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 (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 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