator-independent
remodeling of nuc-1 is observed. We propose a model for CHA1 activation based on
promoter remodeling through interactions of Cha4p with chromatin components
other than basal factors and associated proteins.
PMID: 9774346 [PubMed - indexed for MEDLINE]
905: Proc Natl Acad Sci U S A 1998 Oct 13;95(21):12486-91
Rap1 protein regulates telomere turnover in yeast.
Krauskopf A, Blackburn EH.
Department of Microbiology and Immunology, University of California, San
Francisco, CA 94143-0414, USA.
Telomere length is maintained through a dynamic balance between addition and
loss of the terminal telomeric DNA. Normal telomere length regulation requires
telomerase as well as a telomeric protein-DNA complex. Previous work has
provided evidence that in the budding yeasts Kluyveromyces lactis and
Saccharomyces cerevisiae, the telomeric double-stranded DNA binding protein
Rap1p negatively regulates telomere length, in part by nucleating, by its
C-terminal tail, a higher-order DNA binding protein complex that presumably
limits access of telomerase to the chromosome end. Here we show that in K.
lactis, truncating the Rap1p C-terminal tail (Rap1p-DeltaC mutant) accelerates
telomeric repeat turnover in the distal region of the telomere. In addition,
combining the rap1-DeltaC mutation with a telomerase template mutation
(ter1-kpn), which directs the addition of mutated telomeric DNA repeats to
telomeres, synergistically caused an immediate loss of telomere length
regulation. Capping of the unregulated telomeres of these double mutants with
functionally wild-type repeats restored telomere length control. We propose that
the rate of terminal telomere turnover is controlled by Rap1p specifically
through its interactions with the most distal telomeric repeats.
PMID: 9770512 [PubMed - indexed for MEDLINE]
906: RNA 1998 Oct;4(10):1239-50
Corrected and republished in:
RNA 1998 Dec;4(12):1675-86
Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae.
Dix I, Russell CS, O'Keefe RT, Newman AJ, Beggs JD.
Institute of Cell and Molecular Biology, University of Edinburgh, United
Kingdom.
We present here the first insights into the organization of proteins on the RNA
in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly
labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting
proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller
proteins requiring an intact Sm site. Site-specific crosslinking showed that
Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with
five different regions of the 5' stem-loop, but not with the Sm site or 3'
stem-loop. Both internal loops in the 5' domain are essential for Prp8p to
associate with the snRNP, but the conserved loop 1 is not, although this is the
region to which Prp8p crosslinks most strongly. The extensive contacts between
Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in
spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing
that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p
may be the principal anchoring factor for exons in the spliceosome. This and the
close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p
support and extend the proposal that Snu114p mimics U5 loop 1 during a
translocation event in the spliceosome.
PMID: 9769098 [PubMed - indexed for MEDLINE]
907: J Virol 1998 Nov;72(11):9318-22
A map of interactions between the proteins of a retrotransposon.
Steele SJ, Levin HL.
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
The yeast two-hybrid system and in vitro binding assays were used to
characterize 54 potential interactions between the proteins of Tf1, an
LTR-retrotransposon found in Schizosaccharomyces pombe. The Tf1 integrase (IN)
protein was found to interact strongly with itself and not with other control
proteins. In addition, the IN core domain interacted strongly with itself and
full-length IN. Interestingly, the two-hybrid analysis detected an interaction
between the RNase H domain of reverse transcriptase and IN. The biological
implications of these interactions are discussed.
PMID: 9765482 [PubMed - indexed for MEDLINE]
908: J Virol 1998 Nov;72(11):9192-200
Mutations in the N terminus of the brome mosaic virus polymerase affect genetic
RNA-RNA recombination.
Figlerowicz M, Nagy PD, Tang N, Kao CC, Bujarski JJ.
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
Previously, we have observed that mutations in proteins 1a and 2a, the two
virally encoded components of the brome mosaic virus (BMV) replicase, can affect
the frequency of recombination and the locations of RNA recombination sites (P.
D. Nagy, A. Dzianott, P. Ahlquist, and J. J. Bujarski, J. Virol. 69:2547-2556,
1995; M. Figlerowicz, P. D. Nagy, and J. J. Bujarski, Proc. Natl. Acad. Sci. USA
94:2073-2078, 1997). Also, it was found before that the N-terminal domain of 2a,
the putative RNA polymerase protein, participates in the interactions between 1a
and 2a (C. C. Kao, R. Quadt, R. P. Hershberger, and P. Ahlquist, J. Virol.
66:6322-6329, 1992; E. O'Reilly, J. Paul, and C. C. Kao, J. Virol. 71:7526-7532,
1997). In this work, we examine how mutations within the N terminus of 2a
influence RNA recombination in BMV. Because of the likely electrostatic
character of 1a-2a interactions, five 2a mutants, MF1 to MF5, were generated by
replacing clusters of acidic amino acids with their neutral counterparts. MF2
and MF5 retained nearly wild-type levels of 1a-2a interaction and were
infectious in Chenopodium quinoa. However, compared to that in wild-type virus,
the frequency of nonhomologous recombination in both MF2 and MF5 was markedly
decreased. Only in MF2 was the frequency of homologous recombination reduced and
the occurrence of imprecise homologous recombination increased. In MF5 there was
also a 3' shift in the positions of homologous crossovers. The observed effects
of MF2 and MF5 reveal that the 2a N-terminal domain participates in different
ways in homologous and in nonhomologous BMV RNA recombination. This work maps
specific locations within the N terminus involved in 1a-2a interaction and in
recombination and further suggests that the mechanisms of the two types of
crossovers in BMV are different.
PMID: 9765466 [PubMed - indexed for MEDLINE]
909: Mol Biol Cell 1998 Oct;9(10):2803-17
A late mitotic regulatory network controlling cyclin destruction in
Saccharomyces cerevisiae.
Jaspersen SL, Charles JF, Tinker-Kulberg RL, Morgan DO.
Department of Physiology, University of California, San Francisco, California
94143-0444, USA.
Exit from mitosis requires the inactivation of mitotic cyclin-dependent
kinase-cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis.
Cyclin destruction is regulated by a ubiquitin ligase known as the
anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae,
members of a large class of late mitotic mutants, including cdc15, cdc5, cdc14,
dbf2, and tem1, arrest in anaphase with a phenotype similar to that of cells
expressing nondegradable forms of mitotic cyclins. We addressed the possibility
that the products of these genes are components of a regulatory network that
governs cyclin proteolysis. We identified a complex array of genetic
interactions among these mutants and found that the growth defect in most of the
mutants is suppressed by overexpression of SPO12, YAK1, and SIC1 and is
exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late
mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is
accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1.
Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15,
Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late
mitosis but are not required for maintenance of that activity in G1.
PMID: 9763445 [PubMed - indexed for MEDLINE]
910: Mol Biol Cell 1998 Oct;9(10):2729-38
The role of glucosidase I (Cwh41p) in the biosynthesis of cell wall
beta-1,6-glucan is indirect.
Abeijon C, Chen LY.
Department of Molecular and Cell Biology, Boston University-Goldman School of
Dental Medicine, Boston, Massachusetts 02118, USA. cabeijon@bu.edu
CWH41, a gene involved in the assembly of cell wall beta-1,6-glucan, has
recently been shown to be the structural gene for Saccharomyces cerevisiae
glucosidase I that is responsible for initiating the trimming of terminal
alpha-1,2-glucose residue in the N-glycan processing pathway. To distinguish
between a direct or indirect role of Cwh41p in the biosynthesis of
beta-1,6-glucan, we constructed a double mutant, alg5Delta (lacking
dolichol-P-glucose synthase) cwh41Delta, and found that it has the same
phenotype as the alg5Delta single mutant. It contains wild-type levels of cell
wall beta-1,6-glucan, shows moderate underglycosylation of N-linked
glycoproteins, and grows at concentrations of Calcofluor White (which interferes
with cell wall assembly) that are lethal to cwh41Delta single mutant. The strong
genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in
the beta-1,6-glucan biosynthetic pathway, disappear in the absence of
dolichol-P-glucose synthase (alg5Delta). The triple mutant
alg5Deltacwh41Deltakre6Delta is viable, whereas the double mutant
cwh41Deltakre6Delta in the same genetic background is not. The severe slow
growth phenotype and 75% reduction in cell wall beta-1,6-glucan, characteristic
of the cwh41Deltakre1Delta double mutant, are not observed in the triple mutant
alg5Deltacwh41Deltakre1Delta. Kre6p, a putative Golgi glucan synthase, is
unstable in cwh41Delta strains, and its overexpression renders these cells
Calcofluor White resistant. These results demonstrate that the role of
glucosidase I (Cwh41p) in the biosynthesis of cell wall beta-1,6-glucan is
indirect and that dolichol-P-glucose is not an intermediate in this pathway.
PMID: 9763440 [PubMed - indexed for MEDLINE]
911: J Cell Biol 1998 Oct 5;143(1):49-63
Characterization of the kinetochore binding domain of CENP-E reveals
interactions with the kinetochore proteins CENP-F and hBUBR1.
Chan GK, Schaar BT, Yen TJ.
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia,
Pennsylvania 19111, USA.
We have identified a 350-amino acid domain in the kinetochore motor CENP-E that
specifies kinetochore binding in mitosis but not during interphase. The
kinetochore binding domain was used in a yeast two-hybrid screen to isolate
interacting proteins that included the kinetochore proteins CENP-E, CENP-F, and
hBUBR1, a BUB1-related kinase that was found to be mutated in some colorectal
carcinomas (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Wilson, S.D.
Markowitz, K.W. Kinzler, and B. Vogelstein. 1998. Nature. 392:300-303). CENP-F,
hBUBR1, and CENP-E assembled onto kinetochores in sequential order during late
stages of the cell cycle. These proteins therefore define discrete steps along
the kinetochore assembly pathway. Kinetochores of unaligned chromosome exhibited
stronger hBUBR1 and CENP-E staining than those of aligned chromosomes. CENP-E
and hBUBR1 remain colocalized at kinetochores until mid-anaphase when hBUBR1
localized to portions of the spindle midzone that did not overlap with CENP-E.
As CENP-E and hBUBR1 can coimmunoprecipitate with each other from HeLa cells,
they may function as a motor-kinase complex at kinetochores. However, the
complex distribution pattern of hBUBR1 suggests that it may regulate multiple
functions that include the kinetochore and the spindle midzone.
PMID: 9763420 [PubMed - indexed for MEDLINE]
912: Biochemistry 1998 Oct 6;37(40):14257-66
Kinetics of dimerization and interactions of p13suc1 with cyclin-dependent
kinases.
Morris MC, Heitz F, Divita G.
Centre de Recherches de Biochimie Macromoleculaire, CNRS, Montpellier, France.
The impact of p13suc1 on the conformation and regulation of cyclin-dependent
kinases (cdks) and cyclins was investigated by spectroscopic and rapid kinetic
approaches. In the absence of phosphorylation on cdks, p13suc1 formed stable
complexes, mainly stabilized by hydrophobic interactions, specifically with cdk2
and cdc2. The presence of cyclin A, associated with cdk2 or cdc2, increased the
stability of the interaction between cdk2 and p13suc1 by a factor of 2. However,
cyclin A did not modify the association rate of p13suc1 to cdk2, but the
dissociation rate, which was decreased 3-fold. Moreover, binding of p13suc1 to
cdk2 resulted in a 2-fold decrease in the release of nucleotide from cdk2,
indicating that p13suc1 induces a marked change in the structure of the
nucleotide binding site of cdks. On the basis of the structure of cdk2/CksHs1
complex and on our kinetic results, we propose that the binding of Cks proteins
to C-lobe of cdk2 is stabilized by the presence of cyclin A and that it may
modify the orientation of the loop carrying residues 14 and 15 and their
consequent access for dephosphorylation by cdc25 phosphatases. Finally, we have
shown that dimerization of p13suc1 in the presence of zinc abolishes its
interaction with cdks, which suggests that the binding of p13suc1 to cdk2 or
cdk2/cyclin A may be regulated by dimerization of p13suc1 in vivo.
PMID: 9760264 [PubMed - indexed for MEDLINE]
913: EMBO J 1998 Oct 1;17(19):5796-804
Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and
Cox3p depends on targeting information in untranslated portions of their mRNAs.
Sanchirico ME, Fox TD, Mason TL.
Department of Biochemistry and Molecular Biology and The Graduate Program in
Molecular and Cellular Biology, University of Massachusetts, Amherst, MA
01003-4505, USA.
The essential products of the yeast mitochondrial translation system are seven
hydrophobic membrane proteins and Var1p, a hydrophilic protein in the small
ribosomal subunit. Translation of the membrane proteins depends on nuclearly
encoded, mRNA-specific translational activators that recognize the
5'-untranslated leaders of their target mRNAs. These translational activators
are themselves membrane associated and could therefore tether translation to the
inner membrane. In this study, we tested whether chimeric mRNAs with the
untranslated sequences normally present on the mRNA encoding soluble Var1p, can
direct functional expression of coding sequences specifying the integral
membrane proteins Cox2p and Cox3p. DNA sequences specifying these chimeric mRNAs
were inserted into mtDNA at the VAR1 locus and expressed in strains containing a
nuclearly localized plasmid that supplies a functional form of Var1p, imported
from the cytoplasm. Although cells expressing these chimeric mRNAs actively
synthesized both membrane proteins, they were severely deficient in cytochrome c
oxidase activity and in the accumulation of Cox2p and Cox3p, respectively. These
data strongly support the physiological importance of interactions between
membrane-bound mRNA-specific translational activators and the native
5'-untranslated leaders of the COX2 and COX3 mRNAs for localizing productive
synthesis of Cox2p and Cox3p to the inner membrane.
PMID: 9755179 [PubMed - indexed for MEDLINE]
914: EMBO J 1998 Oct 1;17(19):5679-88
The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent
hyperphosphorylation and interacts with Rad53 after DNA damage.
Vialard JE, Gilbert CS, Green CM, Lowndes NF.
Imperial Cancer Research Fund, Clare Hall Laboratories, CDC Laboratory, South
Mimms, Hertfordshire EN6 3LD, UK.
The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient
cell-cycle arrests and transcriptional induction of DNA repair genes in response
to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized
several polypeptides in asynchronous cultures, and in cells arrested in S or
G2/M phases while a single form was observed in G1-arrested cells. Treatment
with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane
sulfonate, resulted in the appearance of hypermodified forms of the protein. All
modifications detected during a normal cell cycle and after DNA damage were
sensitive to phosphatase treatment, indicating that they resulted from
phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with
checkpoint functions (cell-cycle arrest and transcriptional induction) and was
cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9
hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and
ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required
functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating
cell-cycle stage specificity of the checkpoint genes in this response to DNA
damage. Analysis of checkpoint protein interactions after DNA damage revealed
that Rad9 physically associates with Rad53.
PMID: 9755168 [PubMed - indexed for MEDLINE]
915: EMBO J 1998 Oct 1;17(19):5525-8
DNA damage checkpoint in budding yeast.
Longhese MP, Foiani M, Muzi-Falconi M, Lucchini G, Plevani P.
Dipartimento di Genetica e Biologia dei Microrganismi, Via Celoria 26, 20133
Milano, Italy.
Eukaryotic cells have evolved a network of control mechanisms, known as
checkpoints, which coordinate cell-cycle progression in response to internal and
external cues. The yeast Saccharomyces cerevisiae has been invaluable in
dissecting genetically the DNA damage checkpoint pathway. Recent results on
posttranslational modifications and protein-protein interactions of some key
factors provide new insights into the architecture of checkpoint protein
complexes and their order of function.
Publication Types:
Review
Review, Tutorial
PMID: 9755152 [PubMed - indexed for MEDLINE]
916: Nucleic Acids Res 1998 Oct 15;26(20):4771-7
Interaction of myocyte enhancer factor 2 (MEF2) with a mitogen-activated protein
kinase, ERK5/BMK1.
Yang CC, Ornatsky OI, McDermott JC, Cruz TF, Prody CA.
Department of Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, Ontario, Canada.
Myocyte enhancer factor 2 (MEF2) has been implicated in the complex hierarchical
regulation of muscle-specific gene expression and differentiation. While the
MyoD family members are able to initiate the skeletal muscle differentiation
program, whether MEF2 is sufficient in directing skeletal muscle differentiation
is still controversial. Furthermore, how MEF2 transactivates its target genes is
not fully understood. It has been suggested that the interactions of MEF2 with
other factors modify its transcriptional activity. Therefore, the identification
of MEF2-interacting factors may be important in understanding the mechanism by
which MEF2 activates its target genes. In this study, a mitogen-activated
protein kinase (MAP kinase), ERK5/BMK1 was found to interact with MEF2 in a
yeast two hybrid screen. The interaction was confirmed by a glutathione S
-transferase-pull down assay and a co-immunoprecipitation study indicating that
endogenous ERK5 and MEF2 interact with each other in vivo . The interacting
domain of MEF2 was mapped to the N-terminus which contains the highly conserved
MADS and MEF2 domains. Functionally, ERK5/BMK1 was able to phosphorylate MEF2 in
vitro . Furthermore, when cotransfected with ERK5/BMK1, the transactivation
capacity of MEF2 was enhanced. These results suggest that the functions of MEF2
could be regulated through ERK5/BMK1.
PMID: 9753748 [PubMed - indexed for MEDLINE]
917: Biochem Biophys Res Commun 1998 Sep 18;250(2):212-6
Protein-protein interactions of the yeast Golgi t-SNARE Sed5 protein distinct
from its neural plasma membrane cognate syntaxin 1.
Kosodo Y, Noda Y, Yoda K.
Department of Biotechnology, University of Tokyo, Japan.
Targeting of vesicles to the acceptor membrane in protein transport depends on
membrane proteins called SNAREs. Saccharomyces cerevisiae Golgi t-SNARE Sed5
protein and its neural cognate syntaxin 1 have similar three alpha-helices which
are predicted to form coiled coils. We dissected the helices of Sed5 and found
several characteristics unexpectedly distinct from those of syntaxin 1. Most
importantly, only the N-terminal helix is responsible for the binding of Sly1
protein while almost the entire molecule of syntaxin is necessary for the
binding of the cognate, Munc-18. The N-terminal region of Sed5 protein also
binds to the C-terminal helix and Sly1 protein interfered this binding.
PMID: 9753609 [PubMed - indexed for MEDLINE]
918: Proc Natl Acad Sci U S A 1998 Sep 29;95(20):11590-5
Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho
corepressors.
Levanon D, Goldstein RE, Bernstein Y, Tang H, Goldenberg D, Stifani S, Paroush
Z, Groner Y.
Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot
76100, Israel.
