1: Mol Biol Cell 2002 Mar;13(3):854-65 Endoplasmic reticulum dynamics, inheritance, and cytoskeletal interactions in budding yeast. Fehrenbacher KL, Davis D, Wu M, Boldogh I, Pon LA. Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, New York 10032. The endoplasmic reticulum (ER) in Saccharomyces cerevisiae consists of a reticulum underlying the plasma membrane (cortical ER) and ER associated with the nuclear envelope (nuclear ER). We used a Sec63p-green fluorescent protein fusion protein to study motility events associated with inheritance of cortical ER and nuclear ER in living yeast cells. During M phase before nuclear migration, we observed thick, apparently rigid tubular extensions emanating from the nuclear ER that elongate, undergo sweeping motions along the cell cortex, and shorten. Two findings support a role for microtubules in this process. First, extension of tubular structures from the nuclear ER is inhibited by destabilization of microtubules. Second, astral microtubules, structures that undergo similar patterns of extension, cortical surveillance and retraction, colocalize with nuclear ER extensions. During S and G(2) phases of the cell cycle, we observed anchorage of the cortical ER at the site of bud emergence and apical bud growth. Thin tubules of the ER that extend from the anchored cortical ER display undulating, apparently random movement and move into the bud as it grows. Finally, we found that cortical ER morphology is sensitive to a filamentous actin-destabilizing drug, latrunculin-A, and to mutations in the actin-encoding ACT1 gene. Our observations support 1) different mechanisms and cytoskeletal mediators for the inheritance of nuclear and cortical ER elements and 2) a mechanism for cortical ER inheritance that is cytoskeleton dependent but relies on anchorage, not directed movement. PMID: 11907267 [PubMed - in process] 2: Genetics 2002 Mar;160(3):923-34 The Novel Adaptor Protein, Mti1p, and Vrp1p, a Homolog of Wiskott-Aldrich Syndrome Protein-Interacting Protein (WIP), May Antagonistically Regulate Type I Myosins in Saccharomyces cerevisiae. Mochida J, Yamamoto T, Fujimura-Kamada K, Tanaka K. Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, 060-0815, Japan. Type I myosins in yeast, Myo3p and Myo5p (Myo3/5p), are involved in the reorganization of the actin cytoskeleton. The SH3 domain of Myo5p regulates the polymerization of actin through interactions with both Las17p, a homolog of mammalian Wiskott-Aldrich syndrome protein (WASP), and Vrp1p, a homolog of WASP-interacting protein (WIP). Vrp1p is required for both the localization of Myo5p to cortical patch-like structures and the ATP-independent interaction between the Myo5p tail region and actin filaments. We have identified and characterized a new adaptor protein, Mti1p (Myosin tail region-interacting protein), which interacts with the SH3 domains of Myo3/5p. Mti1p co-immunoprecipitated with Myo5p and Mti1p-GFP co-localized with cortical actin patches. A null mutation of MTI1 exhibited synthetic lethal phenotypes with mutations in SAC6 and SLA2, which encode actin-bundling and cortical actin-binding proteins, respectively. Although the mti1 null mutation alone did not display any obvious phenotype, it suppressed vrp1 mutation phenotypes, including temperature-sensitive growth, abnormally large cell morphology, defects in endocytosis and salt-sensitive growth. These results suggest that Mti1p and Vrp1p antagonistically regulate type I myosin functions. PMID: 11901111 [PubMed - in process] 3: J Mol Biol 2002 Mar 8;316(5):1071-81 Mechanistic Implications for Escherichia coli Cofactor-dependent Phosphoglycerate Mutase Based on the High-resolution Crystal Structure of a Vanadate Complex. Bond CS, White MF, Hunter WN. Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, University of Dundee, Dundee, DD1 5EH, UK The structure of Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), complexed with the potent inhibitor vanadate, has been determined to a resolution of 1.30A (R-factor 0.159; R-free 0.213). The inhibitor is present in the active site, principally as divanadate, but with evidence of additional vanadate moieties at either end, and representing a different binding mode to that observed in the structural homologue prostatic acid phosphatase. The analysis reveals the enzyme-ligand interactions involved in inhibition of the mutase activity by vanadate and identifies a water molecule, observed in the native E.coli dPGM structure which, once activated by vanadate, may dephosphorylate the active protein. Rather than reflecting the active conformation previously observed for E.coli dPGM, the inhibited protein's conformation resembles that of the inactive dephosphorylated Saccharomyces cerevisiae dPGM. The provision of a high-resolution structure of both active and inactive forms of dPGM from a single organism, in conjunction with computational modelling of substrate molecules in the active site provides insight into the binding of substrates and the specific interactions necessary for three different activities, mutase, synthase and phosphatase, within a single active site. The sequence similarity of E.coli and human dPGMs allows us to correlate stucture with clinical pathology. Copyright 2002 Elsevier Science Ltd. PMID: 11884145 [PubMed - in process] 4: J Mol Biol 2002 Mar 1;316(4):955-68 Implications for the Ubiquitination Reaction of the Anaphase-promoting Complex from the Crystal Structure of the Doc1/Apc10 Subunit. Au SW, Leng X, Harper JW, Barford D. Section of Structural Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK The anaphase-promoting complex (APC) is a multi-subunit E3 protein ubiquitin ligase that is responsible for the metaphase to anaphase transition and the exit from mitosis. One of the subunits of the APC that is required for its ubiquitination activity is Doc1/Apc10, a protein composed of a Doc1 homology domain that has been identified in a number of diverse putative E3 ubiquitin ligases. Here, we present the crystal structure of Saccharomyces cerevisiae Doc1/Apc10 at 2.2A resolution. The Doc1 homology domain forms a beta-sandwich structure that is related in architecture to the galactose-binding domain of galactose oxidase, the coagulation factor C2 domain and a domain of XRCC1. Residues that are invariant amongst Doc1/Apc10 sequences, including a temperature-sensitive mitotic arrest mutant, map to a beta-sheet region of the molecule, whose counterpart in galactose oxidase, the coagulation factor C2 domains and XRCC1, mediate bio-molecular interactions. This finding suggests the identification of the functionally important and conserved region of Doc1/Apc10 and, since invariant residues of Doc1/Apc10 colocalise with conserved residues of other Doc1 homology domains, we propose that the Doc1 homology domains perform common ubiquitination functions in the APC and other E3 ubiquitin ligases. Copyright 2002 Elsevier Science Ltd. PMID: 11884135 [PubMed - in process] 5: Proc Natl Acad Sci U S A 2002 Mar 5;99(5):2684-9 Interactions among the protein and RNA subunits of Saccharomyces cerevisiae nuclear RNase P. Houser-Scott F, Xiao S, Millikin CE, Zengel JM, Lindahl L, Engelke DR. Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606; and Department of Biological Sciences, University of Maryland--Baltimore County, Baltimore, MD 21250. Ribonuclease P (RNase P) is a ubiquitous endoribonuclease that cleaves precursor tRNAs to generate mature 5prime prime or minute termini. Although RNase P from all kingdoms of life have been found to have essential RNA subunits, the number and size of the protein subunits ranges from one small protein in bacteria to at least nine proteins of up to 100 kDa. In Saccharomyces cerevisiae nuclear RNase P, the enzyme is composed of ten subunits: a single RNA and nine essential proteins. The spatial organization of these components within the enzyme is not yet understood. In this study we examine the likely binary protein--protein and protein--RNA subunit interactions by using directed two- and three-hybrid tests in yeast. Only two protein subunits, Pop1p and Pop4p, specifically bind the RNA subunit. Pop4p also interacted with seven of the other eight protein subunits. The remaining protein subunits all showed one or more specific protein--protein interactions with the other integral protein subunits. Of particular interest was the behavior of Rpr2p, the only protein subunit found in RNase P but not in the closely related enzyme, RNase MRP. Rpr2p interacts strongly with itself as well as with Pop4p. Similar interactions with self and Pop4p were also detected for Snm1p, the only unique protein subunit so far identified in RNase MRP. This observation is consistent with Snm1p and Rpr2p serving analogous functions in the two enzymes. This study provides a low-resolution map of the multisubunit architecture of the ribonucleoprotein enzyme, nuclear RNase P from S. cerevisiae. PMID: 11880623 [PubMed - in process] 6: Mol Cell Biol 2002 Mar;22(6):1615-25 Transcription activator interactions with multiple SWI/SNF subunits. Neely KE, Hassan AH, Brown CE, Howe L, Workman JL. Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The Pennsylvania State University, University Park, PA 16802, USA. We have previously shown that the yeast SWI/SNF complex stimulates in vitro transcription from chromatin templates in an ATP-dependent manner. SWI/SNF function in this regard requires the presence of an activator with which it can interact directly, linking activator recruitment of SWI/SNF to transcriptional stimulation. In this study, we determine the SWI/SNF subunits that mediate its interaction with activators. Using a photo-cross-linking label transfer strategy, we show that the Snf5, Swi1, and Swi2/Snf2 subunits are contacted by the yeast acidic activators, Gcn4 and Hap4, in the context of the intact native SWI/SNF complex. In addition, we show that the same three subunits can interact individually with acidic activation domains, indicating that each subunit contributes to binding activators. Furthermore, mutations that reduce the activation potential of these activators also diminish its interaction with each of these SWI/SNF subunits. Thus, three distinct subunits of the SWI/SNF complex contribute to its interactions with activation domains. PMID: 11865042 [PubMed - indexed for MEDLINE] 7: J Mol Biol 2001 Nov 30;314(3):563-75 Solution structures of two FHA1-phosphothreonine peptide complexes provide insight into the structural basis of the ligand specificity of FHA1 from yeast Rad53. Yuan C, Yongkiettrakul S, Byeon IJ, Zhou S, Tsai MD. Department of Chemistry, The Ohio State University, Columbus OH 43210, USA. Rad53, a yeast checkpoint protein involved in regulating the repair of DNA damage, contains two forkhead-associated domains, FHA1 and FHA2. Previous combinatorial