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 <Long id=1>endoplasmic reticulum</Long> (<Short id=1>ER</Short>) 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 <Long id=2>Wiskott-Aldrich
Syndrome Protein-Interacting Protein</Long> (<Short id=2>WIP</Short>), 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, <Long id=3>Myo3p and Myo5p</Long> (<Short id=3>Myo3/5p</Short>), 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 <Long id=4>Wiskott-Aldrich syndrome protein</Long> (<Short id=4>WASP</Short>), and Vrp1p, a homolog of
<Long id=5>WASP-interacting protein</Long> (<Short id=5>WIP</Short>). 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, <Short id=6>Mti1p</Short> (<Long id=6>Myosin tail region-interacting
protein</Long>), 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 <Long id=7>cofactor-dependent phosphoglycerate mutase</Long>
(<Short id=7>dPGM</Short>), 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 <Long id=8>anaphase-promoting complex</Long> (<Short id=8>APC</Short>) 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.

<Long id=9>Ribonuclease P</Long> (<Short id=9>RNase P</Short>) 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 <Long id=10>type 1 protein phosphatase</Long>
(<Short id=10>PP1(C)</Short>) 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
<Long id=11>matrix-assisted laser desorption/ionization</Long> (<Short id=11>MALDI</Short>) mass spectrometry. To
confirm the accuracy of our identifications, peptides from some of the proteins
were also sequenced using <Long id=12>high-performance liquid chromatography</Long> (<Short id=11>HPLC</Short>) 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 <Short id=13>CPF</Short> (<Long id=13>cleavage and
polyadenylation factor</Long>) 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 <Long id=14>components of the yeast</Long> (<Short id=14>CPF</Short>) 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 <Long id=15>estrogen receptor alpha</Long> (<Short id=15>ER alpha</Short>) <Long id=16>ligand-binding domain</Long> (<Short id=16>LBD</Short>).
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 <Long id=17>high-throughput mass spectrometric protein complex
identification</Long> (<Short id=17>HMS-PCI</Short>). 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 <Long id=18>tandem-affinity purification</Long> (<Short id=18>TAP</Short>) 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 <Long id=19>protein phosphatase 1</Long> (<Short id=19>PP1(C)</Short>) 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). <Long id=20>Temperature-sensitive</Long>
(<Short id=20>Ts(-)</Short>) 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 <Short id=21>SUISEKI</Short> (<Long id=21>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 <Long id=22>general inhibitor</Long> (<Short id=22>RI</Short>) 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 <Long id=23>serine residues</Long> (<Short id=23>RS domains</Short>) 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 <Long id=24>postmeiotic
segregation</Long> (<Short id=24>PMS</Short>). 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 <Long id=25>protein kinase C</Long>
(<Short id=25>PKC</Short>) 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
<Long id=26>trabecular meshwork</Long> (<Short id=26>TM</Short>) 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
<Long id=27>regulatory light chain</Long> (<Short id=27>RLC</Short>). 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 <Long id=28>Rho GDP dissociation inhibitor</Long> (<Short id=28>RhoGDI</Short>) 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 <Long id=29>topoisomerase I</Long> (<Short id=29>Top1p</Short>) catalyzes topological changes in DNA and is the
cellular target of the antitumor agent <Long id=30>camptothecin</Long> (<Short id=30>CPT</Short>). 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 <Long id=31>heat-shock protein</Long> (<Short id=31>Hsp</Short>) 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.

<Long id=32>Heat-shock protein 40</Long> (<Short id=32>Hsp40</Short>) 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.

<Long id=33>Nitric oxide</Long> (<Short id-33>NO</Short>) 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 <Long id=34>synthetic genetic
array</Long> (<Short id=34>SGA</Short>) 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
<Long id=35>Wiskott-Aldrich Syndrome protein</Long> (<Short id=35>WASP</Short>) 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 <Long id=36>regulatory particle</Long> (<Short id=36>RP</Short>), 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 <Long id=37>COP9 signalosome</Long> (<Short id=37>CSN</Short>) 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 <Long id=38>decapping enzyme complex</Long> (<Short id=38>Dcp1p</Short> 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 <Long id=39>methyl methanesulfonate</Long> (<Short id=39>MMS</Short>) 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 <Long id=40>cytochrome bc1 complex</Long> (<Short id=40>QCR</Short>), 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 <Long id=41>inner centromere protein</long>
(<Short id=41>INCENP</Short>)-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 <Long id=42>inner centromere protein</Long> (<Short id=42>INCENP</Short>).
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 <Long id=42>general transcription factor</Long> (<Short>GTF</Short>) that has also
been implicated in the mechanism of action of certain promoter-specific
activators (see, for examples, [1-11]). TFIIB enters the <Long id=43>preinitiation complex</Long>
(<Short id=43>PIC</Short>) primarily through contact with the <Long id=44>TATA box binding protein</Long> (<Short id=44>TBP</Short>), 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 <Short id=45>UAS</Short> (<Long id=45>upstream activating
sequence</Long>), 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 <Short id=46>TALE</Short> (<Long id=46>three-amino acid loop extension</Long>) 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 <Long id=47>Arabidopsis BELL1</Long> (<Short id=47>BEL1</Short>) 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 <Long id=48>SHOOT
MERISTEMLESS</Long> (<Short id=48>STM</Short>) 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
<Long id=49>actin-regulating Ser/Thr kinase</Long> (<Short id=49>ARK</Short>) 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 <Short id=50>GGAs</Short> (<Long id=50>Golgi-localized, gamma-ear-containing, ARF-binding proteins</Long>) 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
<Long id=50>carboxypeptidase Y</Long> (<Short id=50>CPY</Short>) 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 <Long id=51>human immunodeficiency virus type 1</Long> (<Short id=51>HIV-1</Short>) proviral DNA in the
nuclear genome is catalyzed by the retroviral <Long id=52>integrase</Long> (<Short id=52>IN</Short>). 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.

<Long id=53>Yeast pyruvate kinase</Long> (<Short id=53>YPK</Short>) is regulated by intermediates of the glycolytic
pathway [e.g., <Long id=54>phosphoenolpyruvate</Long> (<Short id=54>PEP</Short>), <Long id=55>fructose 1,6-bisphosphate</Long> (<Short id=55>FBP</Short>), 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., <Long id=56>His46Cys</Long>
(<Short id=56>H46C</Short>) and <Long id=57>His120Cys</Long> (<Short id=57>H120C</Short>), 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 <Long id=58>C-terminal heptapeptide repeat domain</Long> (<Short id=58>CTD</Short>) 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 <Long id=59>haloacid dehalogenase</Long> (<Short id=59>HAD</Short>) 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 <Long id=60>activation function 1 domain</Long> (<Short id=60>AF-1</Short>), domain C as the
DNA-binding domain, domain D as a hinge domain and domain E/F as the
<Long id=61>ligand-dependent transcriptional domain</Long> (<Short id=61>AF-2</Short>). 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 <Long id=61>G-protein signaling</Long> (<Short id=61>RGS</Short>) 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: <Long id=62>Ubiquitin-conjugating enzymes</Long> (<Short id=62>E2s</Short>) are central enzymes involved in
ubiquitin-mediated protein degradation. During this process, <Long id=63>ubiquitin</Long> (<Short id=63>Ub</Short>) 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 <Long id=64>adaptor complex</Long> <Short id=64>AP-1</Short> 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 <Long id=65>tyrosine-based sorting signal</Long> (<Short id=65>YLPL</Short>) 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
S