The mammalian AML/CBFalpha runt domain (RD) transcription factors regulate
hematopoiesis and osteoblast differentiation. Like their Drosophila
counterparts, most mammalian RD proteins terminate in a common pentapeptide,
VWRPY, which serves to recruit the corepressor Groucho (Gro). Using a yeast
two-hybrid assay, in vitro association and pull-down experiments, we demonstrate
that Gro and its mammalian homolog TLE1 specifically interact with AML1 and
AML2. In addition to the VWRPY motif, other C-terminal sequences are required
for these interactions with Gro/TLE1. TLE1 inhibits AML1-dependent
transactivation of the T cell receptor (TCR) enhancers alpha and beta, which
contain functional AML binding sites, in transfected Jurkat T cells. LEF-1 is an
additional transcription factor that mediates transactivation of TCR enhancers.
LEF-1 and its Drosophila homolog Pangolin (Pan) are involved in the Wnt/Wg
signaling pathway through interactions with the coactivator beta-catenin and its
highly conserved fly homolog Armadillo (Arm). We show that TLE/Gro interacts
with LEF-1 and Pan, and inhibits LEF-1:beta-catenin-dependent transcription.
These data indicate that, in addition to their activity as transcriptional
activators, AML1 and LEF-1 can act, through recruitment of the corepressor TLE1,
as transcriptional repressors in TCR regulation and Wnt/Wg signaling.
PMID: 9751710 [PubMed - indexed for MEDLINE]
919: Biochim Biophys Acta 1998 Oct 1;1400(1-3):3-18
Structure of DNA topoisomerases.
Berger JM.
Division of Biochemistry and Molecular Biology, Department of Molecular and
Cellular Biology, 229 Stanley Hall, University of California, Berkeley,
Berkeley, CA 94720, USA.
Over the last several years topoisomerases have finally begun to yield to
high-resolution structural studies. These models have greatly aided our
understanding of the mechanisms of topoisomerase catalysis and drug
interactions. This review will cover advances in the structural biology of
topoisomerases and discuss their implications for topoisomerase function.
Publication Types:
Review
Review, Tutorial
PMID: 9748476 [PubMed - indexed for MEDLINE]
920: Mol Gen Genet 1998 Aug;259(2):142-9
The chromatin structure of the GAL1 promoter forms independently of Reb1p in
Saccharomyces cerevisiae.
Reagan MS, Majors JE.
Department of Biochemistry and Molecular Biophysics, Washington University
School of Medicine, Saint Louis, MO 63110, USA. mreagan@csbsju.edu
Positive and negative regulation of the GAL1 promoter of the yeast Saccharomyces
cerevisiae results from a network of interactions between transcription factors
and chromatin. In this study we used footprinting procedures to characterize
these interactions in vivo. DNase I analysis of the GAL1 upstream activating
sequence (UAS(GAL1/10)) showed expected Gal4 activator protein binding during
growth in galactose, and also revealed binding of the Reb1 protein (Reb1p)
during growth in glucose. In addition, we mapped to nucleotide resolution a
positioned nucleosome that, in the inactive promoter, packages DNA between the
UAS(GAL1/10) and the GAL1 TATA sequence, leaving both of these elements
nucleosome free. The nucleosome footprint was lost when the promoter was
activated. Surprisingly, mutation of the Reb1p binding site had no effect on
nucleosome positioning or on the kinetics or extent of activation or repression
of either the GAL1 or GAL10 promoters under any of the conditions assayed.
PMID: 9747705 [PubMed - indexed for MEDLINE]
921: Curr Biol 1998 Aug 27;8(17):959-62
The WASp homologue Las17p functions with the WIP homologue End5p/verprolin and
is essential for endocytosis in yeast.
Naqvi SN, Zahn R, Mitchell DA, Stevenson BJ, Munn AL.
Institute of Molecular Agrobiology, National University of Singapore, Republic
of Singapore.
Several end mutations that block the internalisation step of endocytosis in
Saccharomyces cerevisiae also affect the cortical actin cytoskeleton [1]. END5
encodes a proline-rich protein (End5p or verprolin) required for a polarised
cortical actin cytoskeleton and endocytosis [2,3]. End5p interacts with actin
[4], but its exact function is not yet known. To help elucidate End5p function,
we sought other End5p-interacting proteins and identified the LAS17/BEE1 gene
(encoding the yeast homologue of the human Wiskott-Aldrich Syndrome protein,
WASp) as a high-copy-number suppressor of the temperature-sensitive growth and
endocytic defects of end5-1 cells (carrying a frameshift mutation affecting the
last 213 residues of End5p). LAS17 is unable to suppress a full deletion of END5
(end5 delta), however, suggesting that the defective End5-1p in end5-1 mutants
may be stabilised by Las17p. The amino terminus of Las17p interacts with the
carboxyl terminus of End5p in the yeast two-hybrid system and similar
interactions have been shown between WASp and a mammalian End5p homologue,
WASp-interacting protein (WIP) [5]. As las17 delta deletion mutants are blocked
in endocytosis, we conclude that Las17p and End5p interact and are essential for
endocytosis.
PMID: 9742397 [PubMed - indexed for MEDLINE]
922: Mol Cell Biol 1998 Oct;18(10):6110-20
Functional characterization of the N terminus of Sir3p.
Gotta M, Palladino F, Gasser SM.
Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges/Lausanne,
Switzerland.
Silent information regulator 3 is an essential component of the Saccharomyces
cerevisiae silencing complex that functions at telomeres and the silent
mating-type loci, HMR and HML. We show that expression of the N- and
C-terminal-encoding halves of SIR3 in trans partially complements the mating
defect of the sir3 null allele, suggesting that the two domains have distinct
functions. We present here a functional characterization of these domains. The
N-terminal domain (Sir3N) increases both the frequency and extent of
telomere-proximal silencing when expressed ectopically in SIR+ yeast strains,
although we are unable to detect interaction between this domain and any known
components of the silencing machinery. In contrast to its effect at telomeres,
Sir3N overexpression derepresses transcription of reporter genes inserted in the
ribosomal DNA (rDNA) array. Immunolocalization of Sir3N-GFP and Sir2p suggests
that Sir3N directly antagonizes nucleolar Sir2p, releasing an rDNA-bound
population of Sir2p so that it can enhance repression at telomeres.
Overexpression of the C-terminal domain of either Sir3p or Sir4p has a
dominant-negative effect on telomeric silencing. In strains overexpressing the
C-terminal domain of Sir4p, elevated expression of either full-length Sir3p or
Sir3N restores repression and the punctate pattern of Sir3p and Rap1p
immunostaining. The similarity of Sir3N and Sir3p overexpression phenotypes
suggests that Sir3N acts as an allosteric effector of Sir3p, either enhancing
its interactions with other silencing components or liberating the full-length
protein from nonfunctional complexes.
PMID: 9742128 [PubMed - indexed for MEDLINE]
923: Mol Cell Biol 1998 Oct;18(10):5942-51
Processivity of the Saccharomyces cerevisiae poly(A) polymerase requires
interactions at the carboxyl-terminal RNA binding domain.
Zhelkovsky A, Helmling S, Moore C.
Department of Molecular Biology and Microbiology, Tufts University School of
Medicine, Boston, Massachusetts 02111-1800, USA.
The interaction of the Fip1 subunit of polyadenylation factor I with the
Saccharomyces cerevisiae poly(A) polymerase (PAP) was assayed in vivo by
two-hybrid analysis and was found to involve two separate regions on PAP,
located at opposite ends of the protein sequence. In vitro, Fip1 blocks access
of the RNA primer to an RNA binding site (RBS) that overlaps the Fip1
carboxy-terminal interaction region and, in doing so, shifts PAP to a
distributive mode of action. Partial truncation of this RBS has the same effect,
indicating that this site is required for processivity. A comparison of the
utilization of ribo- and deoxyribonucleotides as substrates indicates the
existence on PAP of a second RBS which recognizes the last three nucleotides at
the 3' end of the primer. This site discriminates against deoxyribonucleotides
at the 3' end, and interactions at this site are not affected by Fip1. Further
analysis revealed that the specificity of PAP for adenosine is not simply a
function of the ATP binding site but also reflects interactions with bases at
the 3' end of the primer and at another contact site 14 nucleotides upstream of
the 3' end. These results suggest that the unique specificity of PAP for ribose
and base, and thus the extent and type of activity with different substrates,
depends on interactions at multiple nucleotide binding sites.
PMID: 9742111 [PubMed - indexed for MEDLINE]
924: J Biol Chem 1998 Sep 25;273(39):25041-4
Ribosomal P-protein stalk function is targeted by sordarin antifungals.
Gomez-Lorenzo MG, Garcia-Bustos JF.
Research Department, Glaxo Wellcome, S. A., Severo Ochoa 2, 28760 Tres Cantos,
Spain.
Sordarin derivatives are remarkably selective inhibitors of fungal protein
synthesis. Available evidence points to a binding site for these inhibitors on
elongation factor 2, but high affinity binding requires the presence of
ribosomes. The gene mutated in one of the two isolated complementation groups of
Saccharomyces cerevisiae mutants resistant to the sordarin derivative GM193663
has now been identified. It is RPP0, encoding the essential protein of the large
ribosomal subunit stalk rpP0. Resistant mutants are found to retain most of the
binding capacity for the drug, indicating that mutations in rpP0 endow the
ribosome with the capacity to perform translation elongation in the presence of
the inhibitor. Other proteins of the ribosomal stalk influence the expression of
resistance, pointing to a wealth of interactions between stalk components and
elongation factors. The involvement of multiple elements of the translation
machinery in the mode of action of sordarin antifungals may explain the large
selectivity of these compounds, even though the individual target components are
highly conserved proteins.
PMID: 9737960 [PubMed - indexed for MEDLINE]
925: J Mol Biol 1998 Sep 25;282(3):525-41
Mutant alleles of the MRS2 gene of yeast nuclear DNA suppress mutations in the
catalytic core of a mitochondrial group II intron.
Schmidt U, Maue I, Lehmann K, Belcher SM, Stahl U, Perlman PS.
Department of Microbiology and Genetics, University of Technology, Berlin,
D-13355, Germany. schm1534@mailszrz.zrz.tu-berlin.de
Previous studies show that some yeast strains carrying point mutations of domain
5 that block splicing of a mitochondrial group II intron yield spontaneous
revertants in which splicing is partially restored by dominant mutations of
nuclear genes. Here we cloned and sequenced the suppressor allele of one such
gene, and found it to be a missense mutation of the MRS2 gene (MRS2-L232F). The
MRS2 gene was first implicated in group II intron splicing by the finding that
overexpression of the wild-type gene weakly suppresses the splicing defect of a
mutation of another intron. Tetrad analysis showed that independently isolated
suppressors of two other domain 5 mutations are also allelles of the MRS2 gene
and DNA sequencing identified a new missense mutation in each strain (MRS2-T230I
and MRS2-L213M). All three suppressor mutations cause a temperature-sensitive
respiration defect that is dominant negative in heterozygous diploids, but those
strains splice the mutant intron at the elevated temperature. The three
mutations are in a domain of the protein that is likely to be a helix-turn-helix
region, so that effects of the mutations on protein-protein interactions may
contribute to these phenotypes. These mutations suppress the splicing defect of
many, but not all, of the available splicing defective mutations of aI5gamma,
including mutations of several intron domains. Protein and RNA blot experiments
show that the level of the protein encoded by the MRS2 gene, but not the mRNA,
is elevated by these mutations. Interestingly, overexpression of the wild-type
protein restores much lower levels of splicing than were obtained with similar
elevated levels of the mutated Mrs2 proteins. The splicing phenotypes of these
strains suggest a direct role for Mrs2 protein on group II intron splicing, but
an indirect effect is not yet ruled out. Copyright 1998 Academic Press.
PMID: 9737920 [PubMed - indexed for MEDLINE]
926: EMBO J 1998 Sep 15;17(18):5438-48
L-arginine recognition by yeast arginyl-tRNA synthetase.
Cavarelli J, Delagoutte B, Eriani G, Gangloff J, Moras D.
UPR 9004 Biologie Structurale, Institut de Genetique et de Biologie Moleculaire
et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch Cedex, France.
The crystal structure of arginyl-tRNA synthetase (ArgRS) from Saccharomyces
cerevisiae, a class I aminoacyl-tRNA synthetase (aaRS), with L-arginine bound to
the active site has been solved at 2.75 A resolution and refined to a
crystallographic R-factor of 19.7%. ArgRS is composed predominantly of
alpha-helices and can be divided into five domains, including the class
I-specific active site. The N-terminal domain shows striking similarity to some
completely unrelated proteins and defines a module which should participate in
specific tRNA recognition. The C-terminal domain, which is the putative
anticodon-binding module, displays an all-alpha-helix fold highly similar to
that of Escherichia coli methionyl-tRNA synthetase. While ArgRS requires tRNAArg
for the first step of the aminoacylation reaction, the results show that its
presence is not a prerequisite for L-arginine binding. All H-bond-forming
capability of L-arginine is used by the protein for the specific recognition.
The guanidinium group forms two salt bridge interactions with two acidic
residues, and one H-bond with a tyrosine residue; these three residues are
strictly conserved in all ArgRS sequences. This tyrosine is also conserved in
other class I aaRS active sites but plays several functional roles. The ArgRS
structure allows the definition of a new framework for sequence alignments and
subclass definition in class I aaRSs.
PMID: 9736621 [PubMed - indexed for MEDLINE]
927: Methods 1998 Jul;15(3):207-23
Using genetic means to dissect homologous and heterologous protein-protein
interactions of PKR, the interferon-induced protein kinase.
Tan SL, Katze MG.
School of Medicine, University of Washington, Seattle, Washington, 98195, USA.
The interferon-induced protein kinase, PKR, is a pivotal component of interferon
(IFN)-induced cellular antiviral and antiproliferative response. The
identification and characterization of proteins, of both viral and cellular
origins, that interact with PKR have proven to be a valuable probe for
unraveling the cellular regulation and function of PKR. Several studies have
demonstrated that PKR forms dimers and that dimerization is likely to be
required for activation and/or catalytic function. It is therefore important to
elucidate the mechanism of PKR dimer formation and the role of PKR effectors in
modulating kinase dimerization. Herein we describe the use of the two genetic
approaches, the lambda repressor fusion and the yeast two-hybrid systems, to
detect and analyze homo- and heterotypic interactions with PKR. We also describe
several biochemical methodologies commonly used in our laboratory to validate
the genetic results. Although the examples in this article focus on PKR, the
techniques can easily be adapted to investigate protein-protein associations in
a variety of experimental systems. Finally, given the important role of PKR as a
mediator of IFN-induced antiviral and antiproliferative effects, these studies
may provide clues to the development of reagents that target PKR to enhance the
therapeutic use of IFN in the treatment of disease. Copyright 1998 Academic
Press.
PMID: 9735306 [PubMed - indexed for MEDLINE]
928: Genes Cells 1998 Jun;3(6):357-69
Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA
polymerase delta and replication factor C.
Oku T, Ikeda S, Sasaki H, Fukuda K, Morioka H, Ohtsuka E, Yoshikawa H, Tsurimoto
T.
Faculty of Biological Science, Nara Institute of Science and Technology,
Takayama, Ikoma, Japan.
BACKGROUND: PCNA, an eukaryotic DNA sliding clamp interacts with replication
factors and the cell cycle protein, p21(Cip1/Waf1) and functions as a molecular
switch for DNA elongation. To understand how DNA replication is regulated
through PCNA, elucidation of the precise mechanisms of these protein
interactions is necessary. RESULTS: Loop-region mutants in which human PCNA
sequences were substituted with the corresponding Saccharomyces cerevisiae PCNA
regions were prepared. Analysis of their functions, along with previously
prepared alanine scanning mutants, demonstrated that some loops interact with
DNA polymerase delta (pol delta) and replication factor C (RFC). The p21 binding
sites of PCNA, mapped by affinity measurement of the mutant forms, found to be
located within a distinct structure of the PCNA monomer, overlap with RFC- and
pol delta-interaction sites. Competition between p21 and pol delta or RFC for
binding to PCNA results in efficient inhibition of its stimulation of pol delta
DNA synthesis and RFC ATPase but not of PCNA loading on DNA by RFC. CONCLUSIONS:
Semi-saturated amounts of p21 selectively block formation of the active pol
delta complex but not the RFC-PCNA complex at 3'-ends of DNA primers. This
differential effect may explain the specific inhibition of DNA replication by
p21.
PMID: 9734782 [PubMed - indexed for MEDLINE]
929: Genes Cells 1998 Jun;3(6):347-55
Defect in cytokinesis of fission yeast induced by mutation in the WD40 repeat
motif of a TFIID subunit.
Yamamoto T, Horikoshi M.
Department of Cellular Biology, Institute of Molecular and Cellular Biosciences,
The University of Tokyo, Japan.
BACKGROUND: TBP-associated factors contain a variety of structural motifs and
their related in vivo significance has remained unclear. We have attempted to
identify specific biological phenomena linked to a particular domain of a TAF by
analysing domain-exchanged chimeric mutants between Schizosaccharomyces pombe
(Sp) and Saccharomyces cerevisiae (Sc) counterparts. RESULTS: Contrary to the
case of TBP, Sp TAF containing the WD40 repeat cannot be exchanged for its Sc
counterpart, despite their highly conserved primary structures. This
'species-specific' function locates in the N-terminal region. The C-terminal
region, largely consisting of the WD40 repeat, is exchangeable for the
corresponding region of its Sc counterpart. Growth of the strain harbouring this
C-terminal chimeric mutant is temperature-sensitive. The chimeric gene product
did not disappear at a restrictive temperature, a finding which strongly
suggests that the growth defect is caused by an aberration in the interactions
through the WD40 repeat structural motif. With temperature elevation, the
chimeric mutants underwent drastic morphological changes due to a defect in
cytokinesis. CONCLUSIONS: The WD40 repeat of TAF is primarily involved in
reactions which might regulate cytokinesis in Sp.
PMID: 9734781 [PubMed - indexed for MEDLINE]
930: J Virol 1998 Oct;72(10):8332-7
CREB-2, a cellular CRE-dependent transcription repressor, functions in
association with Tax as an activator of the human T-cell leukemia virus type 1
promoter.
Gachon F, Peleraux A, Thebault S, Dick J, Lemasson I, Devaux C, Mesnard JM.
Laboratoire Infections Retrovirales et Signalisation Cellulaire, CRBM-CNRS UPR
1086, Institut de Biologie, 34060 Montpellier, France.
The Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) has been
implicated in human T-cell immortalization. The primary function of Tax is to
transcriptionally activate the HTLV-1 promoter, but Tax is also known to
stimulate expression of cellular genes. It has been reported to associate with
several transcription factors, as well as proteins not involved in
transcription. To better characterize potential cellular targets of Tax present
in infected cells, a Saccharomyces cerevisiae two-hybrid screening was performed
with a cDNA library constructed from the HTLV-1-infected MT2 cell line. From
this study, we found 158 positive clones representing seven different cDNAs. We
focused our attention on the cDNA encoding the transcription factor CREB-2.