library screening has shown that FHA1 strongly selects peptides containing a pTXXD motif. Subsequent location of this motif within the sequence of Rad9, the target protein, coupled with spectroscopic analysis has led to identification of a tight binding sequence that is likely the binding site of FHA1: (188)SLEV(pT)EADATFVQ(200). We present solution structures of FHA1 in complex with this pT-peptide and with another Rad9-derived pT-peptide that has ca 30-fold lower affinity, (148)KKMTFQ(pT)PTDPLE(160). Both complexes showed intermolecular NOEs predominantly between three peptide residues (pT, +1, and +2 residues) and five FHA1 residues (S82, R83, S85, T106, and N107). Furthermore, the following interactions were implicated on the basis of chemical shift perturbations and structural analysis: the phosphate group of the pT residue with the side-chain amide group of N86 and the guanidino group of R70, and the carboxylate group of Asp (at the +3 position) with the guanidino group of R83. The generated structures revealed a similar binding mode adopted by these two peptides, suggesting that pT and the +3 residue Asp are the major contributors to binding affinity and specificity, while +1 and +2 residues could provide additional fine-tuning. It was also shown that FHA1 does not bind to the corresponding pS-peptides or a related pY-peptide. We suggest that differentiation between pT and pS-peptides by FHA1 can be attributed to hydrophobic interactions between the methyl group of the pT residue and the aliphatic protons of R83, S85, and T106 from FHA1. Copyright 2001 Academic Press. PMID: 11846567 [PubMed - indexed for MEDLINE] 8: Nucleic Acids Res 2002 Feb 15;30(4):1029-37 Functional and physical interactions between components of the Prp19p-associated complex. Chen CH, Yu WC, Tsao TY, Wang LY, Chen HR, Lin JY, Tsai WY, Cheng SC. Institute of Molecular Biology, Academia Sinica, Nankang, Taiwan, Republic of China. The Prp19p-associated complex is essential for the yeast pre-mRNA splicing reaction. The complex consists of at least eight protein components, but is not tightly associated with spliceosomal snRNAs. By a combination of genetic and biochemical methods we previously identified four components of this complex, Ntc25p, Ntc85p, Ntc30p and Ntc20p, all of them being novel splicing factors. We have now identified three other components of the complex, Ntc90p, Ntc77p and Ntc31p. These three proteins were also associated with the spliceosome during the splicing reaction in the same manner as Prp19p, concurrently with or immediately after dissociation of U4 snRNA. Two-hybrid analysis revealed that none of these proteins interacted with Prp19p or Ntc25p, but all interacted with Ntc85p. An interaction network between the identified components of the Prp19p-associated complex is demonstrated. Biochemical analysis revealed that Ntc90p, Ntc31p, Ntc30p and Ntc20p form a subcomplex, which, through interacting with Ntc85p and Ntc77p, can associate with Prp19p and Ntc25p to form the Prp19p-associated complex. Genetic analysis suggests that Ntc31p, Ntc30p and Ntc20p may play roles in modulating the function of Ntc90p. PMID: 11842115 [PubMed - indexed for MEDLINE] 9: Biochemistry 2002 Feb 19;41(7):2409-20 Novel interactions of Saccharomyces cerevisiae type 1 protein phosphatase identified by single-step affinity purification and mass spectrometry. Walsh EP, Lamont DJ, Beattie KA, Stark MJ. School of Life Sciences Biocentre, University of Dundee, Dundee DD1 5EH, UK. The catalytic subunit of Saccharomyces cerevisiae type 1 protein phosphatase (PP1(C)) is encoded by the essential gene GLC7 and is involved in regulating diverse cellular processes. To identify potential regulatory or targeting subunits of yeast PP1(C), we tagged Glc7p at its amino terminus with protein A and affinity-purified Glc7p protein complexes from yeast. The purified proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by peptide mass fingerprint analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. To confirm the accuracy of our identifications, peptides from some of the proteins were also sequenced using high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry. Only four of the Glc7p-associated proteins that we identified (Mhp1p, Bni4p, Ref2p, and Sds22p) have previously been shown to interact with Glc7p, and multiple components of the CPF (cleavage and polyadenylation factor) complex involved in messenger RNA 3'-end processing were present as major components in the Glc7p-associated protein fraction. To confirm the interaction of Glc7p with this complex, we used the same approach to purify and characterize the components of the yeast CPF complex using protein A-tagged Pta1p. Six known components of the yeast (CPF) complex, together with Glc7p, were identified among the Pta1p-associated polypeptides using peptide mass fingerprint analysis. Thus Glc7p is a novel component of the CPF complex and may therefore be involved regulating mRNA 3'-end processing. PMID: 11841235 [PubMed - in process] 10: Biotechnol Prog 2002 Jan-Feb;18(1):116-23 Recovery of recombinant cutinase using detergent foam. Fernandes S, Mattiasson B, Hatti-Kaul R. Department of Biotechnology, Center for Chemistry & Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden. Foam generated by vigorous stirring of a nonionic detergent, Triton X-114, was used for the recovery of recombinant cutinase expressed by Saccharomyces cerevisiae. The enzyme with a hydrophobic fusion tag, (Trp-Pro)(4), was recovered with a higher yield as compared to the wild-type cutinase, indicating the involvement of hydrophobic interactions in protein isolation with the foam. The influence of various factors including volume, dilution, pH, different additives, and cell concentration in the medium on enzyme recovery was investigated. Interaction of the enzyme with detergent was monitored using fluorescence spectroscopy. No significant changes in protein conformation after the isolation procedure were observed using circular dichroism. PMID: 11822909 [PubMed - in process] 11: Proc Natl Acad Sci U S A 2002 Feb 5;99(3):1253-8 Probing protein conformational changes in living cells by using designer binding proteins: application to the estrogen receptor. Koide A, Abbatiello S, Rothgery L, Koide S. Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA. A challenge in understanding the mechanism of protein function in biology is to establish the correlation between functional form in the intracellular environment and high-resolution structures obtained with in vitro techniques. Here we present a strategy to probe conformational changes of proteins inside cells. Our method involves: (i) engineering binding proteins to different conformations of a target protein, and (ii) using them to sense changes in the surface property of the target in cells. We probed ligand-induced conformational changes of the estrogen receptor alpha (ER alpha) ligand-binding domain (LBD). By using yeast two-hybrid techniques, we first performed combinatorial library screening of "monobodies" (small antibody mimics using the scaffold of a fibronectin type III domain) for clones that bind to ER alpha and then characterized their interactions with ER alpha in the nucleus, the native environment of ER alpha, in the presence of various ligands. A library using a highly flexible loop yielded monobodies that specifically recognize a particular ligand complex of ER alpha, and the pattern of monobody specificity was consistent with the structural differences found in known crystal structures of ER alpha-LBD. A more restrained loop library yielded clones that bind both agonist- and antagonist-bound ER alpha. Furthermore, we found that a deletion of the ER alpha F domain that is C-terminally adjacent to the LBD increased the crossreactivity of monobodies to the apo-ER alpha-LBD, suggesting a dynamic nature of the ER alpha-LBD conformation and a role of the F domain in restraining the LBD in an inactive conformation. PMID: 11818562 [PubMed - indexed for MEDLINE] 12: J Mol Biol 2002 Jan 25;315(4):809-18 Protein-protein interactions of hCsl4p with other human exosome subunits. Raijmakers R, Noordman YE, van Venrooij WJ, Pruijn GJ. Department of Biochemistry, University of Nijmegen, Nijmegen, The Netherlands. The exosome is a complex of 3'-->5' exoribonucleases, which functions in a variety of cellular processes, all requiring the processing or degradation of RNA. We demonstrate that the two human proteins hCsl4p and hRrp42p, which have been identified on the basis of their sequence homology with Saccharomyces cerevisiae proteins, are associated with the human exosome. By mammalian two-hybrid and GST pull-down assays, we show that the hCsl4p protein interacts directly with two other exosome proteins, hRrp42p and hRrp46p. Mutants of hCsl4p that fail to interact with either hRrp42p or hRrp46p are also not able to associate with exosome complexes in vivo. These results indicate that the association of hCsl4p with the exosome is mediated by protein-protein interactions with hRrp42p and hRrp46p. Copyright 2002 Academic Press. PMID: 11812149 [PubMed - indexed for MEDLINE] 13: Nature 2002 Jan 10;415(6868):180-3 Comment in: Nature. 2002 Jan 10;415(6868):123-4. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L, Adams SL, Millar A, Taylor P, Bennett K, Boutilier K, Yang L, Wolting C, Donaldson I, Schandorff S, Shewnarane J, Vo M, Taggart J, Goudreault M, Muskat B, Alfarano C, Dewar D, Lin Z, Michalickova K, Willems AR, Sassi H, Nielsen PA, Rasmussen KJ, Andersen JR, Johansen LE, Hansen LH, Jespersen H, Podtelejnikov A, Nielsen E, Crawford J, Poulsen V, Sorensen BD, Matthiesen J, Hendrickson RC, Gleeson F, Pawson T, Moran MF, Durocher D, Mann M, Hogue CW, Figeys D, Tyers M. MDS Proteomics, 251 Attwell Drive, Toronto, Canada M9W 7H4, and Staermosegaardsvej 6, DK-5230 Odense M, Denmark. The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks. PMID: 11805837 [PubMed - indexed for MEDLINE] 14: Nature 2002 Jan 10;415(6868):141-7 Comment in: Nature. 2002 Jan 10;415(6868):123-4. Functional organization of the yeast proteome by systematic analysis of protein complexes. Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, Bauer A, Schultz J, Rick JM, Michon AM, Cruciat CM, Remor M, Hofert C, Schelder M, Brajenovic M, Ruffner H, Merino A, Klein K, Hudak M, Dickson D, Rudi T, Gnau V, Bauch A, Bastuck S, Huhse B, Leutwein C, Heurtier MA, Copley RR, Edelmann A, Querfurth E, Rybin V, Drewes G, Raida M, Bouwmeester T, Bork P, Seraphin B, Kuster B, Neubauer G, Superti-Furga G. Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany. anne-claude.gavin@cellzome.com Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery. PMID: 11805826 [PubMed - indexed for MEDLINE] 15: Mol Cell 2002 Jan;9(1):31-44 Comment in: Mol Cell. 2002 Jan;9(1):8-9. Composition and functional characterization of the yeast spliceosomal penta-snRNP. Stevens SW, Ryan DE, Ge HY, Moore RE, Young MK, Lee TD, Abelson J. California Institute of Technology, Division of Biology 147-75, Pasadena, CA 91125, USA. Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs. PMID: 11804584 [PubMed - indexed for MEDLINE] 16: J Cell Sci 2002 Jan 1;115(Pt 1):195-206 Essential functions of Sds22p in chromosome stability and nuclear localization of PP1. Peggie MW, MacKelvie SH, Bloecher A, Knatko EV, Tatchell K, Stark MJ. Division of Gene Regulation and Expression, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, UK. Sds22p is a conserved, leucine-rich repeat protein that interacts with the catalytic subunit of protein phosphatase 1 (PP1(C)) and which has been proposed to regulate one or more functions of PP1(C) during mitosis. Here we show that Saccharomyces cerevisiae Sds22p is a largely nuclear protein, most of which is present as a sTable 1:1 complex with yeast PP1(C) (Glc7p). Temperature-sensitive (Ts(-)) S. cerevisiae sds22 mutants show profound chromosome instability at elevated growth temperatures but do not confer a cell cycle stage-specific arrest. In the sds22-6 Ts(-) mutant, nuclear Glc7p is both reduced in level and aberrantly localized at 37 degrees C and the interaction between Glc7p and Sds22p in vitro is reduced at higher temperatures, consistent with the in vivo Ts(-) growth defect. Like some glc7 mutations, sds22-6 can suppress the Ts(-) growth defect associated with ipl1-2, a loss of function mutation in a protein kinase that is known to work in opposition to PP1 on at least two nuclear substrates. This, together with reciprocal genetic interactions between GLC7 and SDS22, suggests that Sds22p functions positively with Glc7p to promote dephosphorylation of nuclear substrates required for faithful transmission of chromosomes during mitosis, and this role is at least partly mediated by effects of Sds22p on the nuclear distribution of Glc7p PMID: 11801737 [PubMed - in process] 17: Genome Inform Ser Workshop Genome Inform 2001;12:123-34 The potential use of SUISEKI as a protein interaction discovery tool. Blaschke C, Valencia A. Protein Design Group, CNB/CSIC, Campus Universidad Autonoma, 28049 Madrid, Spain. blaschke@cnb.uam.es Relevant information about protein interactions is stored in textual sources. This sources are commonly used not only as archives of what is already known but also as information for generating new knowledge, particularly to pose hypothesis about new possible interactions that can be inferred from the existing ones. This task is the more creative part of scientific work in experimental systems. We present a large-scale analysis for the prediction of new interactions based on the interaction network for the ones already known and detected automatically in the literature. During the last few years it has became clear that part of the information about protein interactions could be extracted with automatic tools, even if these tools are still far from perfect and key problems such as detection of protein names are not completely solved. We have developed a integrated automatic approach, called SUISEKI (System for Information Extraction on Interactions), able to extract protein interactions from collections of Medline abstracts. Previous experiments with the system have shown that it is able to extract almost 70% of the interactions present in relatively large text corpus, with an accuracy of approximately 80% (for the best defined interactions) that makes the system usable in real scenarios, both at the level of extraction of protein names and at the level of extracting interaction between them. With the analysis of the interaction map of Saccharomyces cerevisiae we show that interactions published in the years 2000/2001 frequently correspond to proteins or genes that were already very close in the interaction network deduced from the literature published before these years and that they are often connected to the same proteins. That is, discoveries are commonly done among highly connected entities. Some biologically relevant examples illustrate how interactions described in the year 2000 could have been proposed as reasonable working hypothesis with the information previously available in the automatically extracted network of interactions. PMID: 11791231 [PubMed - in process] 18: Biochem Biophys Res Commun 2002 Jan 18;290(2):676-81 Saccharomyces cerevisiae Pra1p/Yip3p interacts with Yip1p and Rab proteins. Calero M, Collins RN. Department of Molecular Medicine, Cornell University, Ithaca, New York 14853-6401, USA. The regulation of membrane traffic involves the Rab family of Ras-related GTPases, of which there are a total of 11 members in the yeast Saccharomyces cerevisiae. Previous work has identified PRA1 as a dual prenylated Rab GTPase and VAMP2 interacting protein [Martinic et al. (1999) J. Biol. Chem. 272, 26991-26998]. In this study we demonstrate that the yeast counterpart of PRA1 interacts with Rab proteins and with Yip1p, a membrane protein of unknown function that has been reported to interact specifically with the Rab proteins Ypt1p and Ypt31p. Yeast Pra1p/Yip3p is a factor capable of biochemical interaction with a panel of different Rab proteins and does not show in vitro specificity for any particular Rab. The interactions between Pra1p/Yip3p and Rab proteins are dependent on the presence of the Rab protein C-terminal cysteines and require C-terminal prenylation. PMID: 11785952 [PubMed - indexed for MEDLINE] 19: Plant Mol Biol 2001 Dec;47(6):771-83 Identification of a S-ribonuclease-binding protein in Petunia hybrida. Sims TL, Ordanic M. Department of Biological Sciences and Plant Molecular Biology Center, Northern Illinois University, DeKalb, 60115-2861, USA. tsmis@niu.edu To investigate protein-protein interactions in gametophytic self-incompatibility, we used a yeast two-hybrid assay to identify proteins that could interact with the S-ribonuclease protein. These assays identified a pollen-expressed protein, which we have named PhSBP1, that appears to bind with a high degree of specificity to the Petunia hybrida S-ribonuclease. Although PhSBP1 activates reporter gene expression only when expressed in tandem with a S-RNAse bait protein, binding is not allele-specific. Sequence analysis demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that includes a RING-HC domain. Because many RING-finger domain proteins appear to function as E3 ubiquitin ligases, our results suggest that ubiquitination and protein degradation may play a role in regulating self-incompatibility interactions. Together, these results suggest that PhSBPI may be a candidate for the recently proposed general inhibitor (RI) of self-incompatibility ribonucleases. PMID: 11785938 [PubMed - indexed for MEDLINE] 20: Mol Cell Biol 2002 Feb;22(3):927-34 The Sur7p family defines novel cortical domains in Saccharomyces cerevisiae, affects sphingolipid metabolism, and is involved in sporulation. Young ME, Karpova TS, Brugger B, Moschenross DM, Wang GK, Schneiter R, Wieland FT, Cooper JA. Department of Cell Biology and Physiology, Washington University, St. Louis, Missouri 63110, USA. We have discovered a novel cortical patch structure in Saccharomyces cerevisiae defined by a family of integral plasma membrane proteins, including Sur7p, Ynl194p, and Ydl222p. Sur7p-family patches localized as cortical patches that were immobile and stable. These patches were polarized to regions of the cell with a mature cell wall; they were absent from small buds and the tips of many medium-sized buds. These patches were distinct from other known cortical structures. Digestion of the cell wall caused Sur7p patches to disassemble, indicating that Sur7p requires cell wall-dependent extracellular interactions for its localization as patches. sur7Delta, ydl222Delta, and ynl194Delta mutants had reduced sporulation efficiencies. SUR7 was originally described as a multicopy suppressor of rvs167, whose product is an actin patch component. This suppression is probably mediated by sphingolipids, since deletion of SUR7, YDL222, and YNL194 altered the sphingolipid content of the yeast plasma membrane, and other SUR genes suppress rvs167 via effects on sphingolipid synthesis. In particular, the sphingoid base length and number of hydroxyl groups in inositol phosphorylceramides were altered in sur7Delta, ydl222Delta, and yne194Delta strains. PMID: 11784867 [PubMed - indexed for MEDLINE] 21: Mol Cell Biol 2002 Feb;22(3):693-703 Histone-dependent association of Tup1-Ssn6 with repressed genes in vivo. Davie JK, Trumbly RJ, Dent SY. Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. The Tup1-Ssn6 complex regulates diverse classes of genes in Saccharomyces cerevisiae and serves as a model for corepressor functions in many organisms. Tup1-Ssn6 does not directly bind DNA but is brought to target genes through interactions with sequence-specific DNA binding factors. Full repression by Tup1-Ssn6 appears to require interactions with both the histone tails and components of the general transcription machinery, although the relative contribution of these two pathways is not clear. Here, we map Tup1 locations on two classes of Tup1-Ssn6-regulated genes in vivo via chromatin immunoprecipitations. Distinct profiles of Tup1 are observed on a cell-specific genes and DNA damage-inducible genes, suggesting that alternate repressive architectures may be created on different classes of repressed genes. In both cases, decreases in acetylation of histone H3 colocalize with Tup1. Strikingly, although loss of the Srb10 mediator protein had no effect on Tup1 localization, both histone tail mutations and histone deacetylase mutations crippled the association of Tup1 with target loci. Together with previous findings that Tup1-Ssn6 physically associates with histone deacetylase activities, these results indicate that the repressor complex alters histone modification states to facilitate interactions with histones and that these interactions are required to maintain a stable repressive state. PMID: 11784848 [PubMed - indexed for MEDLINE] 22: RNA 2001 Dec;7(12):1693-701 A genome-wide survey of RS domain proteins. Boucher L, Ouzounis CA, Enright AJ, Blencowe BJ. Banting and Best Department of Medical Research, C.H. Best Institute, University of Toronto, Ontario, Canada. Domains rich in alternating arginine and serine residues (RS domains) are frequently found in metazoan proteins involved in pre-mRNA splicing. The RS domains of splicing factors associate with each other and are important for the formation of protein-protein interactions required for both constitutive and regulated splicing. The prevalence of the RS domain in splicing factors suggests that it might serve as a useful signature for the identification of new proteins that function in pre-mRNA processing, although it remains to be determined whether RS domains also participate in other cellular functions. Using database search and sequence clustering methods, we have identified and categorized RS domain proteins encoded within the entire genomes of Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae. This genome-wide survey revealed a surprising complexity of RS domain proteins in metazoans with functions associated with chromatin structure, transcription by RNA polymerase II, cell cycle, and cell structure, as well as pre-mRNA processing. Also identified were RS domain proteins in S. cerevisiae with functions associated with cell structure, osmotic regulation, and cell cycle progression. The results thus demonstrate an effective strategy for the genomic mining of RS domain proteins. The identification of many new proteins using this strategy has provided a database of factors that are candidates for forming RS domain-mediated interactions associated with different steps in pre-mRNA processing, in addition to other cellular functions. PMID: 11780626 [PubMed - indexed for MEDLINE] 23: Genetics 2001 Dec;159(4):1539-45 (CA/TG) microsatellite sequences escape the inhibition of recombination by mismatch repair in Saccharomyces cerevisiae. Gendrel CG, Dutreix M. UMR-CNRS 2027, Institut Curie-section de Recherche, Universite Paris-Sud, F-91405 Orsay, France. Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system. In the yeast Saccharomyces cerevisiae, repair of mismatches results in gene conversion or restoration, whereas failure to repair mismatches results in postmeiotic segregation (PMS). By examining the conversion and PMS in yeast strains deficient in various MMR genes and heterozygous for large inserts (107 bp) with either a mixed sequence or a 39 (CA/TG) repetitive microsatellite sequence, we demonstrate that: (1) the inhibition of conversion by large inserts depends upon a complex containing both Msh2 and Pms1 proteins; (2) conversion is not inhibited if the single-stranded DNA loop in the heteroduplex is the microsatellite sequence; and (3) large heteroduplex loops with random sequence or repetitive sequence might be repaired by two complexes, containing either Msh2 or Pms1. Our results suggest that inhibition of recombination by heterologous inserts and large loop repair are not processed by the same MMR complexes. We propose that the inhibition of conversion by large inserts is due to recognition by the Msh2/Pms1 complex of mismatches created by intrastrand interactions in the heteroduplex loop. PMID: 11779795 [PubMed - indexed for MEDLINE] 24: Genetics 2001 Dec;159(4):1435-48 Overactivation of the protein kinase C-signaling pathway suppresses the defects of cells lacking the Rho3/Rho4-GAP Rgd1p in Saccharomyces cerevisiae. de Bettignies G, Thoraval D, Morel C, Peypouquet MF, Crouzet M. Laboratoire de Biologie Moleculaire et de Sequencage, UMR CNRS 5095, Bordeaux Cedex, France. The nonessential RGD1 gene encodes a Rho-GTPase activating protein for the Rho3 and Rho4 proteins in Saccharomyces cerevisiae. Previous studies have revealed genetic interactions between RGD1 and the SLG1 and MID2 genes, encoding two putative sensors for cell integrity signaling, and VRP1 encoding an actin and myosin interacting protein involved in polarized growth. To better understand the role of Rgd1p, we isolated multicopy suppressor genes of the cell lethality of the double mutant rgd1Delta mid2Delta. RHO1 and RHO2 encoding two small GTPases, MKK1 encoding one of the MAP-kinase kinases in the protein kinase C (PKC) pathway, and MTL1, a MID2-homolog, were shown to suppress the rgd1Delta defects strengthening the functional links between RGD1 and the cell integrity pathway. Study of the transcriptional activity of Rlm1p, which is under the control of Mpk1p, the last kinase of the PKC pathway, and follow-up of the PST1 transcription, which is positively regulated by Rlm1p, indicate that the lack of RGD1 function diminishes the PKC pathway activity. We hypothesize that the rgd1Delta inactivation, at least through the hyperactivation of the small GTPases Rho3p and Rho4p, alters the secretory pathway and/or the actin cytoskeleton and decreases activity of the PKC pathway. PMID: 11779787 [PubMed - indexed for MEDLINE] 25: Invest Ophthalmol Vis Sci 2002 Jan;43(1):176-82 Protein interactions with myocilin. Wentz-Hunter K, Ueda J, Yue BY. Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612, USA. PURPOSE: To identify factors that interact in vivo with myocilin, a glaucoma gene product. METHODS: The yeast two-hybrid system with myocilin as the bait and a human skeletal muscle cDNA library as the prey was used to identify potential factors that interact with myocilin. Interactions were also examined in bovine trabecular meshwork (TM) cells through a mammalian two-hybrid system. Biochemical coimmunoprecipitation from both human TM cell lysate and in vitro translated proteins was also used to confirm results obtained from yeast analysis. RESULTS: Twenty positive clones isolated through yeast two-hybrid screening were deemed potential myocilin partners. Sequence analysis determined that two of them encoded for myocilin from amino acids 64 to 268. Myocilin was also found to interact with a component of the myosin motor protein, myosin regulatory light chain (RLC). The myocilin-myocilin and myocilin-RLC interactions revealed by the yeast system were further confirmed and demonstrated in cultured TM cells, by means of a mammalian two-hybrid system, and through biochemical coimmunoprecipitation, subcellular fractionation, immunofluorescence, and immunogold double labeling. CONCLUSIONS: These results indicate that myocilin can form homomultimers in vivo, independent of the olfactomedin-like domain. Further analysis established that the leucine zipper motif of myocilin may be necessary for the myocilin-RLC interaction. The interaction of myocilin with RLC, a component of the myosin motor protein complex, implies a role for myocilin in the actomyosin system, linking in turn this novel protein to functional status of the TM. PMID: 11773029 [PubMed - indexed for MEDLINE] 26: Biochem J 2002 Jan 15;361(Pt 2):243-54 Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho GDP dissociation inhibitor and their extraction from biological membranes. Forget MA, Desrosiers RR, Gingras D, Beliveau R. Laboratoire de medecine moleculaire, Hopital Sainte-Justine-Universite du Quebec a Montreal, P.O. Box 8888, Centre-ville station, Montreal, Quebec, Canada H3C 3P8. The Rho GDP dissociation inhibitor (RhoGDI) regulates the activation-inactivation cycle of Rho small GTPases, such as Cdc42 and RhoA, by extracting them from the membrane. To study the roles of Mg(2+), phosphatidylinositol 4,5-bisphosphate (PIP(2)), ionic strength and phosphorylation on the interactions of RhoGDI with Cdc42 and RhoA, we developed a new, efficient and reliable method to produce prenylated Rho proteins using the yeast Saccharomyces cerevisiae. It has been previously reported that protein kinase A (PKA)-treatment of isolated membranes increased RhoA extraction from membranes by RhoGDI [Lang, Gesbert, Delespine-Carmagnat, Stancou, Pouchelet and Bertoglio (1996) EMBO J. 16, 510-519]. In the present study, we used an in vitro affinity chromatography system to show that phosphorylation of RhoA and Cdc42 significantly increased their interaction with RhoGDI under physiological conditions of ionic strength. This increase was independent of the nucleotide (GDP or guanosine 5'-[gamma-thio]triphosphate) loaded on to the Rho proteins, as well as of Mg(2+) and PIP(2). Moreover, dephosphorylation of rat brain membranes by alkaline phosphatase significantly decreased the extraction of RhoA and Cdc42 by RhoGDI. Subsequent re-phosphorylation by PKA restored the extraction levels, indicating the reversibility of this process. These results clearly demonstrate that the phosphorylation states of Cdc42 and RhoA regulate their interactions with RhoGDI and, consequently, their extraction from rat brain membranes. We therefore suggest that phosphorylation is a mechanism of regulation of Cdc42 and RhoA activity that is independent of GDP-GTP cycling. PMID: 11772396 [PubMed - indexed for MEDLINE] 27: J Biol Chem 2002 Feb 8;277(6):3813-22 Active site mutations in DNA topoisomerase I distinguish the cytotoxic activities of camptothecin and the indolocarbazole, rebeccamycin. Woo MH, Vance JR, Marcos AR, Bailly C, Bjornsti MA. Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development. However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast. Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA complexes. Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722. The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1delta, rad52delta yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular target of R-3 and indicate that distinct drug-enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p. PMID: 11733535 [PubMed - indexed for MEDLINE] 28: Biochemistry 2001 Dec 25;40(51):15562-9 Identification of yeast cofilin residues specific for actin monomer and PIP2 binding. Ojala PJ, Paavilainen V, Lappalainen P. Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland. Cofilin/ADF is a ubiquitous actin-binding protein that is important for rapid actin dynamics in vivo. The long alpha-helix (helix 3 in yeast cofilin) forms the most highly conserved region in cofilin/ADF proteins, and residues in the NH2-terminal half of this alpha-helix have been shown to be essential for actin binding in cofilin/ADF. Recent studies also suggested that the basic residues in the COOH-terminal half of this alpha-helix would play an important role in F-actin binding. In contrast to these studies, we show here that the charged residues in the COOH-terminal half of helix 3 are not important for actin filament binding in yeast cofilin. Mutations in these residues, however, result in a small defect in actin monomer interactions. We also show that yeast cofilin can differentiate between various phosphatidylinositides, and mapped the PI(4,5)P2 binding site by using a collection of cofilin mutants. The PI(4,5)P2 binding site of yeast cofilin is a large positively charged surface that consists of residues in helix 3 as well as residues in other parts of the cofilin molecule. This suggests that cofilin/ADF proteins probably interact simultaneously with more than one PI(4,5)P2 molecule. The PI(4,5)P2-binding site overlaps with areas that are important for F-actin binding, explaining why the actin-related activities of cofilin/ADF are inhibited by PI(4,5)P2. The biological roles of actin and PI(4,5)P2 interactions of cofilin are discussed in light of phenotypes of specific yeast strains carrying mutations in residues that are important for actin