CREB-2 is an unconventional member of the ATF/CREB family in that it lacks a
protein kinase A (PKA) phosphorylation site and has been reported to negatively
regulate transcription from the cyclic AMP response element of the human
enkephalin promoter. In this study, we demonstrate that CREB-2 cooperates with
Tax to enhance viral transcription and that its basic-leucine zipper C-terminal
domain is required for both in vitro and in vivo interactions with Tax. Our
results confirm that the activation of the HTLV-1 promoter through Tax and
factors of the ATF/CREB family is PKA independent.
PMID: 9733879 [PubMed - indexed for MEDLINE]
931: J Biol Chem 1998 Sep 18;273(38):24963-71
Transmembrane protein insertion orientation in yeast depends on the charge
difference across transmembrane segments, their total hydrophobicity, and its
distribution.
Harley CA, Holt JA, Turner R, Tipper DJ.
Department of Molecular Genetics and Microbiology, University of Massachusetts
Medical School, Worcester, Massachusetts 01655, USA.
The determinants of transmembrane protein insertion orientation at the
endoplasmic reticulum have been investigated in Saccharomyces cerevisiae using
variants of a Type III (naturally exofacial N terminus (Nexo)) transmembrane
fusion protein derived from the N terminus of Ste2p, the alpha-factor receptor.
Small positive and negative charges adjacent to the transmembrane segment had
equal and opposite effects on orientation, and this effect was independent of N-
or C-terminal location, consistent with a purely electrostatic interaction with
response mechanisms. A 3:1 bias toward Nexo insertion, observed in the absence
of a charge difference, was shown to reflect the Nexo bias conferred by longer
transmembrane segments. Orientation correlated best with total hydrophobicity
rather than length, but it was also strongly affected by the distribution of
hydrophobicity within the transmembrane segment. The most hydrophobic terminus
was preferentially translocated. Insertion orientation thus depends on
integration of responses to at least three parameters: charge difference across
a transmembrane segment, its total hydrophobicity, and its hydrophobicity
gradient. Relative signal strengths were estimated, and consequences for
topology prediction are discussed. Responses to transmembrane sequence may
depend on protein-translocon interactions, but responses to charge difference
may be mediated by the electrostatic field provided by anionic phospholipids.
PMID: 9733804 [PubMed - indexed for MEDLINE]
932: J Mol Biol 1998 Sep 11;282(1):13-24
Missense translation errors in Saccharomyces cerevisiae.
Stansfield I, Jones KM, Herbert P, Lewendon A, Shaw WV, Tuite MF.
Research School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ,
UK.
We describe the development of a novel plasmid-based assay for measuring the in
vivo frequency of misincorporation of amino acids into polypeptide chains in the
yeast Saccharomyces cerevisiae. The assay is based upon the measurement of the
catalytic activity of an active site mutant of type III chloramphenicol acetyl
transferase (CATIII) expressed in S. cerevisiae. A His195(CAC)-->Tyr195(UAC)
mutant of CATIII is completely inactive, but catalytic activity can be restored
by misincorporation of histidine at the mutant UAC codon. The average error
frequency of misincorporation of histidine at this tyrosine UAC codon in
wild-type yeast strains was measured as 0. 5x10(-5) and this frequency was
increased some 50-fold by growth in the presence of paromomycin, a known
translational-error-inducing antibiotic. A detectable frequency of
misincorporation of histidine at a mutant Ala195 GCU codon was also measured as
2x10(-5), but in contrast to the Tyr195-->His195 misincorporation event, the
frequency of histidine misincorporation at Ala195 GCU was not increased by
paromomycin, inferring that this error did not result from miscognate
codon-anticodon interaction. The His195 to Tyr195 missense error assay was used
to demonstrate increased frequencies of missense error at codon 195 in SUP44 and
SUP46 mutants. These two mutants have previously been shown to exhibit a
translation termination error phenotype and the sup44+ and sup46+ genes encode
the yeast ribosomal proteins S4 and S9, respectively. These data represent the
first accurate in vivo measurement of a specific mistranslation event in a
eukaryotic cell and directly confirm that the eukaryotic ribosome plays an
important role in controlling missense errors arising from non-cognate
codon-anticodon interactions. Copyright 1998 Academic Press.
PMID: 9733638 [PubMed - indexed for MEDLINE]
933: Mol Pharmacol 1998 Sep;54(3):591-8
Pharmacological analysis of sterol delta8-delta7 isomerase proteins with
[3H]ifenprodil.
Moebius FF, Reiter RJ, Bermoser K, Glossmann H, Cho SY, Paik YK.
Institut fur Biochemische Pharmakologie, Universitat Innsbruck, Peter Mayr Str.
1, A-6020 Innsbruck, Austria.
Sterol Delta8-Delta7 isomerases (SIs) catalyze the shift of the double bond from
C8-9 to C7-8 in the B-ring of sterols. Surprisingly, the isoenzymes in fungi
(ERG2p) and vertebrates [emopamil binding protein (EBP)] are structurally
completely unrelated, whereas the sigma1 receptor, a mammalian protein of
unknown function, bears significant similarity with the yeast ERG2p. Here, we
compare the drug binding properties of SIs and related proteins with
[3H]ifenprodil as a common high affinity radioligand (Kd = 1.4-19 nM),
demonstrating an intimate pharmacological relationship among ERG2p, sigma1
receptor, and EBP. This renders SIs a remarkable example for structurally
diverse enzymes with similar pharmacological profiles and the propensity to bind
drugs from different chemical groups with high affinity. We identified a variety
of experimental drugs with nanomolar affinity for the human EBP (Ki = 0.5-14 nM)
such as MDL28815, AY9944, triparanol, and U18666A. These compounds, as well as
the fungicide tridemorph and the clinically used drugs tamoxifen, clomiphene,
amiodarone, and opipramol, inhibit the in vitro activity of the recombinant
human EBP (IC50 = 0.015-54 microM). The high affinity of the human EBP for
3H-tamoxifen (Kd = 3 +/- 2 nM) implies that the EBP carries the previously
described microsomal antiestrogen binding site. Interactions of the EBP with
structurally diverse lipophilic amines suggest that novel compounds of related
structure should be counterscreened for inhibition of the enzyme to avoid
interference with sterol Delta8-Delta7 isomerization.
PMID: 9730919 [PubMed - indexed for MEDLINE]
934: Biochemistry 1998 Sep 8;37(36):12496-506
The 32- and 14-kilodalton subunits of replication protein A are responsible for
species-specific interactions with single-stranded DNA.
Sibenaller ZA, Sorensen BR, Wold MS.
Department of Biochemistry, University of Iowa College of Medicine, Iowa City
52242, USA.
Replication protein A (RPA) is a multisubunit single-stranded DNA-binding
(ssDNA) protein that is required for cellular DNA metabolism. RPA homologues
have been identified in all eukaryotes examined. All homologues are
heterotrimeric complexes with subunits of approximately 70, approximately 32,
and approximately 14 kDa. While RPA homologues are evolutionarily conserved,
they are not functionally equivalent. To gain a better understanding of the
functional differences between RPA homologues, we analyzed the DNA-binding
parameters of RPA from human cells and the budding yeast Saccharomyces
cerevisiae (hRPA and scRPA, respectively). Both yeast and human RPA bind ssDNA
with high affinity and low cooperativity. However, scRPA has a larger occluded
binding site (45 nucleotides versus 34 nucleotides) and a higher affinity for
oligothymidine than hRPA. Mutant forms of hRPA and scRPA containing the
high-affinity DNA-binding domain from the 70-kDa subunit had nearly identical
DNA binding properties. In contrast, subcomplexes of the 32- and 14-kDa subunits
from both yeast and human RPA had weak ssDNA binding activity. However, the
binding constants for the yeast and human subcomplexes were 3 and greater than 6
orders of magnitude lower than those for the RPA heterotrimer, respectively. We
conclude that differences in the activity of the 32- and 14-kDa subunits of RPA
are responsible for variations in the ssDNA-binding properties of scRPA and
hRPA. These data also indicate that hRPA and scRPA have different modes of
binding to ssDNA, which may contribute to the functional disparities between the
two proteins.
PMID: 9730822 [PubMed - indexed for MEDLINE]
935: J Biol Chem 1998 Sep 11;273(37):23781-5
Erratum in:
J Biol Chem 1998 Oct 16;273(42):27755
A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor
IID.
Martinez E, Kundu TK, Fu J, Roeder RG.
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University,
New York, New York 10021, USA.
In yeast, SPT3 is a component of the multiprotein SPT-ADA-GCN5 acetyltransferase
(SAGA) complex that integrates proteins with transcription coactivator/adaptor
functions (ADAs and GCN5), histone acetyltransferase activity (GCN5), and core
promoter-selective functions (SPTs) involving interactions with the TATA-binding
protein (TBP). In particular, yeast SPT3 has been shown to interact directly
with TBP. Here we report the molecular cloning of a cDNA encoding a human
homologue of yeast SPT3. Amino acid sequence comparisons between human SPT3
(hSPT3) and its counterparts in different yeast species reveal three highly
conserved domains, with the most conserved 92-amino acid N-terminal domain being
25% identical with human TAFII18. Despite the significant sequence similarity
with TAFII18, native hSPT3 is not a bona fide TAFII because it is not associated
in vivo either with human TBP/TFIID or with a TFIID-related TBP-free TAFII
complex. However, we present evidence that hSPT3 is associated in vivo with
TAFII31 and the recently described longer form of human GCN5 (hGCN5-L) in a
novel human complex that has histone acetyltransferase activity. We propose that
the human SPT3-TAFII31-GCN5-L acetyltransferase (STAGA) complex is a likely
homologue of the yeast SAGA complex.
PMID: 9726987 [PubMed - indexed for MEDLINE]
936: Mol Biol Cell 1998 Sep;9(9):2349-60
An alpha-tubulin mutant destabilizes the heterodimer: phenotypic consequences
and interactions with tubulin-binding proteins.
Vega LR, Fleming J, Solomon F.
Department of Biology and Center for Cancer Research, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, USA.
Many effectors of microtubule assembly in vitro enhance the polymerization of
subunits. However, several Saccharomyces cerevisiae genes that affect cellular
microtubule-dependent processes appear to act at other steps in assembly and to
affect polymerization only indirectly. Here we use a mutant alpha-tubulin to
probe cellular regulation of microtubule assembly. tub1-724 mutant cells arrest
at low temperature with no assembled microtubules. The results of several assays
reported here demonstrate that the heterodimer formed between Tub1-724p and
beta-tubulin is less stable than wild-type heterodimer. The unstable heterodimer
explains several conditional phenotypes conferred by the mutation. These include
the lethality of tub1-724 haploid cells when the beta-tubulin-binding protein
Rbl2p is either overexpressed or absent. It also explains why the TUB1/tub1-724
heterozygotes are cold sensitive for growth and why overexpression of Rbl2p
rescues that conditional lethality. Both haploid and heterozygous tub1-724 cells
are inviable when another microtubule effector, PAC2, is overexpressed. These
effects are explained by the ability of Pac2p to bind alpha-tubulin, a complex
we demonstrate directly. The results suggest that tubulin-binding proteins can
participate in equilibria between the heterodimer and its components.
PMID: 9725898 [PubMed - indexed for MEDLINE]
937: Nucleic Acids Res 1998 Sep 15;26(18):4137-45
Cooperative interaction of branch signals in the actin intron of Saccharomyces
cerevisiae.
Castanotto D, Rossi JJ.
Molecular Biology Department, Beckman Research Institute of the City of Hope,
1450 East Duarte Road, Duarte, CA 91010, USA.
In pre-mRNA splicing, specific spliceosomal components recognize key intron
sequences, but the mechanisms by which splice sites are selected arenot
completely understood. In the Saccharomyces cerevisiae actin intron a silent
branch point-like sequence (UACUAAG) is located 7 nt upstream of the canonical
sequence. Mutation of the canonicalUACUAAC sequence to UAAUAAC reduces
utilization of this signal and activates the cryptic UACUAAG. Splicing-dependent
beta-galactosidase assays have shown that these two splice signals cooperate to
enhance splicing. Analyses of several variants of this double branch point
intron demonstrate that the upstream UACUAAG sequence significantly increases
usage of the UAAUAAC as a site of lariat formation. This activation is
sequence-specific and unidirectional. However the ability of the UACUAAG signal
to activate the downstream branch point is dependent on the presence of a short
non-conserved sequence located a few nucleotides upstream of the UACUAAG.
Mutation of this sequence leads to the disappearance of the cooperative
interactions between the two branch signals. Our results show that this
non-conserved sequence and the UACUAAG signal must both be present to achieve
activation of the downstream branch point and suggest that a specific structure
may be necessary to allow efficient recognition of the UAAUAAC.
PMID: 9722632 [PubMed - indexed for MEDLINE]
938: J Cell Biochem 1998 Sep 1;70(3):366-75
Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene
regulation.
Grayson J, Bassel-Duby R, Williams RS.
Department of Internal Medicine, University of Texas Southwestern Medical
Center, Dallas 75235-8573, USA.
Previous investigations have demonstrated synergistic interactions in vivo
between CCAC and A/T-rich nucleotide sequence motifs as functional components of
muscle-specific transcriptional enhancers. Using CCAC and A/T-rich elements from
the myoglobin and muscle creatine kinase (MCK) gene enhancers, Sp1 and
myocyte-specific enhancer factor-2 (MEF-2) were identified as cognate binding
proteins that recognize these sites. Physical interactions between Sp1 and MEF-2
were demonstrated by immunological detection of both proteins in DNA binding
complexes formed in vitro by nuclear extracts in the presence of only the A/T
sequence motif, by coprecipitation of recombinant MEF-2 in the presence of a
glutathione-S-transferase-Sp1 fusion protein bound to glutathione beads, and by
a two-hybrid assay in Saccharomyces cerevisiae. The interaction with Sp1 in
vitro and in vivo is specific for MEF-2 and was not observed with serum response
factor, a related MADS domain protein. Forced expression of Sp1 and MEF-2 in
insect cells otherwise lacking these factors promotes synergistic
transcriptional activation of a promoter containing binding sites for both
proteins. These data expand the repertoire of functional and physical
interactions between lineage-restricted (MEF-2) and ubiquitous (Sp1)
transcription factors that may be important for myogenic differentiation.
PMID: 9706874 [PubMed - indexed for MEDLINE]
939: Mol Endocrinol 1998 Aug;12(8):1172-83
Functional interactions of the AF-2 activation domain core region of the human
androgen receptor with the amino-terminal domain and with the transcriptional
coactivator TIF2 (transcriptional intermediary factor2).
Berrevoets CA, Doesburg P, Steketee K, Trapman J, Brinkmann AO.
Department of Endocrinology and Reproduction, Erasmus University, Rotterdam, The
Netherlands.
Previous studies in yeast and mammalian cells showed a functional interaction
between the amino-terminal domain and the carboxy-terminal, ligand-binding
domain (LBD) of the human androgen receptor (AR). In the present study, the AR
subdomains involved in this in vivo interaction were determined in more detail.
Cotransfection experiments in Chinese hamster ovary (CHO) cells and two-hybrid
experiments in yeast revealed that two regions in the NH2-terminal domain are
involved in the functional interaction with the LBD: an interacting domain at
the very NH2 terminus, located between amino acid residues 3 and 36, and a
second domain, essential for transactivation, located between residues 370 and
494. Substitution of glutamic acid by glutamine at position 888 (E888Q) in the
AF-2 activation domain (AD) core region in the LBD, markedly decreased the
interaction with the NH2-terminal domain. This mutation neither influenced
hormone binding nor LBD homodimerization, suggesting a role of the AF-2 AD core
region in the functional interaction between the NH2-terminal domain and the
LBD. The AF-2 AD core region was also involved in the interaction with the
coactivator TIF2 (transcriptional intermediary factor 2), as the E888Q mutation
decreased the stimulatory effect of TIF2 on AR AF-2 activity. Cotransfection of
TIF2 and the AR NH2-terminal domain expression vectors did not result in synergy
between both factors in the induction of AR AF-2 activity. TIF2 highly induced
AR AF-2 activity on a complex promoter [mouse mammary tumor virus (MMTV)], but
it was hardly active on a minimal promoter (GRE-TATA). In contrast, the AR
NH2-terminal domain induced AR AF-2 activity on both promoter constructs. These
data indicate that both the AR NH2-terminal domain and the coactivator TIF2
functionally interact, either directly or indirectly, with the AF-2 AD core
region in the AR-LBD, but the level of transcriptional response induced by TIF2
depends on the promoter context.
PMID: 9717843 [PubMed - indexed for MEDLINE]
940: Mycoses 1998;41 Suppl 1:32-8
Cytochromes P450 in fungi.
Vanden Bossche H, Koymans L.
Department of Anti-infectives Research, Janssen Research Foundation, Beerse,
Belgium.
The article gives an overview on the history of the discovery of P450
cytochromes and on their occurrence in nature, especially on their interactions
with metabolic pathways in fungi. The significance of the P450 cytochromes in
the ergosterol synthesis as well as in the inhibitory mechanisms caused by
imidazole and triazole antimycotics is described in detail.
Publication Types:
Review
Review, Tutorial
PMID: 9717384 [PubMed - indexed for MEDLINE]
941: Genes Dev 1998 Aug 15;12(16):2587-97
Cdc34 and the F-box protein Met30 are required for degradation of the
Cdk-inhibitory kinase Swe1.
Kaiser P, Sia RA, Bardes EG, Lew DJ, Reed SI.
The Scripps Research Institute (TSRI), La Jolla, California 92037 USA.
Ubiquitin-mediated proteolysis controls the abundance of many cell cycle
regulatory proteins. Recent work in Saccharomyces cerevisiae suggests that a
complex consisting of Cdc53, Skp1, and a third component known as an F-box
protein (termed SCF) in combination with Cdc34 specifically targets regulatory
proteins for degradation, and that substrate specificity is likely to be
mediated by the F-box subunit. A screen for genetic interactions with a cdc34
mutation yielded MET30, which encodes an F-box protein. MET30 is an essential
gene required for cell cycle progression and met30 mutations interact
genetically with mutations in SCF components. Furthermore, physical interactions
between Met30, Cdc53, Cdc34, and Skp1 in vivo provide evidence for an SCFMet30
complex. We demonstrate the involvement of Met30 in the degradation of the
Cdk-inhibitory kinase Swe1. Swe1 is stabilized in met30 mutants and GST-Met30
pull-down experiments reveal that Met30 specifically binds Swe1 in vivo.
Furthermore, extracts prepared from cdc34 or met30 mutants are defective in
polyubiquitination of Swe1. Taken together, these data suggest that SCF-mediated
proteolysis may contribute to the regulation of entry into mitosis. Our data, in
combination with previously published results, also provide evidence for
distinct SCF complexes in vivo and support the idea that their F-box subunits
mediate SCF substrate specificity.
PMID: 9716410 [PubMed - indexed for MEDLINE]
942: Genes Dev 1998 Aug 15;12(16):2574-86
Telomere-mediated chromosome pairing during meiosis in budding yeast.
Rockmill B, Roeder GS.
Howard Hughes Medical Institute, Yale University, New Haven, Connecticut
06520-8103 USA.
Certain haploid strains of Saccharomyces cerevisiae can undergo meiosis, but
meiotic prophase progression and subsequent nuclear division are delayed if
these haploids carry an extra chromosome (i. e., are disomic). Observations
indicate that interactions between homologous chromosomes cause a delay in
meiotic prophase, perhaps to allow time for interhomolog interactions to be
completed. Analysis of meiotic mutants demonstrates that the relevant aspect of
homolog recognition is independent of meiotic recombination and synaptonemal
complex formation. A disome in which the extra chromosome is circular sporulates
without a delay, indicating that telomeres are important for homolog
recognition. Consistent with this hypothesis, fluorescent in situ hybridization
demonstrates that a circular chromosome has a reduced capacity to pair with its
homolog, and a telomere-associated meiotic protein (Ndj1) is required to delay
sporulation in disomes. A circular dimer containing two copies of the same
chromosome delays meiosis to the same extent as two linear homologs, implying
that physical proximity bypasses the requirement for telomeres in homolog
pairing. Analysis of a disome carrying two linear permuted chromosomes suggests
that even nonhomologous chromosome ends can promote homolog pairing to a limited
extent. We speculate that telomere-mediated chromosome movement and/or telomere
clustering promote homolog pairing.
PMID: 9716409 [PubMed - indexed for MEDLINE]
943: Biochim Biophys Acta 1998 Aug 10;1366(1-2):127-37
Bcl-2 family proteins and mitochondria.
Reed JC, Jurgensmeier JM, Matsuyama S.
The Burnham Institute, Program on Apoptosis and Cell Death Research, 10901 North
Torrey Pines Road, La Jolla, CA 92037, USA. jreed@burnham-institute.org
The Bcl-2 family of proteins plays a pivotal role in regulating cell life and
death. Many of these proteins reside in the outer mitochondrial membrane,
oriented towards the cytosol. Cytoprotective Bcl-2 family proteins such as Bcl-2
and Bcl-XL prevent mitochondrial permeability transition pore opening and
release of apoptogenic proteins from mitochondria under many circumstances that
would otherwise result in either apoptosis or necrosis. In contrast, some
pro-apoptotic members of this family such as Bax can induce these destructive
changes in mitochondria in both mammalian cells and when expressed exogenously
in yeast. The mechanisms by which Bcl-2 family proteins control cell life and
death remain elusive, but may include both the ability to form ion channels or
pores in membranes and physical interactions with a variety of proteins
implicated in apoptosis regulation.
PMID: 9714773 [PubMed - indexed for MEDLINE]
944: J Biol Chem 1998 Aug 28;273(35):22589-94
Yeast transcript elongation factor (TFIIS), structure and function. I: NMR
structural analysis of the minimal transcriptionally active region.
Olmsted VK, Awrey DE, Koth C, Shan X, Morin PE, Kazanis S, Edwards AM,
Arrowsmith CH.
Ontario Cancer Institute and Department of Medical Biophysics, University of
Toronto, Toronto, Ontario M5G 2M9, Canada.
TFIIS is a general transcription elongation factor that helps arrested RNA
polymerase II elongation complexes resume transcription. We have previously
shown that yeast TFIIS (yTFIIS) comprises three structural domains (I-III). The
three-dimensional structures of domain II and part of domain III have been
previously reported, but neither domain can autonomously stimulate transcription
elongation. Here we report the NMR structural analysis of residues 131-309 of
yTFIIS which retains full activity and contains all of domains II and III. We
confirm that the structure of domain II in the context of fully active yTFIIS is
the same as that determined previously for a shorter construct. We have
determined the structure of the C-terminal zinc ribbon domain of active yTFIIS
and shown that it is similar to that reported for a shorter construct of human
TFIIS. The region linking domain II with the zinc ribbon of domain III appears
to be conformationally flexible and does not adopt a single defined tertiary
structure. NMR analysis of inactive mutants of yTFIIS support a role for the
linker region in interactions with the transcription elongation complex.
PMID: 9712887 [PubMed - indexed for MEDLINE]
945: J Immunol 1998 Aug 15;161(4):1728-37
Interaction of p59fyn kinase with the dynein light chain, Tctex-1, and
colocalization during cytokinesis.
Campbell KS, Cooper S, Dessing M, Yates S, Buder A.
Basel Institute for Immunology, Switzerland. ks_campbell@fccc.edu
The protein tyrosine kinase p59fyn (Fyn) plays important roles in both
lymphocyte Ag receptor signaling and cytokinesis of proB cells. We utilized
yeast two-hybrid cloning to identify the product of the tctex-1 gene as a
protein that specifically interacts with Fyn, but not with other Src family
kinases. Tctex-1 was recently identified as a component of the dynein
cytoskeletal motor complex. The capacity of a Tctex-1-glutathione S-transferase
fusion protein to effectively bind Fyn from cell lysates confirmed the
authenticity of this interaction. Tctex-1 binding required the first 19 amino
acids of Fyn and integrity of two lysine residues within this sequence that were
previously shown to be important for Fyn interactions with the immunoreceptor
tyrosine-based activation motifs (ITAMs) of lymphocyte Ag receptors. Expression
of tctex-1 mRNA and protein was observed in all lymphoma lines analyzed, and
immunofluorescence confocal microscopy localized the protein to the perinuclear
region. Analysis of a T cell hybridoma revealed prominent colocalization of
Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in cells undergoing
cytokinesis. Our results provide a unique insight into a mechanism by which
Tctex-1 might mediate specific recruitment of Fyn to the dynein complex in
lymphocytes, which may be a critical event in mediating the previously defined
role of Fyn in cytokinesis.
PMID: 9712037 [PubMed - indexed for MEDLINE]
946: Mol Cell Biol 1998 Sep;18(9):5392-403
High-resolution structural analysis of chromatin at specific loci: Saccharomyces
cerevisiae silent mating type locus HMLalpha.
Weiss K, Simpson RT.
Department of Biochemistry and Molecular Biology, The Center for Gene
Regulation, The Pennsylvania State University, University Park, Pennsylvania
16802, USA.
Genetic studies have suggested that chromatin structure is involved in
repression of the silent mating type loci in Saccharomyces cerevisiae. Chromatin
mapping at nucleotide resolution of the transcriptionally silent HMLalpha and
the active MATalpha shows that unique organized chromatin structure
characterizes the silent state of HMLalpha. Precisely positioned nucleosomes
abutting the silencers extend over the alpha1 and alpha2 coding regions. The HO
endonuclease recognition site, nuclease hypersensitive at MATalpha, is protected
at HMLalpha. Although two precisely positioned nucleosomes incorporate
transcription start sites at HMLalpha, the promoter region of the alpha1 and
alpha2 genes is nucleosome free and more nuclease sensitive in the repressed
than in the transcribed locus. Mutations in genes essential for HML silencing
disrupt the nucleosome array near HML-I but not in the vicinity of HML-E, which
is closer to the telomere of chromosome III. At the promoter and the HO site,
the structure of HMLalpha in Sir protein and histone H4 N-terminal deletion
mutants is identical to that of the transcriptionally active MATalpha. The
discontinuous chromatin structure of HMLalpha contrasts with the continuous
array of nucleosomes found at repressed a-cell-specific genes and the
recombination enhancer. Punctuation at HMLalpha may be necessary for
higher-order structure or karyoskeleton interactions. The unique chromatin
architecture of HMLalpha may relate to the combined requirements of
transcriptional repression and recombinational competence.
PMID: 9710623 [PubMed - indexed for MEDLINE]
947: Mol Cell Biol 1998 Sep;18(9):5308-19
Vam7p, a SNAP-25-like molecule, and Vam3p, a syntaxin homolog, function together
in yeast vacuolar protein trafficking.
Sato TK, Darsow T, Emr SD.
Division of Cellular and Molecular Medicine and Department of Biology, Howard
Hughes Medical Institute, University of California at San Diego School of
Medicine, La Jolla, California 92093-0668, USA.
A genetic screen to isolate gene products required for vacuolar morphogenesis in
the yeast Saccharomyces cerevisiae identified VAM7, a gene which encodes a
protein containing a predicted coiled-coil domain homologous to the coiled-coil
domain of the neuronal t-SNARE, SNAP-25 (Y. Wada and Y. Anraku, J. Biol. Chem.
267:18671-18675, 1992; T. Weimbs, S. H. Low, S. J. Chapin, K. E. Mostov, P.
Bucher, and K. Hofmann, Proc. Natl. Acad. Sci. USA 94:3046-3051, 1997). Analysis
of a temperature-sensitive-for-function (tsf) allele of VAM7 (vam7(tsf))
demonstrated that the VAM7 gene product directly functions in vacuolar protein
transport. vam7(tsf) mutant cells incubated at the nonpermissive temperature
displayed rapid defects in the delivery of multiple proteins that traffic to the
vacuole via distinct biosynthetic pathways. Examination of vam7(tsf) cells at
the nonpermissive temperature by electron microscopy revealed the accumulation
of aberrant membranous compartments that may represent unfused transport
intermediates. A fraction of Vam7p was localized to vacuolar membranes.
Furthermore, VAM7 displayed genetic interactions with the vacuolar syntaxin
homolog, VAM3. Consistent with the genetic results, Vam7p physically associated
in a complex containing Vam3p, and this interaction was enhanced by inactivation
of the yeast NSF (N-ethyl maleimide-sensitive factor) homolog, Sec18p. In
addition to the coiled-coil domain, Vam7p also contains a putative NADPH oxidase
p40(phox) (PX) domain. Changes in two conserved amino acids within this domain
resulted in synthetic phenotypes when combined with the vam3(tsf) mutation,
suggesting that the PX domain is required for Vam7p function. This study
provides evidence for the functional and physical interaction between Vam7p and
Vam3p at the vacuolar membrane, where they function as part of a t-SNARE complex
required for the docking and/or fusion of multiple transport intermediates
destined for the vacuole.
PMID: 9710615 [PubMed - indexed for MEDLINE]
948: Mol Cell Biol 1998 Sep;18(9):5189-98
Genetic, physical, and functional interactions between the triphosphatase and
guanylyltransferase components of the yeast mRNA capping apparatus.
Ho CK, Schwer B, Shuman S.
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021,
USA.
We have characterized an essential Saccharomyces cerevisiae gene, CES5, that
when present in high copy, suppresses the temperature-sensitive growth defect
caused by the ceg1-25 mutation of the yeast mRNA guanylyltransferase (capping
enzyme). CES5 is identical to CET1, which encodes the RNA triphosphatase
component of the yeast capping apparatus. Purified recombinant Cet1 catalyzes
hydrolysis of the gamma phosphate of triphosphate-terminated RNA at a rate of 1
s-1. Cet1 is a monomer in solution; it binds with recombinant Ceg1 in vitro to
form a Cet1-Ceg1 heterodimer. The interaction of Cet1 with Ceg1 elicits >10-fold
stimulation of the guanylyltransferase activity of Ceg1. This stimulation is the
result of increased affinity for the GTP substrate. A truncated protein,
Cet1(201-549), has RNA triphosphatase activity, heterodimerizes with and
stimulates Ceg1 in vitro, and suffices when expressed in single copy for cell
growth in vivo. The more extensively truncated derivative Cet1(246-549) also has
RNA triphosphatase activity but fails to stimulate Ceg1 in vitro and is lethal
when expressed in single copy in vivo. These data suggest that the Cet1-Ceg1
interaction is essential but do not resolve whether the triphosphatase activity
is also necessary. The mammalian capping enzyme Mce1 (a bifunctional
triphosphatase-guanylyltransferase) substitutes for Cet1 in vivo. A mutation of
the triphosphatase active-site cysteine of Mce1 is lethal. Hence, an RNA
triphosphatase activity is essential for eukaryotic cell growth. This work
highlights the potential for regulating mRNA cap formation through
protein-protein interactions.
PMID: 9710603 [PubMed - indexed for MEDLINE]
949: Mol Cell 1998 Jul;2(1):135-40
Circularization of mRNA by eukaryotic translation initiation factors.
Wells SE, Hillner PE, Vale RD, Sachs AB.
Department of Molecular and Cell Biology, University of California, Berkeley
94720, USA.
Communication between the 5' cap structure and 3' poly(A) tail of eukaryotic
mRNA results in the synergistic enhancement of translation. The cap and poly(A)
tail binding proteins, eIF4E and Pab1p, mediate this effect in the yeast S.
cerevisiae through their interactions with different parts of the translation
factor eIF4G. Here, we demonstrate the reconstitution of an eIF4E/eIF4G/Pab1p
complex with recombinant proteins, and show by atomic force microscopy that the
complex can circularize capped, polyadenylated RNA. Our results suggest that
formation of circular mRNA by translation factors could contribute to the
control of mRNA expression in the eukaryotic cell.
PMID: 9702200 [PubMed - indexed for MEDLINE]
950: Mol Vis 1998 Aug 11;4:13
Interaction of phosducin and phosducin isoforms with a 26S proteasomal subunit,
SUG1.
Zhu X, Craft CM.
Doheny Eye Institute and Department of Cell & Neurobiology, University of
Southern California School of Medicine, Los Angeles, CA 90033, USA.
PURPOSE: Retinal phosducin (Phd) and phosducin-like protein 1 (PhLP1)
selectively bind G-protein beta/gamma subunits (Gbetagamma). Our laboratory has
recently identified two phosducin-like orphan proteins (PhLOP1 and PhLOP2) that
lack the ability to interact with Gbetagamma. In search of potential functional
protein partner(s) for these phosducin orphans, we examined their
protein-protein interactions using a yeast two-hybrid screen. METHODS: A bovine
retina yeast expression cDNA library was screened with the GAL4 DNA binding
domain (BD) fusion of PhLOP1. Quantitative analysis of the selected positives
with PhLOP1 and other Phd isoforms was assessed by growth and beta-galactosidase
activity. Further molecular, biochemical, and immunological detection methods
utilizing glutathione S-transferase (GST)-Phd isoform fusion proteins and the
potential partner were also performed. RESULTS: A member of the superfamily of
putative ATPases was selected in the yeast two hybrid screen. Further
characterization identified a direct interaction of this putative ATPase with
PhLOP1, as well as Phd and PhLP1, but not with PhLOP2. A database search
verified this ATPase as a bovine orthologue of the yeast SUG1 (ySUG1), a
putative transcriptional mediator and a subunit of the 26S proteasome complex.
Our experiments reveal that the carboxy-terminus of PhLOP1 is essential for the
protein-protein interaction with SUG1, but it alone is not sufficient to mediate
SUG1 interaction. CONCLUSIONS: Based on these experimental results, Phd, PhLP1
and PhLOP1 have protein-protein interaction with SUG1. PhLOP1, a truncated
amino-terminal splice variant of Phd, is the best candidate for the interaction
with SUG1 among the four Phd isoforms studied, which suggests a potential
function for PhLOP1.
PMID: 9701609 [PubMed - indexed for MEDLINE]
951: Biochemistry 1998 Aug 11;37(32):11171-81
Structure determination and characterization of Saccharomyces cerevisiae
profilin.
Eads JC, Mahoney NM, Vorobiev S, Bresnick AR, Wen KK, Rubenstein PA, Haarer BK,
Almo SC.
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York
10461, USA.
The structure of profilin from the budding yeast Saccharomyces cerevisiae has
been determined by X-ray crystallography at 2.3 A resolution. The overall fold
of yeast profilin is similar to the fold observed for other profilin structures.
The interactions of yeast and human platelet profilins with rabbit skeletal
muscle actin were characterized by titration microcalorimetry, fluorescence
titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for
rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of
human platelet profilin for rabbit actin (0.1 microM), and the relative
contributions of entropic and enthalpic terms to the overall free energy of
binding are different for the two profilins. The titration of pyrene-labeled
rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to
the Kd of 2.0 microM for the interaction between yeast profilin and
pyrene-labeled yeast actin. The binding data are discussed in the context of the
known crystal structures of profilin and actin, and the residues present at the
actin-profilin interface. The affinity of yeast profilin for poly-L-proline was
determined from fluorescence measurements and is similar to the reported
affinity of Acanthamoeba profilin for poly-L-proline. Yeast profilin was shown
to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of
magnitude less efficiently than human profilin and rabbit skeletal muscle actin.
The in vivo and in vitro properties of yeast profilin mutants with altered
poly-L-proline and actin binding sites are discussed in the context of the
crystal structure.
PMID: 9698363 [PubMed - indexed for MEDLINE]
952: Pac Symp Biocomput 1998;:264-78
A computational "genome walk" technique to identify regulatory interactions in
gene networks.
Wagner A.
Santa Fe Institute, NM 87501, USA. aw@santafe.edu
To delineate the astronomical number of possible interactions of all genes in a
genome is a task for which conventional experimental techniques are ill-suited.
Sorely needed are rapid and inexpensive methods that identify candidates for
interacting genes, candidates that can be further investigated by experiment.
The subject of this paper is the application of a novel method to the genome of
the yeast Saccharomyces cerevisiae. The method applies to an important class of
gene interactions, that is, transcriptional regulation via transcription factors
(TFs) that bind to specific enhancer or silencer sites on DNA. The method
addresses the question: which of the genes in a genome are likely to be
regulated by one or more TFs with known DNA binding specificity? It takes
advantage of the fact that many TFs show cooperativity in transcriptional
activation which manifests itself in closely spaced TF binding sites. Such
"clusters" of binding sites are very unlikely to occur by chance alone, as
opposed to individual sites, which are often abundant both in the genome and in
promoter regions. Statistical information about binding site clusters in the
genome, can be complemented by information about (i) known biochemical functions
of the TF, (ii) the structure of its binding site, and (iii) function of the
genes near the cluster, to identify genes likely to be regulated by a given
transcription factor. Previously, binding sites of well characterized
transcription factors in Saccharomyces cerevisiae were analyzed. Here, the
method is applied to a somewhat different situation: the yeast DNA binding
activity yE2F, similar to the mammalian transcription factor E2F. yE2F has a DNA
binding specificity identical to E2F, and its binding site shows UAS activity in
a GAL1-based promoter construct. However, despite its high conservation, the in
vivo function of yE2F is unknown. The analysis carried out the here suggests
candidate genes for regulation by yE2F.
PMID: 9697188 [PubMed - indexed for MEDLINE]
953: Cell 1998 Jul 24;94(2):217-27
Degradation signal masking by heterodimerization of MATalpha2 and MATa1 blocks
their mutual destruction by the ubiquitin-proteasome pathway.
Johnson PR, Swanson R, Rakhilina L, Hochstrasser M.
Department of Biochemistry and Molecular Biology, University of Chicago,
Illinois 60637, USA.
Proteolysis by the ubiquitin-proteasome pathway is often regulated, but the
mechanisms underlying such regulation remain ill-defined. In Saccharomyces
cerevisiae, cell type is controlled by the MAT transcription factors. The alpha2
repressor is a known ubiquitin pathway substrate in alpha haploid cells. We show
that a1 is rapidly degraded in a haploids. In a/alpha diploids, alpha2 and a1
are stabilized by heterodimerization. Association depends on N-terminal
coiled-coil interactions between a1 and alpha2. Residues in alpha2 important for
these interactions overlap a critical determinant of an alpha2 degradation
signal, which we delimit by extensive mutagenesis. Our data provide a detailed
description of a natural ubiquitin-dependent degradation signal and point to a
molecular mechanism for regulated turnover in which proteolytic signals are
differentially masked in alternative multiprotein complexes.
PMID: 9695950 [PubMed - indexed for MEDLINE]
954: Mol Biol Cell 1998 Aug;9(8):2201-16
A genetic analysis of interactions with Spc110p reveals distinct functions of
Spc97p and Spc98p, components of the yeast gamma-tubulin complex.
Nguyen T, Vinh DB, Crawford DK, Davis TN.
Molecular and Cellular Biology Program, University of Washington, Seattle,
Washington 98195, USA.
The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the
microtubule-organizing center. Spc110p is an essential structural component of
the SPB and spans between the central and inner plaques of this multilamellar
organelle. The amino terminus of Spc110p faces the inner plaque, the
substructure from which spindle microtubules radiate. We have undertaken a
synthetic lethal screen to identify mutations that enhance the phenotype of the
temperature-sensitive spc110-221 allele, which encodes mutations in the amino
terminus. The screen identified mutations in SPC97 and SPC98, two genes encoding
components of the Tub4p complex in yeast. The spc98-63 allele is synthetic
lethal only with spc110 alleles that encode mutations in the N terminus of
Spc110p. In contrast, the spc97 alleles are synthetic lethal with spc110 alleles
that encode mutations in either the N terminus or the C terminus. Using the
two-hybrid assay, we show that the interactions of Spc110p with Spc97p and
Spc98p are not equivalent. The N terminus of Spc110p displays a robust
interaction with Spc98p in two different two-hybrid assays, while the
interaction between Spc97p and Spc110p is not detectable in one strain and gives
a weak signal in the other. Extra copies of SPC98 enhance the interaction
between Spc97p and Spc110p, while extra copies of SPC97 interfere with the
interaction between Spc98p and Spc110p. By testing the interactions between
mutant proteins, we show that the lethal phenotype in spc98-63 spc110-221 cells
is caused by the failure of Spc98-63p to interact with Spc110-221p. In contrast,
the lethal phenotype in spc97-62 spc110-221 cells can be attributed to a
decreased interaction between Spc97-62p and Spc98p. Together, these studies
provide evidence that Spc110p directly links the Tub4p complex to the SPB.
Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p
is a critical component of the linkage, whereas the interaction between Spc97p
and Spc110p is dependent on Spc98p.
PMID: 9693376 [PubMed - indexed for MEDLINE]
955: J Endocrinol 1998 Jun;157(3):361-71
Post-transcriptional gene regulatory mechanisms in eukaryotes: an overview.
Day DA, Tuite MF.
Department of Biosciences, University of Kent, Canterbury, UK.
Expression of a gene can be controlled at many levels, including transcription,
mRNA splicing, mRNA stability, translation and post-translational events such as
protein stability and modification. The majority of studies to date have focused
on transcriptional control mechanisms, but the importance of
post-transcriptional mechanisms in regulating gene expression in eukaryotes is
becoming increasingly clear. In this short review, selected examples of
post-transcriptional gene regulatory mechanisms operating in both lower and
higher eukaryotes will be used to highlight the plethora of such mechanisms
already identified. The underlying theme is that post-transcriptional gene
regulation relies on specific RNA-protein interactions that either result in the
targeted degradation of the mRNA or prevent access of the ribosome to the
translation start codon. Such interactions can occur in the 5' or 3'
untranslated regions of an mRNA or within the decoded portion of the molecule.
The importance of these regulatory mechanisms in a range of biological systems
is also illustrated.
Publication Types:
Review
Review, Academic
PMID: 9691970 [PubMed - indexed for MEDLINE]
956: Genetics 1998 Aug;149(4):1717-27
Sro7p, a Saccharomyces cerevisiae counterpart of the tumor suppressor l(2)gl
protein, is related to myosins in function.
Kagami M, Toh-e A, Matsui Y.
Department of Biological Sciences, Graduate School of Science, University of
Tokyo, Tokyo 113, Japan.
Yeast SRO7 was identified as a multicopy suppressor of a defect in Rho3p, a
small GTPase that maintains cell polarity. Sro7p and Sro77p, a homologue of
Sro7p, possess domains homologous to the protein that are encoded by the
Drosophila tumor suppressor gene lethal (2) giant larvae [l(2)gl]. sro7Delta
sro77Delta mutants showed a partial defect of organization of the polarized
actin cytoskeleton and a cold-sensitive growth phenotype. A human counterpart of
l(2)gl could suppress the sro7Delta sro77Delta defect. Similar to the l(2)gl
protein, Sro7p formed a complex with Myo1p, a type II myosin. These results
indicate that Sro7p and Sro77p are the yeast counterparts of the l(2)gl protein.
Our genetic analysis revealed that deletion of SRO7 and SRO77 showed reciprocal
suppression with deletion of MYO1 (i.e., the sro7Delta sro77Delta defect was
suppressed by myo1Delta and vice versa). In addition, SRO7 showed genetic
interactions with MYO2, encoding an essential type V myosin: Overexpression of
SRO7 suppressed a defect in MYO2 and, conversely, overexpression of MYO2
suppressed the cold-sensitive phenotype of sro7Delta sro77Delta mutants. These
results indicate that Sro7 function is closely related to both Myo1p and Myo2p.
We propose a model in which Sro7 function is involved in the targeting of the
myosin proteins to their intrinsic pathways.
PMID: 9691031 [PubMed - indexed for MEDLINE]
957: Science 1998 Jul 31;281(5377):698-700
Nucleation of COPII vesicular coat complex by endoplasmic reticulum to Golgi
vesicle SNAREs.
Springer S, Schekman R.
Howard Hughes Medical Institute and Department of Molecular and Cell Biology,
University of California at Berkeley, Berkeley, CA 94720-3202, USA.
Protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus
involves specific uptake into coat protein complex II (COPII)-coated vesicles of
secretory and of vesicle targeting (v-SNARE) proteins. Here, two ER to Golgi
v-SNAREs, Bet1p and Bos1p, were shown to interact specifically with Sar1p,
Sec23p, and Sec24p, components of the COPII coat, in a guanine
nucleotide-dependent fashion. Other v-SNAREs, Sec22p and Ykt6p, might interact
more weakly with the COPII coat or interact indirectly by binding to Bet1p or
Bos1p. The data suggest that transmembrane proteins can be taken up into COPII
vesicles by direct interactions with the coat proteins and may play a structural
role in the assembly of the COPII coat complex.
PMID: 9685263 [PubMed - indexed for MEDLINE]
958: Plant J 1998 Jun;14(6):685-92
Isolation of putative plant transcriptional coactivators using a modified
two-hybrid system incorporating a GFP reporter gene.
Cormack RS, Hahlbrock K, Somssich IE.
Max-Planck-Institut fur Zuchtungsforschung, Abteilung Biochemie, Cologne,
Germany.
Dual hybrid interacting screening in yeast led to the identification of two
proteins from Arabidopsis both exhibiting sequence similarity to a family of
transcriptional coactivators from a diverse range of organisms. Their discovery
constitutes the first description of such plant proteins. A modified yeast
two-hybrid approach utilising the green fluorescent protein (GFP) of Aequora
victoria was developed and used to clone one of the putative plant
transcriptional coactivators from an Arabidopsis cDNA library. The two proteins,
designated KIWI and KELP, can associate both hetero- and homomerically and their
genes were cloned and mapped on the Arabidopsis genome. Both proteins are
believed to play a role in gene activation during pathogen defence and plant
development. The involvement of these proteins in general plant transcription as
well as the advantages of using GFP as a reporter gene for detecting
protein-protein interactions are discussed.
PMID: 9681033 [PubMed - indexed for MEDLINE]
959: J Cell Biol 1998 Jul 27;142(2):443-55
Iqg1p, a yeast homologue of the mammalian IQGAPs, mediates cdc42p effects on the
actin cytoskeleton.
Osman MA, Cerione RA.
Department of Pharmacology, Cornell University, Ithaca, New York 14853, USA.
The Rho-type GTPase Cdc42p has been implicated in diverse cellular functions
including cell shape, cell motility, and cytokinesis, all of which involve the
reorganization of the actin cytoskeleton. Targets of Cdc42p that interface the
actin cytoskeleton are likely candidates for mediating cellular activities. In
this report, we identify and characterize a yeast homologue for the mammalian
IQGAP, a cytoskeletal target for Cdc42p. The yeast IQGAP homologue, designated
Iqg1p, displays a two-hybrid interaction with activated Cdc42p and
coimmunoprecipitates with actin filaments. Deletion of IQG1 results in a
temperature-sensitive lethality and causes aberrant morphologies including
elongated and round multinucleated cells. This together with its localization at
the mother-bud neck, suggest that Iqg1p promotes budding and cytokinesis. At
restrictive temperatures, the vacuoles of the mutant cells enlarge and vesicles
accumulate in the bud. Interestingly, Iqg1p shows two-hybrid interactions with
the ankyrin repeat-containing protein, Akr1p (Kao, L.-R., J. Peterson, J. Ruiru,
L. Bender, and A. Bender. 1996. Mol. Cell. Biol. 16:168-178), which inhibits
pheromone signaling and appears to promote cytokinesis and/or trafficking. We
also show two-hybrid interactions between Iqg1p and Afr1p, a septin-binding
protein involved in projection formation (Konopka, J.B., C. DeMattei, and C.
Davis. 1995. Mol. Cell. Biol. 15:723-730). We propose that Iqg1p acts as a
scaffold to recruit and localize a protein complex involved in actin-based
cellular functions and thus mediates the regulatory effects of Cdc42p on the
actin cytoskeleton.
PMID: 9679143 [PubMed - indexed for MEDLINE]
960: J Cell Biol 1998 Jul 27;142(2):341-54
A functional GTPase domain, but not its transmembrane domain, is required for
function of the SRP receptor beta-subunit.
Ogg SC, Barz WP, Walter P.
Howard Hughes Medical Institute and Department of Biochemistry and Biophysics,
University of California School of Medicine, San Francisco, California
94143-0448, USA.
The signal recognition particle and its receptor (SR) target nascent secretory
proteins to the ER. SR is a heterodimeric ER membrane protein whose subunits,
SRalpha and SRbeta, are both members of the GTPase superfamily. Here we
characterize a 27-kD protein in Saccharomyces cerevisiae (encoded by SRP102) as
a homologue of mammalian SRbeta. This notion is supported (a) by Srp102p's
sequence similarity to SRbeta; (b) by its disposition as an ER membrane protein;
(c) by its interaction with Srp101p, the yeast SRalpha homologue; and (d) by its
role in SRP-dependent protein targeting in vivo. The GTP-binding site in Srp102p
is surprisingly insensitive to single amino acid substitutions that inactivate
other GTPases. Multiple mutations in the GTP-binding site, however, inactivate
Srp102p. Loss of activity parallels a loss of affinity between Srp102p and
Srp101p, indicating that the interaction between SR subunits is important for
function. Deleting the transmembrane domain of Srp102p, the only known membrane
anchor in SR, renders SR soluble in the cytosol, which unexpectedly does not
significantly impair SR function. This result suggests that SR functions as a
regulatory switch that needs to associate with the ER membrane only transiently
through interactions with other components.
PMID: 9679135 [PubMed - indexed for MEDLINE]
961: Biotechnology (N Y) 1995 Dec;13(13):1431-4
Comment on:
Biotechnology (N Y). 1995 Dec;13(13):1474-8.
Fishing for protein interactions with tribrids.
Paul J, Trueheart J.
Cadus Pharmaceutical Corporation, Tarrytown, NY 10591-6704, USA.
75-763-2141@compuserve. com
Publication Types:
Comment
PMID: 9678924 [PubMed - indexed for MEDLINE]
962: Adv Genet 1998;38:185-218
DNA breakage and repair.
Jeggo PA.
MRC Cell Mutation Unit, University of Sussex, Brighton, United Kingdom.
For many years it has been evident that mammalian cells differ dramatically from
yeast and rejoin the majority of their DNA DSBs by a nonhomologous mechanism,
recently termed NHEJ. In the last few years a number of genes and proteins have
been identified that operate in the pathway providing insights into the
mechanism. These proteins include the three components of DNA-PK, DNA ligase IV,
and XRCC4. In yeast Sir2, -3, and -4 proteins are also involved in the process
and therefore are likely to play a role in higher organisms. Studies with yeast
suggest that NHEJ is an error-free mechanism. Although the process is far from
understood, it is likely that the DNA-PK complex or Ku alone acts in a complex
with the Sir proteins possibly protecting the ends and preventing random
rejoining. Further work is required to establish the details of this mechanism
and to determine whether this represents an accurate rejoining process for a
complex break induced by ionizing radiation. It will be intriguing to discover
how the cell achieves efficient and accurate rejoining without the use of
homology. Interactions between the components of DNA-PK and other proteins
playing a central role in damage response mechanisms are beginning to emerge.
Interestingly, there is evidence that DNA repair and damage response mechanisms
overlap in lower organisms. The overlapping defects of the yeast Ku mutants,
tell mutants, and AT cell lines in telomere maintenance further suggest
overlapping functions or interacting mechanisms. A challenge for the future will
be to establish how these different damage response mechanisms overlap and
interact.
Publication Types:
Review
Review, Academic
PMID: 9677708 [PubMed - indexed for MEDLINE]
963: J Biol Chem 1998 Jul 31;273(31):19792-6
A family of Arf effectors defined as suppressors of the loss of Arf function in
the yeast Saccharomyces cerevisiae.
Zhang CJ, Cavenagh MM, Kahn RA.
Department of Biochemistry, Emory University School of Medicine, Atlanta,
Georgia 30322-3050, USA.
Arf proteins are ubiquitous, eukaryotic regulators of virtually every step of
vesicular membrane traffic. ADP-ribosylation factors are essential in yeast and
the lethality resulting from either overexpression or underexpression (deletion)
of Arf genes has previously been ascribed to dysregulation of the secretory
process. We have identified a family of four genes (Suppressors of Arf ts, SAT)
as high copy suppressors of a loss of function allele of ARF1 (arf1-3). Those
proteins with SAT activity were found to contain a minimal consensus motif,
including a C2C2H2 cluster with a novel and specific spacing. Genetic
interactions between members of this family and with ARF1 are consistent with
each sharing a common cellular pathway. Included in this family is Gcs1, a
protein previously described (Poon, P. P., Wang, X., Rotman, M., Huber, I.,
Cukierman, E., Cassel, D., Singer, R. A., and Johnston, G. C. (1996) Proc. Natl.
Acad. Sci. U. S. A. 93, 10074-10077) to possess Arf GTPase-activating protein
(GAP) activity, demonstrating a direct interaction between Arf and at least one
of these suppressors. The suppression of the loss of Arf function by
overexpression of Gcs1 and demonstration of direct, preferential binding of Gcs1
to the activated form of Arf (Arf.GTP) lead us to conclude that the biological
role of Gcs1 is as an effector of the essential function of Arf in mitotic
growth, rather than a down-regulator as implied by the biochemical (Arf GAP)
activity. Suppression of the growth defect of arf1(-3) cells was observed under
conditions that did not alter the secretory defect associated with arf1(-)
mutation, indicating that the essential role of Arf in eukaryotes can be
distinguished from role(s) in the secretory pathway and appear to employ
distinct pathways and effectors.
PMID: 9677411 [PubMed - indexed for MEDLINE]
964: Yeast 1998 Jun 15;14(8):733-46
The C-terminal hydrophobic repeat of Schizosaccharomyces pombe heat shock factor
is not required for heat-induced DNA-binding.
Saltsman KA, Prentice HL, Kingston RE.
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114,
USA.
The C-terminal hydrophobic repeat (CTR) of heat shock transcription factor (HSF)
has been proposed to regulate DNA binding by intramolecular interactions with
the leucine zipper motifs present in the HSF trimerization domain.
Schizosaccharomyces pombe provides a useful model organism for the study of the
regulation of HSF DNA binding because, unlike Saccharomyces cerevisiae, S. pombe
hsf is highly heat shock inducible for DNA binding and contains a clear homology
to the CTR. We examined the role that the CTR plays in the regulation of S.
pombe hsf by constructing isogenic strains bearing deletion and point mutations
in the chromosomal copy of hsf. Surprisingly, we found that point mutation of
key hydrophobic amino acids within the CTR, as well as full deletion of it,
yielded factors that show normal binding at normal growth temperatures and full
levels of heat-induced binding. Deletion of the CTR did, however, slightly lower
the temperature required for maximal activation. In contrast, a large deletion
of the C-terminus, which removes close to a third of the coding sequence, was
deregulated and bound DNA at control temperature. Several of the deletion
mutants were significantly reduced in their level of expression, yet they showed
wild-type levels of DNA binding activity following heat shock. These experiments
demonstrate that appropriate regulation of the DNA binding activity of S. pombe
hsf is not solely dependent upon the CTR, and imply that a feedback mechanism
exists that establishes proper levels of DNA binding following heat shock
despite mutations that significantly alter levels of total hsf.
PMID: 9675818 [PubMed - indexed for MEDLINE]
965: Cell 1998 Jul 10;94(1):73-82
Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously
aggregated proteins.
Glover JR, Lindquist S.
Howard Hughes Medical Institute and Department of Molecular Genetics and Cell
Biology, The University of Chicago, Illinois 60637, USA.
Hsp104 is a stress tolerance factor that promotes the reactivation of
heat-damaged proteins in yeast by an unknown mechanism. Herein, we demonstrate
that Hsp104 functions in this process directly. Unlike other chaperones, Hsp104
does not prevent the aggregation of denatured proteins. However, in concert with
Hsp40 and Hsp70, Hsp104 can reactivate proteins that have been denatured and
allowed to aggregate, substrates refractory to the action of other chaperones.
Hsp104 cooperates with the chaperones present in reticulocyte lysates but not
with DnaK of E. coli. We conclude that Hsp104 has a protein remodeling activity
that acts on trapped, aggregated proteins and requires specific interactions
with conventional chaperones to promote refolding of the intermediates it
produces.
PMID: 9674429 [PubMed - indexed for MEDLINE]
966: Nucleic Acids Res 1998 Aug 1;26(15):3577-83
Assessment of aryl hydrocarbon receptor complex interactions using pBEVY
plasmids: expressionvectors with bi-directional promoters for use in
Saccharomyces cerevisiae.
Miller CA 3rd, Martinat MA, Hyman LE.
Environmental Health Sciences Department and Tulane-Xavier Center for
Bioenvironmental Research,Tulane University School of Public Health and Tropical
Medicine, 1430 Tulane Avenue, New Orleans,LA 70112, USA.
The pBEVY (bi-directional expression vectors for yeast) plasmids were designed
with constitutive and galactose-induced bi-directional promoters to direct the
expression of multiple proteins in Saccharomyces cerevisiae . Using human
estrogen receptor as a test gene, relatively balanced expression levels from
each side of a bi-directional promoter were observed. Expression of a functional
heterodimeric transcription factor composed of human aryl hydrocarbon receptor
(Ahr) and aryl hydrocarbon receptor nuclear translocator (Arnt) proteins was
accomplished using a single pBEVY plasmid. Previous studies suggest that
inhibitory cross-talk between the estrogen receptor and the Ahr/Arnt complex may
occur and that Hsp90-Ahr complex formation is important for Ahr-mediated signal
transduction. Evidence for functional interaction among these proteins was
investigated using pBEVY plasmids in a yeast system. No inhibitory cross-talk
was observed in signaling assays performed with yeast that co-expressed Ahr,
Arnt and estrogen receptor. In contrast, Ahr/Arnt-mediated signal transduction
was reduced by 80% in a temperature-sensitive Hsp90 strain grown under
non-permissive conditions. We conclude that pBEVY plasmids facilitate the
examination of multiple protein interactions in yeast model systems.
PMID: 9671822 [PubMed - indexed for MEDLINE]
967: Mol Cell Biol 1998 Aug;18(8):4899-913
Disruption of PML subnuclear domains by the acidic IE1 protein of human
cytomegalovirus is mediated through interaction with PML and may modulate a RING
finger-dependent cryptic transactivator function of PML.
Ahn JH, Brignole EJ 3rd, Hayward GS.
Molecular Virology Laboratories, Departments of Pharmacology and Molecular
Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland
21205, USA.
Both of the major immediate-early (IE) proteins IE1 and IE2 of human
cytomegalovirus (HCMV) as well as input viral DNA and sites of viral IE
transcription colocalize with or adjacent to punctate PML domains (PML oncogenic
domains [PODs] or nuclear domain 10) in the nucleus within the first few hours
after infection of permissive human fibroblasts. However, colocalization of IE1
and PML in PODs is only transient, with both proteins subsequently
redistributing into a nuclear diffuse form. These processes are believed to
promote efficient viral IE transcription and initiation of DNA synthesis
especially at low multiplicities of infection. To examine the mechanism of PML
displacement by IE1, we carried out indirect immunofluorescence assay
experiments with plasmids expressing intact or deleted forms of PML and IE1 in
DNA-transfected cells. The results demonstrated that deletion of the C-terminal
acidic region of IE1 uncouples the requirements for displacement of both
endogenous and coexpressed PML from those needed to target to the PODs. Mutant
PML proteins containing either a Cys point mutation within the N-terminal RING
finger domain or a small deletion (of positions 281 to 304) within the
coiled-coil region did not localize to the PODs but instead gave a nuclear
diffuse distribution, similar to that produced by intact PML in the presence of
IE1. Endogenous PML also colocalized with IE1 in metaphase chromosomes in HCMV
or recombinant adenovirus type 5-IE1-infected HF cells undergoing mitosis,
implying that there may be a direct physical interaction between IE1 and PML.
Indeed, a specific interaction between IE1 and PML was observed in a yeast
two-hybrid assay, and the strength of this interaction was comparable to that of
IE2 with the retinoblastoma protein. The RING finger mutant form of PML showed a
threefold-lower interaction with IE1 in the yeast system, and deletion of the
N-terminal RING finger domain of PML abolished the interaction. Consistent with
the IFA results, a mutant IE1 protein that lacks the C-terminal acidic region
was sufficient for interaction with PML in the yeast system. The two-hybrid
interaction assay also showed that both the N-terminal RING finger domain and
the intact coiled-coil region of PML are required cooperatively for efficient
self-interactions involving dimerization or oligomerization. Furthermore,
truncated or deleted GAL4/PML fusion proteins that retained the RING finger
domain but lacked the intact coiled-coil region displayed an unmasked cryptic
transactivator function in both yeast and mammalian cells, and the RING finger
mutation abolished this transactivation property of PML. Therefore, we suggest
that a direct interaction between IE1 and the N-terminal RING finger domain of
PML may inhibit oligomerization and protein-protein complex formation by PML,
leading to displacement of PML and IE1 from the PODs, and that this interaction
may also modulate a putative conditional transactivator function of PML.
PMID: 9671498 [PubMed - indexed for MEDLINE]
968: Mol Cell Biol 1998 Aug;18(8):4935-46
Identification of a translation initiation factor 3 (eIF3) core complex,
conserved in yeast and mammals, that interacts with eIF5.
Phan L, Zhang X, Asano K, Anderson J, Vornlocher HP, Greenberg JR, Qin J,
Hinnebusch AG.
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and
Human Development, Bethesda, Maryland 20892, USA.
Only five of the nine subunits of human eukaryotic translation initiation factor
3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae
genome, and only two of these (Prt1p and Tif34p) were identified previously as
subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p
(His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a
complex of approximately 600 kDa composed of six polypeptides whose
copurification was completely dependent on the polyhistidine tag on His-Prt1p.
All five polypeptides associated with His-Prt1p were identified by mass
spectrometry, and four were found to be the other putative homologs of human
eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and
YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation
factor not previously known to interact with eIF3. The purified complex could
rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1
mutant or extracts from which Nip1p had been depleted, indicating that it
possesses a known biochemical activity of eIF3. These findings suggest that
Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex,
conserved between yeast and mammals, that stably interacts with eIF5. Nip1p
bound to eIF5 in yeast two-hybrid and in vitro protein binding assays.
Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have
been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and
Sui1p may be recruited to the 40S ribosomes through physical interactions with
the Nip1p subunit of eIF3.
PMID: 9671501 [PubMed - indexed for MEDLINE]
969: EMBO J 1998 Jul 15;17(14):3981-9
The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the
ribosome-nascent chain complex.
Pfund C, Lopez-Hoyo N, Ziegelhoffer T, Schilke BA, Lopez-Buesa P, Walter WA,
Wiedmann M, Craig EA.
Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI
53706, USA.
The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular
chaperones. The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70
found associated with translating ribosomes. To understand better the function
of Ssb in association with ribosomes, the Ssb-ribosome interaction was
characterized. Incorporation of the aminoacyl-tRNA analog puromycin by
translating ribosomes caused the release of Ssb concomitant with the release of
nascent chains. In addition, Ssb could be cross-linked to nascent chains
containing a modified lysine residue with a photoactivatable cross-linker.
Together, these results suggest an interaction of Ssb with the nascent chain.
The interaction of Ssb with the ribosome-nascent chain complex was stable, as
demonstrated by resistance to treatment with high salt; however, Ssb interaction
with the ribosome in the absence of nascent chain was salt sensitive. We propose
that Ssb is a core component of the translating ribosome which interacts with
both the nascent polypeptide chain and the ribosome. These interactions allow
Ssb to function as a chaperone on the ribosome, preventing the misfolding of
newly synthesized proteins.
PMID: 9670014 [PubMed - indexed for MEDLINE]
970: Biotechniques 1998 Jul;25(1):85-8, 90-2
Development of a yeast trihybrid screen using stable yeast strains and regulated
protein expression.
Fuller KJ, Morse MA, White JH, Dowell SJ, Sims MJ.
Immunology Unit, Glaxo Wellcome Medicines Research Centre, Stevenage,
Hertfordshire, UK.
We describe a yeast trihybrid system that facilitates rapid screening of cDNA
libraries. Novel yeast vectors were developed that direct integration of cDNA
encoding the bait and third protein component into the yeast chromosome. A
recombinant yeast strain is thus generated (screening strain) and is available
for library transformation. Transformation with the library DNA is a single,
efficient transformation event, allowing the cDNA library to be represented in
one step. Recovery of the library plasmid from the yeast is also simplified,
since it is the only episomal plasmid. Assay of trihybrid interaction and
identification of positive clones is facilitated by regulating expression of the
third protein component using the yeast MET3 promoter, which is repressed in the
presence of exogenous methionine. Trihybrid interactions are detected only on
media lacking methionine. This trihybrid system uses the standard E. coli LacZ
and yeast HIS3 reporter genes and is compatible with most available Gal4
activation domain cDNA libraries. We describe the successful application of this
yeast trihybrid system to the study of phosphoprotein interactions involved in
T-cell signaling.
Publication Types:
Technical Report
PMID: 9668981 [PubMed - indexed for MEDLINE]
971: Methods Mol Biol 1998;84:201-22
Two-hybrid analysis of Ras-Raf interactions.
Van Aelst L.
Cold Spring Harbor Laboratory, NY, USA.
PMID: 9666451 [PubMed - indexed for MEDLINE]
972: Gene 1998 Jul 17;215(1):143-52
Construction of a modular yeast two-hybrid cDNA library from human EST clones
for the human genome protein linkage map.
Hua SB, Luo Y, Qiu M, Chan E, Zhou H, Zhu L.
Gene, Net Group, CLONTECH Laboratories Inc., 1020 East Meadow Circle, Palo Alto,
CA 94303, USA. sbhua@clontech.com
Identification of all human protein-protein interactions will lead to a global
human protein linkage map that will provide important information for functional
genomics studies. The yeast two-hybrid system is a powerful molecular genetic
approach for studying protein-protein interactions. To apply this technology to
generate a human protein linkage map, the first step is to construct two-hybrid
cDNA libraries that cover the entire human genome. With a homologous
recombination-mediated approach, we have constructed a modular human EST-derived
yeast two-hybrid library in the Gal4 activation domain-based vector, pACT2.
Quality analysis of this library indicated that the approach of constructing
two-hybrid cDNA libraries from individually arrayed human EST clones is
feasible, and such a two-hybrid library is suitable for detecting
protein-protein interactions. This is also the first time that a comprehensive
two-hybrid system cDNA library has been constructed from a collection of
individually arrayed EST clones.
PMID: 9666106 [PubMed - indexed for MEDLINE]
973: Methods Mol Biol 1998;93:251-61
Analysis of protein interactions between protein phosphatase 1 and noncatalytic
subunits using the yeast two-hybrid assay.
Ramaswamy NT, Dalley BK, Cannon JF.
Department of Molecular Microbiology and Immunology, University of Missouri,
Columbia, USA.
PMID: 9664543 [PubMed - indexed for MEDLINE]
974: J Cell Biol 1998 Jul 13;142(1):39-49
Assembly of the yeast vacuolar H+-ATPase occurs in the endoplasmic reticulum and
requires a Vma12p/Vma22p assembly complex.
Graham LA, Hill KJ, Stevens TH.
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
Three previously identified genes from Saccharomyces cerevisiae, VMA12, VMA21,
and VMA22, encode proteins localized to the endoplasmic reticulum (ER). These
three proteins are required for the biogenesis of a functional vacuolar ATPase
(V-ATPase), but are not part of the final enzyme complex. Subcellular
fractionation and chemical cross-linking studies have revealed that Vma12p and
Vma22p form a stable membrane associated complex. Cross-linking analysis also
revealed a direct physical interaction between the Vma12p/Vma22p assembly
complex and Vph1p, the 100-kD integral membrane subunit of the V-ATPase. The
interaction of the Vma12p/Vma22p complex with Vph1p was transient (half-life of
approximately 5 min), reflecting trafficking of this V-ATPase subunit through
the ER en route to the vacuolar membrane. Analysis of these protein-protein
interactions in ER-blocked sec12 mutant cells indicated that the
Vph1p-Vma12p/Vma22p interactions are quite stable when transport of the V-ATPase
out of the ER is blocked. Fractionation of solubilized membrane proteins on a
density gradient revealed comigration of Vma22p and Vma12p, indicating that they
form a complex even in the absence of cross-linker. Vma12p and Vma22p migrated
to fractions separate from Vma21p. Loss of Vph1p caused the Vma12p/Vma22p
complex to sediment to less dense fractions, consistent with association of
Vma12p/ Vma22p with nascent Vph1p in ER membranes. This is the first evidence
for a dedicated assembly complex in the ER required for the assembly of an
integral membrane protein complex (V-ATPase) as it is transported through the
secretory pathway.
PMID: 9660861 [PubMed - indexed for MEDLINE]
975: J Biol Chem 1998 Jul 17;273(29):18573-85
Complex formation by all five homologues of mammalian translation initiation
factor 3 subunits from yeast Saccharomyces cerevisiae.
Asano K, Phan L, Anderson J, Hinnebusch AG.
Laboratory of Eukaryotic Gene Regulation, NICHD, National Institutes of Health,
Bethesda, Maryland 20892, USA.
The PRT1, TIF34, GCD10, and SUI1 proteins of Saccharomyces cerevisiae were found
previously to copurify with eukaryotic translation initiation factor 3 (eIF3)
activity. Although TIF32, NIP1, and TIF35 are homologous to subunits of human
eIF3, they were not known to be components of the yeast factor. We detected
interactions between PRT1, TIF34, and TIF35 by the yeast two-hybrid assay and in
vitro binding assays. Discrete segments (70-150 amino acids) of PRT1 and TIF35
were found to be responsible for their binding to TIF34. Temperature-sensitive
mutations mapping in WD-repeat domains of TIF34 were isolated that decreased
binding between TIF34 and TIF35 in vitro. The lethal effect of these mutations
was suppressed by increasing TIF35 gene dosage, suggesting that the TIF34-TIF35
interaction is important for TIF34 function in translation. Pairwise in vitro
interactions were also detected between PRT1 and TIF32, TIF32 and NIP1, and NIP1
and SUI1. Furthermore, PRT1, NIP1, TIF34, TIF35, and a polypeptide with the size
of TIF32 were specifically coimmunoprecipitated from the ribosomal salt wash
fraction. We propose that all five yeast proteins homologous to human eIF3
subunits are components of a stable heteromeric complex in vivo and may comprise
the conserved core of yeast eIF3.
PMID: 9660829 [PubMed - indexed for MEDLINE]
976: J Biol Chem 1998 Jul 17;273(29):18490-8
Characterization of Pak2p, a pleckstrin homology domain-containing,
p21-activated protein kinase from fission yeast.
Sells MA, Barratt JT, Caviston J, Ottilie S, Leberer E, Chernoff J.
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
p21-activated kinases (PAKs) bind to and are activated by Rho family GTPases
such as Cdc42 and Rac. Since these GTPases play key roles in regulating cell
polarity, stress responses, and cell cycle progression, the ability of PAK to
affect these processes has been examined. We previously showed that fission
yeast pak1+ encodes an essential protein that affects mating and cell polarity.
Here, we characterize a second pak gene (pak2+) from Schizosaccharomyces pombe.
Like the Saccharomyces cerevisiae proteins Cla4p and Skm1p, fission yeast Pak2p
contains an N-terminal pleckstrin homology domain in addition to a p21-binding
domain and a protein kinase domain that are common to other members of the PAK
family. Unlike pak1+, pak2(+) is not essential for vegetative growth or for
mating in S. pombe. Overexpression of the wild-type pak2+ allele suppresses the
lethal growth defect associated with deletion of pak1+, and this suppression
requires both the pleckstrin homology- and the p21-binding domains of Pak2p, as
well as kinase activity. A substantial fraction of Pak2p is associated with
membranous components, an association mediated both by the pleckstrin homology-
and by the p21-binding domains. These results show that S. pombe encodes at
least two pak genes with distinct functions and suggest that the membrane
localization of Pak2p, directed by its interactions with membrane lipids and
Cdc42p, is critical to its biological activity.
PMID: 9660818 [PubMed - indexed for MEDLINE]
977: J Virol 1998 Aug;72(8):6944-9
Binding of the human immunodeficiency virus type 1 Gag protein to the viral RNA
encapsidation signal in the yeast three-hybrid system.
Bacharach E, Goff SP.
Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical
Institute, Columbia University College of Physicians and Surgeons, New York, New
York 10032, USA.
We have used the yeast three-hybrid system (D. J. SenGupta, B. Zhang, B.
Kraemer, P. Pochart, S. Fields, and M. Wickens, Proc. Natl. Acad. Sci. USA
93:8496-8501, 1996) to study binding of the human immunodeficiency virus type 1
(HIV-1) Gag protein to the HIV-1 RNA encapsidation signal (HIVPsi). Interaction
of these elements results in the activation of a reporter gene in the yeast
Saccharomyces cerevisiae. Using this system, we have shown that the HIV-1 Gag
protein binds specifically to a 139-nucleotide fragment of the HIVPsi signal
containing four stem-loop structures. Mutations in either the Gag protein or the
encapsidation signal that have been shown previously to impair this interaction
reduced the activation of the reporter gene. Interestingly, the nucleocapsid
portion of Gag retained the RNA binding activity but lost its specificity
compared to the full-length Gag. These results demonstrate the utility of this
system and suggest that a variety of genetic analyses could be performed to
study Gag-encapsidation signal interactions.
PMID: 9658151 [PubMed - indexed for MEDLINE]
978: J Virol 1998 Aug;72(8):6732-41
Complete protein linkage map of poliovirus P3 proteins: interaction of
polymerase 3Dpol with VPg and with genetic variants of 3AB.
Xiang W, Cuconati A, Hope D, Kirkegaard K, Wimmer E.
Department of Molecular Genetics and Microbiology, School of Medicine, State
University of New York at Stony Brook, Stony Brook, New York 11794-5222, USA.
Poliovirus has evolved to maximize its genomic information by producing
multifunctional viral proteins. The P3 nonstructural proteins harbor various
activities when paired with different binding partners. These viral polypeptides
regulate host cell macromolecular synthesis and function as proteinases, as RNA
binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the
P3 region is the genome-linked protein VPg that is essential in the initiation
of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze
directly protein-protein interactions among P3 proteins. Sixteen signals of
homo- or heterodimer interactions have been observed and have been divided into
three groups. Of interest is the newly discovered affinity of VPg to 3Dpol that
suggests direct interaction between these molecules in genome replication. A
battery of 3AB variants (eight clustered-charge-to-alanine changes and five
single-amino-acid mutations) has been used to map the binding determinants of
3AB-3AB interaction which were found to differ from the amino acids critical for
the 3AB-3Dpol interaction. The viral proteinase 3Cpro was not found to interact
with other 3Cpro molecules or with any other P3 polypeptide in yeast cells, a
result confirmed by glutaraldehyde cross-linking. The weak apparent interaction
between 3AB and 3CDpro scored in the yeast two-hybrid system was in contrast to
a strong signal by far-Western blotting. The results elucidate, in part,
previous results of biochemical and genetic analyses. The role of the
interactions in RNA replication is addressed.
PMID: 9658121 [PubMed - indexed for MEDLINE]
979: Science 1998 Jul 10;281(5374):262-6
Structure of the Escherichia coli RNA polymerase alpha subunit amino-terminal
domain.
Zhang G, Darst SA.
Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
The 2.5 angstrom resolution x-ray crystal structure of the Escherichia coli RNA
polymerase (RNAP) alpha subunit amino-terminal domain (alphaNTD), which is
necessary and sufficient to dimerize and assemble the other RNAP subunits into a
transcriptionally active enzyme and contains all of the sequence elements
conserved among eukaryotic alpha homologs, has been determined. The alphaNTD
monomer comprises two distinct, flexibly linked domains, only one of which
participates in the dimer interface. In the alphaNTD dimer, a pair of helices
from one monomer interact with the cognate helices of the other to form an
extensive hydrophobic core. All of the determinants for interactions with the
other RNAP subunits lie on one face of the alphaNTD dimer. Sequence alignments,
combined with secondary-structure predictions, support proposals that a
heterodimer of the eukaryotic RNAP subunits related to Saccharomyces cerevisiae
Rpb3 and Rpb11 plays the role of the alphaNTD dimer in prokaryotic RNAP.
PMID: 9657722 [PubMed - indexed for MEDLINE]
980: Biochim Biophys Acta 1998 Jun 29;1385(2):271-86
Subunit structure, function and organisation of pyruvate decarboxylases from
various organisms.
Konig S.
Institut fur Biochemie, Fachbereich Biochemie/Biotechnologie,
Martin-Luther-Universitat Halle-Wittenberg, D-06099 Halle/Saale, Germany.
The nature of the environment of macromolecules influences and determines the
state of their overall structure and the extent of binding of specific
(cofactors, substrates) or unspecific ligands. How these interactions between
enzyme molecules and ligands influence their quaternary structures and, in this
way, the realisation of high catalytic activity will be discussed here for the
enzyme pyruvate decarboxylase from various organisms: brewer's yeast, brewer's
yeast strain, recombinant wild type and site-specific mutants of Saccharomyces
cerevisiae, the recombinant wild type of the bacterium Zymomonas mobilis and
germinating seeds of the plant Pisum sativum from a structural point of view
including both high resolution models from crystal structure analysis and low
resolution models from small angle X-ray solution scattering with synchrotron
radiation.
Publication Types:
Review
Review, Tutorial
PMID: 9655918 [PubMed - indexed for MEDLINE]
981: Eur J Biochem 1998 May 1;253(3):560-75
Identification of Man alpha1-3Man alpha1-2Man and Man-linked phosphate on
O-mannosylated recombinant leech-derived tryptase inhibitor produced by
Saccharomyces cerevisiae and determination of the solution conformation of the
mannosylated polypeptide.
Bergwerff AA, Stark W, Fendrich G, Knecht R, Blommers MJ, Maerki W, Kragten EA,
van Oostrum J.
Core Technology Area, Novartis AG, Basle, Switzerland.
The production of recombinant leech-derived tryptase inhibitor (rLDTI) by two
different strains of Saccharomyces cerevisiae resulted in the secretion of
non-glycosylated and glycosylated rLTDI. Monosaccharide analysis and
a-mannosidase treatment demonstrated that glycosylated rLDTI was exclusively
alpha-mannosylated. A trypsin digest of reduced and S-carboxymethylated
glycosylated rLDTI was separated on a reverse-phase HPLC column. Glycopeptides
identified by a combination of matrix-assisted laser desorption mass
spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed
the presence of different glycoforms. It was found that Ser24, Ser33 and Ser36
were partially glycosylated with a single mannose residue, whereas Thr42 in
glycosylated rLDTI from both strains was fully occupied with
manno-oligosaccharides with a degree of polymerization ranging over 1-3 and 1-13
depending on the yeast strain. In phosphorylated rLDTI a single phosphate group
was predominantly located at the innermost Man residue of units of mannobiose,
mannotriose, mannotetraose and mannopentaose at Thr42. Oligosaccharides released
by alkaline treatment were reduced by sodium borohydride and separated by
high-pH anion-exchange chromatography on a CarboPac MA1 column, and analyzed by
one- and two-dimensional 1H-NMR spectroscopy. Besides the major oligosaccharide
Man alpha1-2Man-ol, the (for yeast protein O-glycosylation) unusual Man
alpha1-3Man alpha1-2Man-ol was determined. The solution conformation of
glycosylated rLDTI was investigated by two-dimensional NMR spectroscopy.
Structure calculations by means of distance geometry showed that glycosylated
rLDTI is compactly folded and contained small secondary structure elements.
Analysis of the chemical shifts showed that amino acids Val32-Ser33, Ser36-Ser39
and Thr42 were affected by the O-mannosylation. In addition, changes in chemical
shift were observed within the beta-hairpin peptide regions Val13-Ser16 and
Gly18-Tyr21 attributed to direct interactions of the mannose residue at Ser36.
Furthermore, the protein-linked oligosaccharides were spatially grouped in a
position opposite of the canonical binding loop.
PMID: 9654051 [PubMed - indexed for MEDLINE]
982: Mol Cell 1998 Jun;1(7):1051-5
A single amino acid change in the yeast retrotransposon Ty5 abolishes targeting
to silent chromatin.
Gai X, Voytas DF.
Department of Zoology and Genetics, Iowa State University, Ames 50011, USA.
Many retrotransposons and retroviruses are thought to select integration sites
through interactions with specific chromosomal proteins. In yeast, the Ty5
retrotransposon integrates preferentially with regions bound by silent
chromatin, namely the telomeres and the HMR and HML mating loci. A Ty5 mutant
(M3) was identified with an approximately 20-fold decrease in targeted
integration as measured by a plasmid-based targeting assay. Often chromosomal
insertions generated by M3, none were located at the telomeres or silent mating
loci. A single amino acid change at the boundary of integrase and reverse
transcriptase is responsible for the mutant phenotype. We predict that this
mutation lies within a targeting domain that mediates Ty5 target choice by
interacting with a component of silent chromatin.
PMID: 9651588 [PubMed - indexed for MEDLINE]
983: Mol Gen Genet 1998 May;258(3):215-21
Expression of the yeast BFR2 gene is regulated at the transcriptional level and
through degradation of its product.
Chabane S, Kepes F.
Service de Biochimie et de Genetique Moleculaire, DBCM/DSV, CEA/Saclay, Gif,
France.
The essential Saccharomyces cerevisiae gene BFR2 has been isolated as a
high-copy suppressor of the growth defects induced by Brefeldin A, a drug that
disrupts the Golgi apparatus and its protein influx. Furthermore, BFR2 has been
found to display genetic interactions with four mutations affecting protein
transport to the Golgi apparatus. Here we show that the level of BFR2 mRNA
rapidly increased over fivefold in response to cold shock, and over threefold
following nutrient replenishment by dilution of cells from exhausted to fresh
minimal medium. During subsequent growth, the transcript level returned to its
basal values, except for a transient drop toward the end of the exponential
phase. The early burst of transcription was not caused by toxic compounds in the
fresh medium, or by synchrony among cells that had simultaneously entered their
first cell cycle. The BFR2 gene product (Bfr2p) was synthesized following the
early burst of mRNA, and was no longer produced when the mRNA was back to basal
level. Bfr2p was finally degraded after growth became limited, and reached
undetectable levels in exhausted medium. Under steady-state conditions of
lengthened exponential phase, the intracellular level of Bfr2p remained
constant. This peculiar pattern of gene expression suggests that Bfr2p is
essential for mass growth or cell proliferation, whereas it is either toxic or
not required during nutrient-limited growth.
PMID: 9645427 [PubMed - indexed for MEDLINE]
984: Arch Virol 1998;143(5):981-96
Erratum in:
Arch Virol 1998;143(10):2064
In vivo interactions among rotavirus nonstructural proteins.
Gonzalez RA, Torres-Vega MA, Lopez S, Arias CF.
Departamento de Genetica y Fisiologia Molecular, Universidad Nacional Autonoma
de Mexico, Cuernavaca, Morelos, Mexico.
The rotavirus genome encodes six nonstructural (NS) proteins, five of which
(NSP1, NSP2, NSP3, NSP5, and NSP6) have been suggested to be involved in a
variety of events, such as genome replication, regulation of gene expression,
and gene assortment. These NS proteins have been found to be associated with
replication complexes that are precursors of the viral core, however, little
information is available about the intermolecular interactions that may exist
among them. Using the yeast two-hybrid system, which allows the detection of
protein-protein interactions in vivo, all possible combinations among the
rotavirus NS proteins were tested, and several interactions were observed. NSP1
interacted with the other four proteins tested; NSP3 associated with itself; and
NSP5 was found to form homodimers and to interact with NSP6.
Co-immunoprecipitation of proteins from rotavirus-infected cells, using
hyperimmune sera monospecific for the NS proteins, showed the same interactions
for NSP1 as those observed in yeast. Immunofluorescence co-localization analysis
of virus-infected epithelial cells revealed that the intracellular distribution
of proteins that were seen to interact in yeast had patterns of distribution
that would allow such intermolecular interactions to occur. These findings
should contribute to the understanding of the role these proteins play in
different aspects of the virus replication cycle.
PMID: 9645203 [PubMed - indexed for MEDLINE]
985: Mol Med 1998 May;4(5):299-323
Convergence and divergence of the signaling pathways for insulin and
phosphoinositolglycans.
Muller G, Wied S, Piossek C, Bauer A, Bauer J, Frick W.
Hoechst Marion Roussel Deutschland GmbH, Frankfurt am Main, Germany.
guenter.mueller@hmrag.com
Phosphoinositolglycan molecules isolated from insulin-sensitive mammalian
tissues have been demonstrated in numerous in vitro studies to exert partial
insulin-mimetic activity on glucose and lipid metabolism in insulin-sensitive
cells. However, their ill-defined structures, heterogeneous nature, and limited
availability have prohibited the analysis of the underlying molecular mechanism.
Phosphoinositolglycan-peptide (PIG-P) of defined and homogeneous structure
prepared in large scale from the core glycan of a
glycosyl-phosphatidylinositol-anchored membrane protein from Saccharomyces
cerevisiae has recently been shown to stimulate glucose transport as well as a
number of glucose-metabolizing enzymes and pathways to up to 90% (at 2 to 10
microns) of the maximal insulin effect in isolated rat adipocytes,
cardiomyocytes, and diaphragms (G. Muller et al., 1997, Endocrinology 138:
3459-3476). Consequently, we used this PIG-P for the present study in which we
compare its intracellular signaling with that of insulin. The activation of
glucose transport by both PIG-P and insulin in isolated rat adipocytes and
diaphragms was found to require stimulation of phosphatidylinositol (PI)
3-kinase but to be independent of functional p70S6kinase and mitogen-activated
protein kinase. The increase in glycerol-3-phosphate acyltransferase activity in
rat adipocytes in response to PIG-P and insulin was dependent on both PI
3-kinase and p70S6kinase. This suggest that the signaling pathways for PIG-P and
insulin to glucose transport and metabolism converage at the level of PI
3-kinase. A component of the PIG-P signaling pathway located up-stream of PI
3-kinase was identified by desensitization of isolated rat adipocytes for PIG-P
action by combined treatment with trypsin and NaCl under conditions that
preserved cell viability and the insulin-mimetic activity of sodium vanadate but
completely blunted the insulin response. Incubation of the cells with either
trypsin or NaCl alone was ineffective. The desensitized adipocytes were
reconstituted for stimulation of lipogenesis by PIG-P by addition of the
concentrated trypsin/salt extract. The reconstituted adipocytes exhibited 65-75%
of the maximal PIG-P response and similar EC50 values for PIG-P (2 to 5 microns)
compared with control cells. A proteinaceous N-ethylmaleimide (NEM)-sensitive
component contained in the trypsin/salt extract was demonstrated to bind in a
functional manner to the adipocyte plasma membrane of desensitized adipocytes
via bipolar interactions. An excess of trypsin/salt extract inhibited PIG-P
action in untreated adipocytes in a competitive fashion compatible with a
receptor function for PIG-P of this protein. The presence of the putative PIG-P
receptor protein in detergent-insoluble complexes prepared from isolated rat
adipocytes suggests that caveolae/detergent-insoluble complexes of the plasma
membrane may play a role in insulin-mimetic signaling by PIG-P. Furthermore,
treatment of isolated rat diaphragms and adipocytes with PIG-P as well as with
other agents exerting partially insulin-mimetic activity, such as PI-specific
phospholipase C (PLC) and the sulfonylurea glimepiride, triggered tyrosine
phosphorylation of the caveolar marker protein caveolin, which was apparently
correlated with stimulation of lipogenesis. Strikingly, in adipocytes subjected
to combined trypsin/salt treatment, PIG-P, PI-specific PLC, and glimepiride
failed completely to provoke insulin-mimetic effects. A working model is
presented for a signaling pathway in insulin-sensitive cells used by PIG(-P)
molecules which involves GPI structures, the trypsin/salt- and NEM-sensitive
receptor protein for PIG-P, and additional proteins located in
caveolae/detergent-insoluble complexes.
PMID: 9642681 [PubMed - indexed for MEDLINE]
986: Mol Cell Biol 1998 Jul;18(7):4400-6
Studies of the interaction between Rad52 protein and the yeast single-stranded
DNA binding protein RPA.
Hays SL, Firmenich AA, Massey P, Banerjee R, Berg P.
Department of Biochemistry, Beckman Center for Molecular and Genetic Medicine,
Stanford University School of Medicine, Stanford University, Stanford,
California 94305, USA.
The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded
DNA binding protein replication protein A (RPA), which is known to play a
critical role in DNA replication. A Saccharomyces cerevisiae strain carrying the
rfa1-44 allele displays a number of impaired recombination and repair
phenotypes, all of which are suppressible by overexpression of RAD52. We
demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placing
RFA1 and RAD52 in the same genetic pathway. Furthermore, two-hybrid analysis
indicates the existence of interactions between Rad52 and all three subunits of
RPA. The nature of this Rad52-RPA interaction was further explored by using two
different mutant alleles of rad52. Both mutations lie in the amino terminus of
Rad52, a region previously defined as being responsible for its DNA binding
ability (U. H. Mortenson, C. Beudixen, I. Sunjeuaric, and R. Rothstein, Proc.
Natl. Acad. Sci. USA 93:10729-10734, 1996). The yeast two-hybrid system was used
to monitor the protein-protein interactions of the mutant Rad52 proteins. Both
of the mutant proteins are capable of self-interaction but are unable to
interact with Rad51. The mutant proteins also lack the ability to interact with
the large subunit of RPA, Rfa1. Interestingly, they retain their ability to
interact with the medium-sized subunit, Rfa2. Given the location of the
mutations in the DNA binding domain of Rad52, a model incorporating the role of
DNA in the protein-protein interactions involved in the repair of DNA
double-strand breaks is presented.
PMID: 9632824 [PubMed - indexed for MEDLINE]
987: Mol Cell Biol 1998 Jul;18(7):3752-61
Interaction of TATA-binding protein with upstream activation factor is required
for activated transcription of ribosomal DNA by RNA polymerase I in
Saccharomyces cerevisiae in vivo.
Steffan JS, Keys DA, Vu L, Nomura M.
Department of Biological Chemistry, University of California-Irvine, Irvine,
California 92697-1700, USA.
Previous in vitro studies have shown that initiation of transcription of
ribosomal DNA (rDNA) in the yeast Saccharomyces cerevisiae involves an
interaction of upstream activation factor (UAF) with the upstream element of the
promoter, forming a stable UAF-template complex; together with TATA-binding
protein (TBP), UAF then recruits an essential factor, core factor (CF), to the
promoter, forming a stable preinitiation complex. TBP interacts with both UAF
and CF in vitro. In addition, a subunit of UAF, Rrn9p, interacts with TBP in
vitro and in the two-hybrid system, suggesting the possible importance of this
interaction for UAF function. Using the yeast two-hybrid system, we have
identified three mutations in RRN9 that abolish the interaction of Rrn9p with
TBP without affecting its interaction with Rrn10p, another subunit of UAF. Yeast
cells containing any one of these individual mutations, L110S, L269P, or L274Q,
did not show any growth defects. However, cells containing a combination of
L110S with one of the other two mutations showed a temperature-sensitive
phenotype, and this phenotype was suppressed by fusing the mutant genes to
SPT15, which encodes TBP. In addition, another mutation (F186S), which disrupts
both Rrn9p-TBP and Rrn9p-Rrn10p interactions in the two-hybrid system, abolished
UAF function in vivo, and this mutational defect was suppressed by fusion of the
mutant gene to SPT15 combined with overexpression of Rrn10p. These experiments
demonstrate that the interaction of UAF with TBP, which is presumably achieved
by the interaction of Rrn9p with TBP, is indeed important for high-level
transcription of rDNA by RNA polymerase I in vivo.
PMID: 9632758 [PubMed - indexed for MEDLINE]
988: J Cell Biol 1998 Jun 15;141(6):1371-81
Interaction between mitochondria and the actin cytoskeleton in budding yeast
requires two integral mitochondrial outer membrane proteins, Mmm1p and Mdm10p.
Boldogh I, Vojtov N, Karmon S, Pon LA.
Department of Anatomy and Cell Biology, Columbia University College of
Physicians and Surgeons, New York, New York 10032, USA.
Transfer of mitochondria to daughter cells during yeast cell division is
essential for viable progeny. The actin cytoskeleton is required for this
process, potentially as a track to direct mitochondrial movement into the bud.
Sedimentation assays reveal two different components required for
mitochondria-actin interactions: (1) mitochondrial actin binding protein(s)
(mABP), a peripheral mitochondrial outer membrane protein(s) with ATP-sensitive
actin binding activity, and (2) a salt-inextractable, presumably integral,
membrane protein(s) required for docking of mABP on the organelle. mABP activity
is abolished by treatment of mitochondria with high salt. Addition of either the
salt-extracted mitochondrial peripheral membrane proteins (SE), or a protein
fraction with ATP-sensitive actin-binding activity isolated from SE, to
salt-washed mitochondria restores this activity. mABP docking activity is
saturable, resistant to high salt, and inhibited by pre-treatment of salt-washed
mitochondria with papain. Two integral mitochondrial outer membrane proteins,
Mmm1p (Burgess, S.M., M. Delannoy, and R.E. Jensen. 1994. J.Cell Biol.
126:1375-1391) and Mdm10p, (Sogo, L.F., and M.P. Yaffe. 1994. J.Cell Biol.
126:1361- 1373) are required for these actin-mitochondria interactions.
Mitochondria isolated from an mmm1-1 temperature-sensitive mutant or from an
mdm10 deletion mutant show no mABP activity and no mABP docking activity.
Consistent with this, mitochondrial motility in vivo in mmm1-1 and mdm10Delta
mutants appears to be actin independent. Depolymerization of F-actin using
latrunculin-A results in loss of long-distance, linear movement and a fivefold
decrease in the velocity of mitochondrial movement. Mitochondrial motility in
mmm1-1 and mdm10Delta mutants is indistinguishable from that in
latrunculin-A-treated wild-type cells. We propose that Mmm1p and Mdm10p are
required for docking of mABP on the surface of yeast mitochondria and coupling
the organelle to the actin cytoskeleton.
PMID: 9628893 [PubMed - indexed for MEDLINE]
989: Mutat Res 1998 Mar;407(2):135-45
Genetic interactions between mutants of the 'error-prone' repair group of
Saccharomyces cerevisiae and their effect on recombination and mutagenesis.
Liefshitz B, Steinlauf R, Friedl A, Eckardt-Schupp F, Kupiec M.
Department of Molecular Microbiology and Biotechnology, Tel Aviv University,
Israel.
We have created an isogenic series of yeast strains that carry genetic systems
to monitor different types of recombination and mutation [B. Liefshitz, A.
Parket, R. Maya, M. Kupiec, The role of DNA repair genes in recombination
between repeated sequences in yeast, Genetics 140 (1995) 1199-1211.]. In the
present study we characterize the effect of mutations in genes of the
'error-prone' or postreplicative repair group on recombination and mutation. We
show that rad5 and rad18 strains have elevated levels of spontaneous
recombination, both of ectopic gene conversion and of recombination between
direct repeats. The increase in recombination levels is similar in both mutants
and in the rad5 rad18 double mutant, suggesting that the RAD5 and RAD18 gene
products act together with respect to spontaneous recombination. In contrast,
RAD5 and RAD18 play alternative roles in mutagenic repair: mutations in each of
these genes elevate spontaneous forward mutation at the CAN1 locus, but when
both genes are deleted, a low level of spontaneous mutagenesis is seen. The
RAD5/RAD18 pathway of mutagenic repair is dependent on the REV3-encoded
translesion polymerase. We analyze the interactions between the RAD5 and RAD18
gene products and other repair genes. The high recombination levels seen in rad5
and rad18 mutants is dependent on the RAD1, RAD51, RAD52, and RAD57 genes. The
Srs2 helicase plays an important role in creating the recombinogenic
substrate(s) processed by the RAD5 and RAD18 gene products.
PMID: 9637242 [PubMed - indexed for MEDLINE]
990: Biotechnology (N Y) 1995 Dec;13(13):1474-8
Comment in:
Biotechnology (N Y). 1995 Dec;13(13):1431-4.
The yeast tribrid system--genetic detection of trans-phosphorylated
ITAM-SH2-interactions.
Osborne MA, Dalton S, Kochan JP.
Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche, Inc., USA.
Protein-protein interactions are often dependent on the post-translational
modification of one component of a complex. To facilitate the study of these
interactions in signal transduction, we have developed the yeast tribrid system,
a modification of the yeast two-hybrid system. We demonstrate that the
interactions are dependent upon the presence of a tyrosine kinase, an SH2 domain
and a tyrosine containing substrate. Using the gamma subunit of the
high-affinity IgE receptor, Fc epsilon RI, this approach has been used to
isolate a novel SH2-containing family member. The mRNA encoding this novel
protein is differentially expressed in rat tissues. The yeast tribrid system can
be readily adapted for the characterization of novel tyrosine kinases or
substrates, as well as the study of protein-protein interactions which involve
other post-translational modifications.
PMID: 9636306 [PubMed - indexed for MEDLINE]
991: EMBO J 1998 May 1;17(9):2494-503
TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle
docking and fusion.
Sacher M, Jiang Y, Barrowman J, Scarpa A, Burston J, Zhang L, Schieltz D, Yates
JR 3rd, Abeliovich H, Ferro-Novick S.
Howard Hughes Medical Institute and the Department of Cell Biology, Yale
University School of Medicine, New Haven, CT 06510, USA.
We previously identified BET3 by its genetic interactions with BET1, a gene
whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport.
To gain insight into the function of Bet3p, we added three c-myc tags to its
C-terminus and immunopurified this protein from a clarified detergent extract.
Here we report that Bet3p is a member of a large complex ( approximately 800
kDa) that we call TRAPP (transport protein particle). We propose that TRAPP
plays a key role in the targeting and/or fusion of ER-to-Golgi transport
vesicles with their acceptor compartment. The localization of Bet3p to the
cis-Golgi complex, as well as biochemical studies showing that Bet3p functions
on this compartment, support this hypothesis. TRAPP contains at least nine other
constituents, five of which have been identified and shown to be highly
conserved novel proteins.
PMID: 9564032 [PubMed - indexed for MEDLINE]
992: Biochemistry 1998 May 26;37(21):7834-43
The structure of the N-terminus of striated muscle alpha-tropomyosin in a
chimeric peptide: nuclear magnetic resonance structure and circular dichroism
studies.
Greenfield NJ, Montelione GT, Farid RS, Hitchcock-DeGregori SE.
Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School,
University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635, USA.
Tropomyosins (TMs) are highly conserved, coiled-coil, actin binding regulatory
proteins found in most eukaryotic cells. The amino-terminal domain of
284-residue TMs is among the most conserved and functionally important regions.
The first nine residues are proposed to bind to the carboxyl-terminal nine
residues to form the "overlap" region between successive TMs, which bind along
the actin filament. Here, the structure of the N-terminus of muscle alpha-TM, in
a chimeric peptide, TMZip, has been solved using circular dichroism (CD) and
two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy.
Residues 1-14 of TMZip are the first 14 N-terminal residues of rabbit striated
alpha-TM, and residues 15-32 of TMZip are the last 18 C-terminal residues of the
yeast GCN4 transcription factor. CD measurements show that TMZip forms a
two-stranded coiled-coil alpha-helix with an enthalpy of folding of -34 +/- 2
kcal/mol. In 2D1H NMR studies at 15 degrees C, pH 6.4, the peptide exhibits 123
sequential and medium range intrachain NOE cross peaks per chain, characteristic
of alpha-helices extending from residue 1 to residue 29, together with 85
long-range NOE cross peaks arising from interchain interactions. The
three-dimensional structure of TMZip has been determined using these data plus
an additional 509 intrachain constraints per chain. The coiled-coil domain
extends to the N-terminus. Amide hydrogen exchange studies, however, suggest
that the TM region is less stable than the GCN4 region. The work reported here
is the first atomic-resolution structure of any region of TM and it allows
insight into the mechanism of the function of the highly conserved N-terminal
domain.
PMID: 9601044 [PubMed - indexed for MEDLINE]
993: J Cell Biol 1998 May 18;141(4):887-94
The beta subunit of the Sec61 complex facilitates cotranslational protein
transport and interacts with the signal peptidase during translocation.
Kalies KU, Rapoport TA, Hartmann E.
Abteilung Biochemie II, Zentrum Biochemie und Molekulare Zellbiologie,
Georg-August-Universitat, 37073 Gottingen, Germany.
The Sec61 complex is the central component of the protein translocation
apparatus of the ER membrane. We have addressed the role of the beta subunit
(Sec61beta) during cotranslational protein translocation. With a reconstituted
system, we show that a Sec61 complex lacking Sec61beta is essentially inactive
when elongation and membrane targeting of a nascent chain occur at the same
time. The translocation process is perturbed at a step where the nascent chain
would be inserted into the translocation channel. However, if sufficient time is
given for the interaction of the nascent polypeptide with the mutant Sec61
complex, translocation is almost normal. Thus Sec61beta kinetically facilitates
cotranslational translocation, but is not essential for it. Using chemical
cross-linking we show that Sec61beta not only interacts with subunits of the
Sec61 complex but also with the 25-kD subunit of the signal peptidase complex
(SPC25), thus demonstrating for the first time a tight interaction between the
SPC and the Sec61 complex. Interestingly, the cross-links between Sec61beta and
SPC25 and between Sec61beta and Sec61alpha depend on the presence of
membrane-bound ribosomes, suggesting that these interactions are induced when
translocation is initiated. We propose that the SPC is transiently recruited to
the translocation site, thus enhancing its activity.
PMID: 9585408 [PubMed - indexed for MEDLINE]
994: Mol Cell Biol 1998 Jun;18(6):3173-81
Role of the negative charges in the cytosolic domain of TOM22 in the import of
precursor proteins into mitochondria.
Nargang FE, Rapaport D, Ritzel RG, Neupert W, Lill R.
Department of Biological Sciences, University of Alberta, Edmonton, Alberta,
Canada T6G 2E9. frank.nargang@ualberta.ca
TOM22 is an essential mitochondrial outer membrane protein required for the
import of precursor proteins into the organelles. The amino-terminal 84 amino
acids of TOM22 extend into the cytosol and include 19 negatively and 6
positively charged residues. This region of the protein is thought to interact
with positively charged presequences on mitochondrial preproteins, presumably
via electrostatic interactions. We constructed a series of mutant derivatives of
TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic
domain were changed to their corresponding amido forms. The mutant constructs
were transformed into a sheltered Neurospora crassa heterokaryon bearing a
tom22::hygromycin R disruption in one nucleus. All constructs restored viability
to the disruption-carrying nucleus and gave rise to homokaryotic strains
containing mutant tom22 alleles. Isolated mitochondria from three representative
mutant strains, including the mutant carrying 15 neutralized residues (strain
861), imported precursor proteins at efficiencies comparable to those for
wild-type organelles. Precursor binding studies with mitochondrial outer
membrane vesicles from several of the mutant strains, including strain 861,
revealed only slight differences from binding to wild-type vesicles. Deletion
mutants lacking portions of the negatively charged region of TOM22 can also
restore viability to the disruption-containing nucleus, but mutants lacking the
entire region cannot. Taken together, these data suggest that an abundance of
negative charges in the cytosolic domain of TOM22 is not essential for the
binding or import of mitochondrial precursor proteins; however, other features
in the domain are required.
PMID: 9584158 [PubMed - indexed for MEDLINE]
995: J Biol Chem 1998 May 15;273(20):12567-75
Jak2-Stat5 interactions analyzed in yeast.
Barahmand-Pour F, Meinke A, Groner B, Decker T.
Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna,
Dr. Bohr-Gasse 9, A-1030 Vienna, Austria.
Many cytokine receptors employ Janus protein tyrosine kinases (Jaks) and signal
transducers and activators of transcription (Stats) for nuclear signaling. Here,
we have established yeast strains in which an autoactivated Jak2 kinase induces
tyrosine phosphorylation, dimerization, nuclear translocation, and DNA binding
of a concomitantly expressed Stat5 protein. Transcriptional activity of Stat5 on
a stably integrated, Stat-dependent reporter gene required the C-terminal fusion
of the VP16 transactivation domain. In such yeast strains, the interaction
between Jak2 and Stat5 was analyzed without interference by other mammalian
proteins involved in regulating Jak-Stat signaling, and mutant versions of both
proteins were analyzed for their ability to productively interact. Complexes
between Jak2 and Stat5 were found to be stable under stringent
co-immunoprecipitation conditions. Deletion of the Jak homology regions 2-7
(JH2-JH7) of Jak2, leaving only the kinase domain (JH1) intact, reduced the
ability of the kinase to phosphorylate Stat5, whereas deletion of the JH2 domain
caused an increased enzymatic activity. A site-directed R618K mutation in the
Stat5 SH2 domain abolished the phosphorylation by Jak2, while deletion of the C
terminus led to Stat5 hyperphosphorylation. A single phosphotyrosine-SH2 domain
interaction was sufficient for the dimerization of Stat5, but such dimers bound
to DNA very inefficiently. Together, our data show that yeast cells are
appropriate tools for studying Jak-Stat or Stat-Stat interactions. Our
mutational analysis suggests that the Stat5 SH2 domain is essential for the
interaction with Jak2 and that the kinase domain of Jak2 is sufficient for
Jak2-Stat5 interaction. Therefore, the Jak kinase domain may be all that is
needed to cause Stat phosphorylation in situations where receptor docking is
dispensable.
PMID: 9575217 [PubMed - indexed for MEDLINE]
996: Nucleic Acids Res 1998 May 1;26(9):2252-3
A recombination based method to rapidly assess specificity of two-hybrid clones
in yeast.
Petermann R, Mossier BM, Aryee DN, Kovar H.
Children's Cancer Research Institute, St. Anna Kinderspital, Kinderspitalgasse
6, A-1090 Vienna, Austria.
The yeast two-hybrid system is frequently used to identify protein-protein
interactions. Confirming the specificity of candidate clones requires separation
and isolation of yeast plasmids, propagation in bacteria and testing
combinations of DNA-binding and activation domain hybrids in yeast. In order to
simplify this procedure, we developed a rapid method based on PCR amplification
of library insert DNAs and in vivo cloning into the activation domain hybrid
vector. Reporter gene activity is assayed in parallel for combinations with
different DNA-binding domain hybrids. Further characterization of inserts does
not require plasmid isolation and intermediate hosts.
PMID: 9547290 [PubMed - indexed for MEDLINE]
997: Curr Opin Struct Biol 1998 Apr;8(2):177-85
Intermediate filament assembly: fibrillogenesis is driven by decisive
dimer-dimer interactions.
Herrmann H, Aebi U.
Division of Cell Biology, German Cancer Research Center, Heidelberg, Germany.
H.Herrmann@DKFZ-Heidelberg.de
Intermediate filaments are built from one to several members of a multigene
family encoding fibrous proteins that share a highly conserved hierarchic
assembly plan for the formation of multistranded filaments from distinctly
structured extended coiled coils. Despite the rather low primary sequence
identity, intermediate filaments form apparently similar filaments with regard
to their spatial dimensions and physical properties. Over the past few years,
substantial progress has been made in the elucidation of the complex expression
patterns and clinically relevant phenotypes of intermediate filaments. The key
question of how these filaments assemble and what the molecular architecture of
their distinct assembly intermediates comprises, however, has still not been
answered to the extent that has been achieved for microfilaments and
microtubules.
Publication Types:
Review
Review, Tutorial
PMID: 9631290 [PubMed - indexed for MEDLINE]
998: Nat Biotechnol 1996 Apr;14(4):481-4
Comment in:
Nat Biotechnol. 1996 Apr;14(4):436.
A fusion protein designed for noncovalent immobilization: stability, enzymatic
activity, and use in an enzyme reactor.
Stempfer G, Holl-Neugebauer B, Kopetzki E, Rudolph R.
Boehringer Mannheim Therapeutics, Pennzberg, Germany.
We have designed a new method for enzyme immobilization using a fusion protein
of yeast alpha-glucosidase containing at its C-terminus a polycationic
hexa-arginine fusion peptide. This fusion protein can be directly adsorbed from
crude cell extracts on polyanionic matrices in a specific, oriented fashion.
Upon noncovalent immobilization by polyionic interactions, the stability of the
fusion protein is not affected by pH-, urea-, or thermal-denaturation.
Furthermore, the enzymatic properties (specific activity at increasing enzyme
concentration, Michaelis constant, or activation energy of the enzymatic
reaction) are not influenced by this noncovalent coupling. The operational
stability of the coupled enzyme under conditions of continuous substrate
conversion is, however, increased significantly compared to the soluble form.
Fusion proteins containing polyionic peptide sequences are proposed as versatile
tools for the production of immobilized enzyme catalysts.
PMID: 9630924 [PubMed - indexed for MEDLINE]
999: Nat Biotechnol 1996 Mar;14(3):329-34
Improved refolding of an immobilized fusion protein.
Stempfer G, Holl-Neugebauer B, Rudolph R.
Boehringer Mannheim Therapeutics, Penzberg, Germany.
Fusion proteins of monomeric alpha-glucosidase from Saccharomyces cerevisiae
containing N- or C-terminal hexa-arginie peptides were expressed in the cytosol
of Escherichia coli in soluble form. The polycationic peptide moieties allow
noncovalent binding of the denatured fusion proteins to a polyanionic solid
support. Upon removal of the denaturant, refolding of the matrix-bound protein
can proceed without perturbation by aggregation. However, nonspecific
interactions of the denatured polypeptide, or of folding intermediates, with the
matrix cause a drastic decrease in renaturation under suboptimal folding
conditions. At low salt concentrations, ionic interactions of the refolding
polypeptide with the matrix result in lower yields of renaturation. At higher
salt concentrations, renaturation is prevented by hydrophobic interactions with
the matrix. Apart from ionic strength, renaturation of the denatured
matrix-bound fusion protein must be optimized with respect to pH, temperature,
cosolvents, and matrix material used. Under optimum conditions, immobilized
alpha-glucosidase can be renatured with a high yield at protein concentrations
up to 5 mg/ml, whereas folding of the wild-type enzyme in solution is feasible
only at an extremely low protein concentration (15 micrograms/ml). Thus, folding
of the immobilized alpha-glucosidase allows an extremely high yield of the
renaturated model protein. The technology should be applicable to other proteins
that tend to aggregate during refolding.
PMID: 9630895 [PubMed - indexed for MEDLINE]
1000: Biochim Biophys Acta 1998 Jun 22;1403(2):158-68
Protein-protein interactions between keratin polypeptides expressed in the yeast
two-hybrid system.
Schnabel J, Weber K, Hatzfeld M.
Department of Biochemistry, Max-Planck-Institute for Biophysical Chemistry,
D-37070 Gottingen, Germany.
Keratin filaments are obligatory heteropolymers of type I and type II keratin
polypeptides. Specific type I/type II pairs are coexpressed in vivo. In
contrast, all type I/type II pairs assemble into filaments in vitro, but the
different pairs have different stabilities as demonstrated by treatment with
increasing concentrations of urea. We have used the yeast two-hybrid system to
analyse type I/type II interactions in a cellular context. We measured
interactions between two different keratin pairs and we confirm the findings
that K6+K17 form very stable heterodimers whereas K8+K18 interactions were
weaker. The deletion of head domains did not reduce the strength of type I/type
II interactions. Rather, the affinities were increased and the differences
between the two pairs were retained in headless mutants. These findings argue
against a major role of the head domains in directing heterodimer interactions
and in defining heterodimer stabilities.
PMID: 9630597 [PubMed - indexed for MEDLINE]