and PI(4,5)P2 binding. PMID: 11747431 [PubMed - indexed for MEDLINE] 29: Biochem J 2002 Jan 1;361(Pt 1):27-34 Direct interactions between molecular chaperones heat-shock protein (Hsp) 70 and Hsp40: yeast Hsp70 Ssa1 binds the extreme C-terminal region of yeast Hsp40 Sis1. Qian X, Hou W, Zhengang L, Sha B. School of Life Sciences, University of Science and Technology of China, Hefei, People's Republic of China 230026. Heat-shock protein 40 (Hsp40) enables Hsp70 to play critical roles in a number of cellular processes, such as protein folding, assembly, degradation and translocation in vivo. Hsp40 recognizes and binds non-native polypeptides and delivers them to Hsp70. Then Hsp40 stimulates the ATPase activity of Hsp70 to fold the polypeptides. By using yeast Hsp40 Sis1 and yeast Hsp70 Ssa1 as our model proteins, we found that the Sis1 peptide-binding fragment interacts directly with the full-length Ssa1 in vitro. Further studies showed that the C-terminal lid domain of Ssa1 could interact with Sis1 peptide-binding domain physically in vitro. The Sis1 peptide-binding fragment forms a stable complex with the Ssa1 C-terminal lid domain in solution. The interactions between these two proteins appear to be charge-charge interactions because high-ionic-strength buffer can dissociate the complex. Further mapping studies showed that the Sis1 peptide-binding fragment binds the extreme C-terminal 15 amino acid residues of Ssa1. A flexible glycine-rich region is followed by these 15 residues in the Ssa1 primary sequence. Atomic force microscopy of the Sis1-Ssa1 complex showed that only one end of the Ssa1 lid domain binds the Sis1 peptide-binding-fragment dimer at the upper level of the huge groove within the Sis1 dimer. Based on the data, we propose an "anchoring and docking" model to illustrate the mechanisms by which Hsp40 interacts with Hsp70 and delivers the non-native polypeptide to Hsp70. PMID: 11743879 [PubMed - indexed for MEDLINE] 30: Chem Res Toxicol 2001 Dec;14(12):1584-9 Mechanisms of nitrogen oxide-mediated disruption of metalloprotein function: an examination of the copper-responsive yeast transcription factor Ace1. Shinyashiki M, Pan CJ, Switzer CH, Fukuto JM. Department of Pharmacology, UCLA School of Medicine, Center for the Health Sciences, Los Angeles, California 90095-1735, USA. Nitric oxide (NO) has been found to inhibit the copper-responsive yeast transcription factor Ace1 in an oxygen-dependent manner. However, the mechanism responsible for NO-dependent inhibition of Ace1 remains unestablished. In the present study, the chemical interaction of nitrogen oxide species with Ace1 was examined using a yeast reporter system. Exposure of yeast to various nitrogen oxides, under a variety of conditions, revealed that the oxygen-dependent inhibition of Ace1 is due to the reaction of NO with O(2). The nitrosating nitrogen oxide species N(2)O(3) is likely to be the disrupter of Ace1 activity. Considering the similarity of metal-thiolate ligation in Ace1 with other mammalian metalloproteins such as metallothionein, metal chaperones, and zinc-finger proteins, these results help to understand the biochemical interactions of NO with those mammalian metalloproteins. PMID: 11743740 [PubMed - indexed for MEDLINE] 31: J Mol Biol 2001 Dec 14;314(5):1053-66 Beyond synexpression relationships: local clustering of time-shifted and inverted gene expression profiles identifies new, biologically relevant interactions. Qian J, Dolled-Filhart M, Lin J, Yu H, Gerstein M. Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, PO Box 208114, New Haven, CT 06520-8114, USA. The complexity of biological systems provides for a great diversity of relationships between genes. The current analysis of whole-genome expression data focuses on relationships based on global correlation over a whole time-course, identifying clusters of genes whose expression levels simultaneously rise and fall. There are, of course, other potential relationships between genes, which are missed by such global clustering. These include activation, where one expects a time-delay between related expression profiles, and inhibition, where one expects an inverted relationship. Here, we propose a new method, which we call local clustering, for identifying these time-delayed and inverted relationships. It is related to conventional gene-expression clustering in a fashion analogous to the way local sequence alignment (the Smith-Waterman algorithm) is derived from global alignment (Needleman-Wunsch). An integral part of our method is the use of random score distributions to assess the statistical significance of each cluster. We applied our method to the yeast cell-cycle expression dataset and were able to detect a considerable number of additional biological relationships between genes, beyond those resulting from conventional correlation. We related these new relationships between genes to their similarity in function (as determined from the MIPS scheme) or their having known protein-protein interactions (as determined from the large-scale two-hybrid experiment); we found that genes strongly related by local clustering were considerably more likely than random to have a known interaction or a similar cellular role. This suggests that local clustering may be useful in functional annotation of uncharacterized genes. We examined many of the new relationships in detail. Some of them were already well-documented examples of inhibition or activation, which provide corroboration for our results. For instance, we found an inverted expression profile relationship between genes YME1 and YNT20, where the latter has been experimentally documented as a bypass suppressor of the former. We also found new relationships involving uncharacterized yeast genes and were able to suggest functions for many of them. In particular, we found a time-delayed expression relationship between J0544 (which has not yet been functionally characterized) and four genes associated with the mitochondria. This suggests that J0544 may be involved in the control or activation of mitochondrial genes. We have also looked at other, less extensive datasets than the yeast cell-cycle and found further interesting relationships. Our clustering program and a detailed website of clustering results is available at http://www.bioinfo.mbb.yale.edu/expression/cluster (or http://www.genecensus.org/expression/cluster). Copyright 2001 Academic Press. PMID: 11743722 [PubMed - indexed for MEDLINE] 32: Science 2001 Dec 14;294(5550):2364-8 Systematic genetic analysis with ordered arrays of yeast deletion mutants. Tong AH, Evangelista M, Parsons AB, Xu H, Bader GD, Page N, Robinson M, Raghibizadeh S, Hogue CW, Bussey H, Andrews B, Tyers M, Boone C. Banting and Best Department of Medical Research, University of Toronto, Toronto ON, Canada M5G 1L6. In Saccharomyces cerevisiae, more than 80% of the approximately 6200 predicted genes are nonessential, implying that the genome is buffered from the phenotypic consequences of genetic perturbation. To evaluate function, we developed a method for systematic construction of double mutants, termed synthetic genetic array (SGA) analysis, in which a query mutation is crossed to an array of approximately 4700 deletion mutants. Inviable double-mutant meiotic progeny identify functional relationships between genes. SGA analysis of genes with roles in cytoskeletal organization (BNI1, ARP2, ARC40, BIM1), DNA synthesis and repair (SGS1, RAD27), or uncharacterized functions (BBC1, NBP2) generated a network of 291 interactions among 204 genes. Systematic application of this approach should produce a global map of gene function. PMID: 11743205 [PubMed - indexed for MEDLINE] 33: Science 2002 Jan 11;295(5553):321-4 Comment in: Science. 2002 Jan 11;295(5553):284-7. A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules. Tong AH, Drees B, Nardelli G, Bader GD, Brannetti B, Castagnoli L, Evangelista M, Ferracuti S, Nelson B, Paoluzi S, Quondam M, Zucconi A, Hogue CW, Fields S, Boone C, Cesareni G. Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5G 1L6. Peptide recognition modules mediate many protein-protein interactions critical for the assembly of macromolecular complexes. Complete genome sequences have revealed thousands of these domains, requiring improved methods for identifying their physiologically relevant binding partners. We have developed a strategy combining computational prediction of interactions from phage-display ligand consensus sequences with large-scale two-hybrid physical interaction tests. Application to yeast SH3 domains generated a phage-display network containing 394 interactions among 206 proteins and a two-hybrid network containing 233 interactions among 145 proteins. Graph theoretic analysis identified 59 highly likely interactions common to both networks. Las17 (Bee1), a member of the Wiskott-Aldrich Syndrome protein (WASP) family of actin-assembly proteins, showed multiple SH3 interactions, many of which were confirmed in vivo by coimmunoprecipitation. PMID: 11743162 [PubMed - indexed for MEDLINE] 34: EMBO J 2001 Dec 17;20(24):7096-107 Subunit interaction maps for the regulatory particle of the 26S proteasome and the COP9 signalosome. Fu H, Reis N, Lee Y, Glickman MH, Vierstra RD. Institute of Botany, Academia Sinica, 128, Sec 2, Academy Road, Taipei, Taiwan 115, Republic of China. hongyong@gate.sinica.edu.tw The 26S proteasome plays a major role in eukaryotic protein breakdown, especially for ubiquitin-tagged proteins. Substrate specificity is conferred by the regulatory particle (RP), which can dissociate into stable lid and base subcomplexes. To help define the molecular organization of the RP, we tested all possible paired interactions among subunits from Saccharomyces cerevisiae by yeast two-hybrid analysis. Within the base, a Rpt4/5/3/6 interaction cluster was evident. Within the lid, a structural cluster formed around Rpn5/11/9/8. Interactions were detected among synonymous subunits (Csn4/5/7/6) from the evolutionarily related COP9 signalosome (CSN) from Arabidopsis, implying a similar quaternary arrangement. No paired interactions were detected between lid, base or core particle subcomplexes, suggesting that stable contacts between them require prior assembly. Mutational analysis defined the ATPase, coiled-coil, PCI and MPN domains as important for RP assembly. A single residue in the vWA domain of Rpn10 is essential for amino acid analog resistance, for degrading a ubiquitin fusion degradation substrate and for stabilizing lid-base association. Comprehensive subunit interaction maps for the 26S proteasome and CSN support the ancestral relationship of these two complexes. PMID: 11742986 [PubMed - indexed for MEDLINE] 35: Mol Cell 2001 Nov;8(5):1075-83 Targeting an mRNA for decapping: displacement of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs. Tharun S, Parker R. Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of Arizona, 1007 E. Lowell, Tucson, AZ 85721, USA. tharun@u.arizona.edu The major pathway of eukaryotic mRNA decay involves deadenylation-dependent decapping followed by 5' to 3' exonucleolytic degradation. By examining interactions among mRNA decay factors, the mRNA, and key translation factors, we have identified a critical transition in mRNP organization that leads to decapping and degradation of yeast mRNAs. This transition occurs after deadenylation and includes loss of Pab1p, eIF4E, and eIF4G from the mRNA and association of the decapping activator complex, Lsm1p-7p, which enhances the coimmunoprecipitation of a decapping enzyme complex (Dcp1p and Dcp2p) with the mRNA. These results define an important rearrangement in mRNP organization and suggest that deadenylation promotes mRNA decapping by both the loss of Pab1p and the recruitment of the Lsm1p-7p complex. PMID: 11741542 [PubMed - indexed for MEDLINE] 36: Genome Res 2001 Dec;11(12):1971-3 Is there a bias in proteome research? Mrowka R, Patzak A, Herzel H. Johannes-Muller-Institut fur Physiologie, Humboldt-Universitat zu Berlin, Berlin, Germany. mrowka@rz.hu-berlin.de Advances in technology have enabled us to take a fresh look at data acquired by traditional single experiments and to compare them with genomewide data. The differences can be tremendous, as we show here, in the field of proteomics. We have compared data sets of protein-protein interactions in Saccharomyces cerevisiae that were detected by an identical underlying technical method, the yeast two-hybrid system. We found that the individually identified protein-protein interactions are considerably different from those identified by two genomewide scans. Interacting proteins in the pooled database from single publications are much more closely related to each other with respect to transcription profiles when compared to genomewide data. This difference may have been introduced by two factors: by a selection process in individual publications and by false positives in the whole-genome scans. If we assume that the differences are a result of false positives in the whole-genome data, the scans would contain 47%, 44%, and 91% of false positives for the UETZ, ITO-core, and ITO-full data, respectively. If, however, the true fraction of false positives is considerably lower than estimated here, the data from hypothesis-driven experiments must have been subjected to a serious selection process. PMID: 11731485 [PubMed - indexed for MEDLINE] 37: Microbiol Mol Biol Rev 2001 Dec;65(4):570-94, table of contents Transport into and out of the nucleus. Macara IG. Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908-0577, USA. imacara@virginia.edu A defining characteristic of eukaryotic cells is the possession of a nuclear envelope. Transport of macromolecules between the nuclear and cytoplasmic compartments occurs through nuclear pore complexes that span the double membrane of this envelope. The molecular basis for transport has been revealed only within the last few years. The transport mechanism lacks motors and pumps and instead operates by a process of facilitated diffusion of soluble carrier proteins, in which vectoriality is provided by compartment-specific assembly and disassembly of cargo-carrier complexes. The carriers recognize localization signals on the cargo and can bind to pore proteins. They also bind a small GTPase, Ran, whose GTP-bound form is predominantly nuclear. Ran-GTP dissociates import carriers from their cargo and promotes the assembly of export carriers with cargo. The ongoing discovery of numerous carriers, Ran-independent transport mechanisms, and cofactors highlights the complexity of the nuclear transport process. Multiple regulatory mechanisms are also being identified that control cargo-carrier interactions. Circadian rhythms, cell cycle, transcription, RNA processing, and signal transduction are all regulated at the level of nucleocytoplasmic transport. This review focuses on recent discoveries in the field, with an emphasis on the carriers and cofactors involved in transport and on possible mechanisms for movement through the nuclear pores. Publication Types: Review Review, Academic PMID: 11729264 [PubMed - indexed for MEDLINE] 38: Genetics 2001 Nov;159(3):1291-8 Probabilistic prediction of unknown metabolic and signal-transduction networks. Gomez SM, Lo SH, Rzhetsky A. Columbia Genome Center, Columbia University, New York, New York 10032, USA. Regulatory networks provide control over complex cell behavior in all kingdoms of life. Here we describe a statistical model, based on representing proteins as collections of domains or motifs, which predicts unknown molecular interactions within these biological networks. Using known protein-protein interactions of Saccharomyces cerevisiae as training data, we were able to predict the links within this network with only 7% false-negative and 10% false-positive error rates. We also use Markov chain Monte Carlo simulation for the prediction of networks with maximum probability under our model. This model can be applied across species, where interaction data from one (or several) species can be used to infer interactions in another. In addition, the model is extensible and can be analogously applied to other molecular data (e.g., DNA sequences). PMID: 11729170 [PubMed - indexed for MEDLINE] 39: EMBO J 2001 Dec 3;20(23):6660-71 Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair. Hartsuiker E, Vaessen E, Carr AM, Kohli J. Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RR, UK. To study the role of Rad50 in the DNA damage response, we cloned and deleted the Schizosaccharomyces pombe RAD50 homologue. The deletion is sensitive to a range of DNA-damaging agents and shows dynamic epistatic interactions with other recombination-repair genes. We show that Rad50 is necessary for recombinational repair of the DNA lesion at the mating-type locus and that rad50Delta shows slow DNA replication. We also find that Rad50 is not required for slowing down S phase in response to hydroxy urea or methyl methanesulfonate (MMS) treatment. Interestingly, in rad50Delta cells, the recombination frequency between two homologous chromosomes is increased at the expense of sister chromatid recombination. We propose that Rad50, an SMC-like protein, promotes the use of the sister chromatid as the template for homologous recombinational repair. In support of this, we found that Rad50 functions in the same pathway for the repair of MMS-induced damage as Rad21, the homologue of the Saccharomyces cerevisiae Scc1 cohesin protein. We speculate that Rad50 interacts with the cohesin complex during S phase to assist repair and possibly re-initiation of replication after replication fork collapse. PMID: 11726502 [PubMed - indexed for MEDLINE] 40: EMBO J 2001 Dec 3;20(23):6591-600 Specific roles of protein-phospholipid interactions in the yeast cytochrome bc1 complex structure. Lange C, Nett JH, Trumpower BL, Hunte C. Max-Planck-Institut fur Biophysik, Heinrich-Hoffmann-Strasse 7, D-60528 Frankfurt, Germany. Biochemical data have shown that specific, tightly bound phospholipids are essential for activity of the cytochrome bc1 complex (QCR), an integral membrane protein of the respiratory chain. However, the structure and function of such phospholipids are not yet known. Here we describe five phospholipid molecules and one detergent molecule in the X-ray structure of yeast QCR at 2.3 A resolution. Their individual binding sites suggest specific roles in facilitating structural and functional integrity of the enzyme. Interestingly, a phosphatidylinositol molecule is bound in an unusual interhelical position near the flexible linker region of the Rieske iron-sulfur protein. Two possible proton uptake pathways at the ubiquinone reduction site have been identified: the E/R and the CL/K pathway. Remarkably, cardiolipin is positioned at the entrance to the latter. We propose that cardiolipin ensures structural integrity of the proton-conducting protein environment and takes part directly in proton uptake. Site-directed mutagenesis of ligating residues confirmed the importance of the phosphatidylinositol- and cardiolipin-binding sites. PMID: 11726495 [PubMed - indexed for MEDLINE] 41: J Cell Biol 2001 Nov 26;155(5):763-74 Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)-related protein Sli15 during chromosome segregation. Kang J, Cheeseman IM, Kallstrom G, Velmurugan S, Barnes G, Chan CS. Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas, Austin, TX 78712, USA. We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1-Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome-spindle interactions or in the movement of kinetochores along microtubules. PMID: 11724818 [PubMed - indexed for MEDLINE] 42: Curr Biol 2001 Nov 13;11(22):1794-8 Promoter-specific activation defects by a novel yeast TBP mutant compromised for TFIIB interaction. Virbasius CM, Holstege FC, Young RA, Green MR. Howard Hughes Medical Institute, Programs in Gene Function and Expression, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA. TFIIB is an RNA polymerase II general transcription factor (GTF) that has also been implicated in the mechanism of action of certain promoter-specific activators (see, for examples, [1-11]). TFIIB enters the preinitiation complex (PIC) primarily through contact with the TATA box binding protein (TBP), an interaction mediated by three TBP residues [12-14]. To study the role of TFIIB in transcription activation in vivo, we randomly mutagenized these three residues in yeast TBP and screened for promoter-specific activation mutants. One mutant bearing a single conservative substitution, TBP-E186D, is the focus of this study. As expected, TBP-E186D binds normally to the TATA box but fails to support the entry of TFIIB into the PIC. Cells expressing TBP-E186D are viable but have a severe slow-growth phenotype. Whole-genome expression analysis indicates that transcription of 17% of yeast genes are compromised by this mutation. Chimeric promoter analysis indicates that the region of the gene that confers sensitivity to the TBP-E186D mutation is the UAS (upstream activating sequence), which contains the activator binding sites. Most interestingly, other TBP mutants that interfere with different interactions (TFIIB, TFIIA, or the TATA box) and a TFIIB mutant defective for interaction with TBP all manifest distinct and selective promoter-specific activation defects. Our results implicate the entry of TFIIB into the PIC as a critical step in the activation of certain promoters and reveal diverse mechanisms of transcription activation. PMID: 11719223 [PubMed - indexed for MEDLINE] 43: J Biol Chem 2002 Feb 1;277(5):3673-9 Differential ATP binding and intrinsic ATP hydrolysis by amino-terminal domains of the yeast Mlh1 and Pms1 proteins. Hall MC, Shcherbakova PV, Kunkel TA. Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. MutL homologs belong to a family of proteins that share a conserved ATP binding site. We demonstrate that amino-terminal domains of the yeast MutL homologs Mlh1 and Pms1 required for DNA mismatch repair both possess independent, intrinsic ATPase activities. Amino acid substitutions in the conserved ATP binding sites concomitantly reduce ATP binding, ATP hydrolysis, and DNA mismatch repair in vivo. The ATPase activities are weak, consistent with the hypothesis that ATP binding is primarily responsible for modulating interactions with other MMR components. Three approaches, ATP hydrolysis assays, limited proteolysis protection, and equilibrium dialysis, provide evidence that the amino-terminal domain of Mlh1 binds ATP with >10-fold higher affinity than does the amino-terminal domain of Pms1. This is consistent with a model wherein ATP may first bind to Mlh1, resulting in events that permit ATP binding to Pms1 and later steps in DNA mismatch repair. PMID: 11717305 [PubMed - indexed for MEDLINE] 44: J Cell Biol 2001 Nov 12;155(4):581-92 Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud. Adamo JE, Moskow JJ, Gladfelter AS, Viterbo D, Lew DJ, Brennwald PJ. Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth. PMID: 11706050 [PubMed - indexed for MEDLINE] 45: Biochemistry 2001 Nov 20;40(46):13933-40 Site-specific mutations in the myosin binding sites of actin affect structural transitions that control myosin binding. Prochniewicz E, Thomas DD. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis 55455, USA. ewa@ddt.biochem.umn.edu We have examined the effects of actin mutations on myosin binding, detected by cosedimentation, and actin structural dynamics, detected by spectroscopic probes. Specific mutations were chosen that have been shown to affect the functional interactions of actin and myosin, two mutations (4Ac and E99A/E100A) in the proposed region of weak binding to myosin and one mutation (I341A) in the proposed region of strong binding. In the absence of nucleotide and salt, S1 bound to both wild-type and mutant actins with high affinity (K(d) < microM), but either ADP or increased ionic strength decreased this affinity. This decrease was more pronounced for actins with mutations that inhibit functional interaction with myosin (E99A/E100A and I341A) than for a mutation that enhances the interaction (4Ac). The mutations E99A/E100A and I341A affected the microsecond time scale dynamics of actin in the absence of myosin, but the 4Ac mutation did not have any effect. The binding of myosin eliminated these effects of mutations on structural dynamics; i.e., the spectroscopic signals from mutant actins bound to S1 were the same as those from wild-type actin. These results indicate that mutations in the myosin binding sites affect structural transitions within actin that control strong myosin binding, without affecting the structural dynamics of the strongly bound actomyosin complex. PMID: 11705383 [PubMed - indexed for MEDLINE] 46: Plant Cell 2001 Nov;13(11):2455-70 The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals. Bellaoui M, Pidkowich MS, Samach A, Kushalappa K, Kohalmi SE, Modrusan Z, Crosby WL, Haughn GW. Botany Department and Biotechnology Laboratory, 6270 University Boulevard, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem. PMID: 11701881 [PubMed - indexed for MEDLINE] 47: Cell 2001 Nov 2;107(3):373-86 Structure of the 80S ribosome from Saccharomyces cerevisiae--tRNA-ribosome and subunit-subunit interactions. Spahn CM, Beckmann R, Eswar N, Penczek PA, Sali A, Blobel G, Frank J. Howard Hughes Medical Institute, Health Research Inc., Albany, NY 12201, USA. A cryo-EM reconstruction of the translating yeast 80S ribosome was analyzed. Computationally separated rRNA and protein densities were used for docking of appropriately modified rRNA models and homology models of yeast ribosomal proteins. The core of the ribosome shows a remarkable degree of conservation. However, some significant differences in functionally important regions and dramatic changes in the periphery due to expansion segments and additional ribosomal proteins are evident. As in the bacterial ribosome, bridges between the subunits are mainly formed by RNA contacts. Four new bridges are present at the periphery. The position of the P site tRNA coincides precisely with its prokaryotic counterpart, with mainly rRNA contributing to its molecular environment. This analysis presents an exhaustive inventory of an eukaryotic ribosome at the molecular level. PMID: 11701127 [PubMed - indexed for MEDLINE] 48: Annu Rev Genet 2001;35:193-208 Chromatin insulators and boundaries: effects on transcription and nuclear organization. Gerasimova TI, Corces VG. Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, UDA. tgerasimova@jhu.edu Chromatin boundaries and insulators are transcriptional regulatory elements that modulate interactions between enhancers and promoters and protect genes from silencing effects by the adjacent chromatin. Originally discovered in Drosophila, insulators have now been found in a variety of organisms, ranging from yeast to humans. They have been found interspersed with regulatory sequences in complex genes and at the boundaries between active and inactive chromatin. Insulators might modulate transcription by organizing the chromatin fiber within the nucleus through the establishment of higher-order domains of chromatin structure. Publication Types: Review Review, Tutorial PMID: 11700282 [PubMed - indexed for MEDLINE] 49: J Mol Biol 2001 Nov 9;313(5):955-63 UBA domains mediate protein-protein interactions between two DNA damage-inducible proteins. Bertolaet BL, Clarke DJ, Wolff M, Watson MH, Henze M, Divita G, Reed SI. Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. The Saccharomyces cerevisiae genes RAD23 and DDI1 were identified in a screen for multicopy suppressors of the temperature-sensitivity of a mutant allele of S. cerevisiae PDS1. Pds1 is a regulator of anaphase that needs to accumulate and then be degraded by the ubiquitin-proteasome pathway at the metaphase-anaphase transition for cells to progress normally through mitosis. Both the Rad23 and Ddi1 pds1 suppression phenotypes depend on a shared motif known as a UBA domain found in a variety of proteins associated with ubiquitin metabolism. UBA domains were found to be essential for homodimerization of Rad23 and heterodimerization between Rad23 and Ddi1, but not for homodimerization of Ddi1. This observation, coupled with the findings that Rad23 and Ddi1 UBA domains bind ubiquitin and that dimerization of Rad23 blocks ubiquitin binding, suggests a possible mechanism for regulating Rad23 and Ddi1 function. Copyright 2001 Academic Press. PMID: 11700052 [PubMed - indexed for MEDLINE] 50: Proc Natl Acad Sci U S A 2001 Nov 20;98(24):13675-80 Yeast Dam1p has a role at the kinetochore in assembly of the mitotic spindle. Jones MH, He X, Giddings TH, Winey M. Department of Molecular, Cellular, and Developmental Biology, Campus Box 347, University of Colorado, Boulder, CO 80309-0347, USA. During mitosis, replicated chromosomes are separated to daughter cells by the microtubule-based mitotic spindle. Chromosomes attach to the mitotic spindle through specialized DNA/protein structures called kinetochores, but the mechanism of attachment is not well understood. We show here that the yeast microtubule-binding protein, Dam1p, associates physically and functionally with kinetochores, suggesting a role in kinetochore attachment to the spindle. An epitope-tagged version of Dam1p colocalizes with the integral kinetochore component Ndc10p/Cbf2p in immunofluorescence analysis of chromosome spreads. In addition, Dam1p is associated preferentially with centromeric DNA as shown by chromatin immunoprecipitation experiments, and this association depends on Ndc10p/Cbf2p. We also demonstrate genetic interactions between DAM1 and CTF19 or SLK19 genes encoding kinetochore proteins. Although the defect caused by the dam1-1 mutation leads to activation of the spindle checkpoint surveillance system and consequent persistence of sister chromatid cohesion, the metaphase arrest spindle abnormally elongates, resulting in virtually complete chromosome missegregation. Execution point experiments indicate that Dam1p has a role in formation of a metaphase spindle and in anaphase spindle elongation. Finally, we have observed that the protein encoded by the dam1-1 allele becomes delocalized at the nonpermissive temperature, correlating with the subsequent onset of the mutant phenotype. Our studies are consistent with a role for Dam1p in attachment of sister chromatids through the kinetochore to the mitotic spindle before chromosome segregation. PMID: 11698664 [PubMed - indexed for MEDLINE] 51: Exp Cell Res 2001 Nov 15;271(1):142-51 Functional conservation of 14-3-3 isoforms in inhibiting bad-induced apoptosis. Subramanian RR, Masters SC, Zhang H, Fu H. Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA. 14-3-3 proteins are a family of homologous eukaryotic molecules with seven distinct isoforms in mammalian cells. Isoforms of 14-3-3 proteins interact with diverse ligands and are involved in the regulation of mitogenesis, cell cycle progression, and apoptosis. However, whether different 14-3-3 isoforms are responsible for distinct functions remains elusive. Here we report that multiple isoforms of 14-3-3 proteins were capable of binding to several ligands, Bad, Raf-1, and Cbl. In a functional assay of 14-3-3 isoforms, all mammalian 14-3-3 isoforms could inhibit Bad-induced apoptosis. Thus, 14-3-3 function in regulating one of its ligands, Bad, is conserved among mammalian isoforms. We addressed whether 14-3-3 isoforms are differentially expressed in tissues, which may in part determine isoform-specific interactions. In situ hybridization revealed that 14-3-3zeta was present in most tissues tested, but sigma was preferentially expressed in epithelial cells. Thus, isoforms of 14-3-3 can interact and control the function of selected protein ligands, and differential tissue distribution of 14-3-3 isoforms may contribute to their specific interactions and subsequent downstream signaling events. Copyright 2001 Academic Press. PMID: 11697890 [PubMed - indexed for MEDLINE] 52: Curr Biol 2001 Oct 30;11(21):1711-5 The DECD box putative ATPase Sub2p is an early mRNA export factor. Jensen TH, Boulay J, Rosbash M, Libri D. Howard Hughes Medical Institute, Department of Biology, Brandeis University, Waltham, MA 02454, USA. thj@mbio.aau.dk Nuclear mRNA metabolism relies on the interplay between transcription, processing, and nuclear export. RNA polymerase II transcripts experience major rearrangements within the nucleus, which include alterations in the structure of the mRNA precursors as well as the addition and perhaps even removal of proteins prior to transport across the nuclear membrane. Such mRNP-remodeling steps are thought to require the activity of RNA helicases/ATPases. One such protein, the DECD box RNA-dependent ATPase Sub2p/UAP56, is involved in both early and late steps of spliceosome assembly. Here, we report a more general function of Saccharomyces cerevisiae Sub2p in mRNA nuclear export. We observe a rapid and dramatic nuclear accumulation of poly(A)(+) RNA in strains carrying mutant alleles of sub2. Strikingly, an intronless transcript, HSP104, also accumulates in nuclei, suggesting that Sub2p function is not restricted to splicing events. The HSP104 transcripts are localized in a single nuclear focus that is suggested to be at or near their site of transcription. Intriguingly, Sub2p shows strong genetic and functional interactions with the RNA polymerase II-associated DNA/DNA:RNA helicase Rad3p as well as the nuclear RNA exosome component Rrp6p, which was independently implicated in the retention of mRNAs at transcription sites. Taken together, our data suggest that Sub2p functions at an early step in the mRNA export process. PMID: 11696331 [PubMed - indexed for MEDLINE] 53: Nat Genet 2001 Dec;29(4):482-6 Correlation between transcriptome and interactome mapping data from Saccharomyces cerevisiae. Ge H, Liu Z, Church GM, Vidal M. Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. Genomic and proteomic approaches can provide hypotheses concerning function for the large number of genes predicted from genome sequences. Because of the artificial nature of the assays, however, the information from these high-throughput approaches should be considered with caution. Although it is possible that more meaningful hypotheses could be formulated by integrating the data from various functional genomic and proteomic projects, it has yet to be seen to what extent the data can be correlated and how such integration can be achieved. We developed a 'transcriptome-interactome correlation mapping' strategy to compare the interactions between proteins encoded by genes that belong to common expression-profiling clusters with those between proteins encoded by genes that belong to different clusters. Using this strategy with currently available data sets for Saccharomyces cerevisiae, we provide the first global evidence that genes with similar expression profiles are more likely to encode interacting proteins. We show how this correlation between transcriptome and interactome data can be used to improve the quality of hypotheses based on the information from both approaches. The strategy described here may help to integrate other functional genomic and proteomic data, both in yeast and in higher organisms. PMID: 11694880 [PubMed - indexed for MEDLINE] 54: Mol Biol Cell 2001 Nov;12(11):3668-79 In vivo role for actin-regulating kinases in endocytosis and yeast epsin phosphorylation. Watson HA, Cope MJ, Groen AC, Drubin DG, Wendland B. Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA. The yeast actin-regulating kinases Ark1p and Prk1p are signaling proteins localized to cortical actin patches, which may be sites of endocytosis. Interactions between the endocytic proteins Pan1p and End3p may be regulated by Prk1p-dependent threonine phosphorylation of Pan1p within the consensus sequence [L/I]xxQxTG. We identified two Prk1p phosphorylation sites within the Pan1p-binding protein Ent1p, a yeast epsin homologue, and demonstrate Prk1p-dependent phosphorylation of both threonines. Converting both threonines to either glutamate or alanine mimics constitutively phosphorylated or dephosphorylated Ent1p, respectively. Synthetic growth defects were observed in a pan1-20 ENT1(EE) double mutant, suggesting that Ent1p phosphorylation negatively regulates the formation/activity of a Pan1p-Ent1p complex. Interestingly, pan1-20 ent2 Delta but not pan1-20 ent1 Delta double mutants had improved growth and endocytosis over the pan1-20 mutant. We found that actin-regulating Ser/Thr kinase (ARK) mutants exhibit endocytic defects and that overexpressing either wild-type or alanine-substituted Ent1p partially suppressed phenotypes associated with loss of ARK kinases, including growth, endocytosis, and actin localization defects. Consistent with synthetic growth defects of pan1-20 ENT1(EE) cells, overexpressing glutamate-substituted Ent1p was deleterious to ARK mutants. Surprisingly, overexpressing the related Ent2p protein could not suppress ARK kinase mutant phenotypes. These results suggest that Ent1p and Ent2p are not completely redundant and may perform opposing functions in endocytosis. These data support the model that, as for clathrin-dependent recycling of synaptic vesicles, yeast endocytic protein phosphorylation inhibits endocytic functions. PMID: 11694597 [PubMed - indexed for MEDLINE] 55: Mol Cell Biol 2001 Dec;21(23):8082-94 Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. Moretti P, Shore D. Department of Microbiology, College of Physicians & Surgeons of Columbia University, New York, New York 10032, USA. Initiation of transcriptional silencing at mating type loci and telomeres in Saccharomyces cerevisiae requires the recruitment of a Sir2/3/4 (silent information regulator) protein complex to the chromosome, which occurs at least in part through its association with the silencer- and telomere-binding protein Rap1p. Sir3p and Sir4p are structural components of silent chromatin that can self-associate, interact with each other, and bind to the amino-terminal tails of histones H3 and H4. We have identified a small region of Sir3p between amino acids 455 and 481 that is necessary and sufficient for association with the carboxyl terminus of Rap1p but not required for Sir complex formation or histone binding. SIR3 mutations that delete this region cause a silencing defect at HMR and telomeres. However, this impairment of repression is considerably less than that displayed by Rap1p carboxy-terminal truncations that are defective in Sir3p binding. This difference may be explained by the ability of the Rap1p carboxyl terminus to interact independently with Sir4p, which we demonstrate by in vitro binding and two-hybrid assays. Significantly, the Rap1p-Sir4p two-hybrid interaction does not require Sir3p and is abolished by mutation of the carboxyl terminus of Rap1p. We propose that both Sir3p and Sir4p can directly and independently bind to Rap1p at mating type silencers and telomeres and suggest that Rap1p-mediated recruitment of Sir proteins operates through multiple cooperative interactions, at least some of which are redundant. The physical separation of the Rap1p interaction region of Sir3p from parts of the protein required for Sir complex formation and histone binding raises the possibility that Rap1p can participate directly in the maintenance of silent chromatin through the stabilization of Sir complex-nucleosome interactions. PMID: 11689698 [PubMed - indexed for MEDLINE] 56: Mol Cell Biol 2001 Dec;21(23):7981-94 Structural requirements for function of yeast GGAs in vacuolar protein sorting, alpha-factor maturation, and interactions with clathrin. Mullins C, Bonifacino JS. Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-5430, USA. The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding proteins) are a family of multidomain adaptor proteins involved in protein sorting at the trans-Golgi network of eukaryotic cells. Here we present results from a functional characterization of the two Saccharomyces cerevisiae GGAs, Gga1p and Gga2p. We show that deletion of both GGA genes causes defects in sorting of carboxypeptidase Y (CPY) and proteinase A to the vacuole, vacuolar morphology, and maturation of alpha-factor. A structure-function analysis reveals a requirement of the VHS, GAT, and hinge for function, while the GAE domain is less important. We identify putative clathrin-binding motifs in the hinge domain of both yeast GGAs. These motifs are shown to mediate clathrin binding in vitro. While mutation of these motifs alone does not block function of the GGAs in vivo, combining these mutations with truncations of the hinge and GAE domains diminishes function, suggesting functional cooperation between different clathrin-binding elements. Thus, these observations demonstrate that the yeast GGAs play important roles in the CPY pathway, vacuole biogenesis, and alpha-factor maturation and identify structural determinants that are critical for these functions. PMID: 11689690 [PubMed - indexed for MEDLINE] 57: J Virol 2001 Dec;75(23):11344-53 Functional interactions of human immunodeficiency virus type 1 integrase with human and yeast HSP60. Parissi V, Calmels C, De Soultrait VR, Caumont A, Fournier M, Chaignepain S, Litvak S. REGER, UMR-5097 Centre National de la Recherche Scientifique (CNRS)-Universite Victor Segalen Bordeaux 2, Bordeaux, France. vincent.parissi@reger.u-bordeaux2.fr Integration of human immunodeficiency virus type 1 (HIV-1) proviral DNA in the nuclear genome is catalyzed by the retroviral integrase (IN). In addition to IN, viral and cellular proteins associated in the high-molecular-weight preintegration complex have been suggested to be involved in this process. In an attempt to define host factors interacting with IN, we used an in vitro system to identify cellular proteins in interaction with HIV-1 IN. The yeast Saccharomyces cerevisiae was chosen since (i) its complete sequence has been established and the primary structure of all the putative proteins from this eucaryote has been deduced, (ii) there is a significant degree of homology between human and yeast proteins, and (iii) we have previously shown that the expression of HIV-1 IN in yeast induces a lethal phenotype. Strong evidences suggest that this lethality is linked to IN activity in infected human cells where integration requires the cleavage of genomic DNA. Using IN-affinity chromatography we identified four yeast proteins interacting with HIV-1 IN, including the yeast chaperonin yHSP60, which is the counterpart of human hHSP60. Yeast lethality induced by HIV-1 IN was abolished when a mutated HSP60 was coexpressed, therefore suggesting that both proteins interact in vivo. Besides interacting with HIV-1 IN, the hHSP60 was able to stimulate the in vitro processing and joining activities of IN and protected this enzyme from thermal denaturation. In addition, the functional human HSP60-HSP10 complex in the presence of ATP was able to recognize the HIV-1 IN as a substrate. PMID: 11689615 [PubMed - indexed for MEDLINE] 58: Hum Mol Genet 2001 Oct 1;10(21):2463-8 The phylogenetic distribution of frataxin indicates a role in iron-sulfur cluster protein assembly. Huynen MA, Snel B, Bork P, Gibson TJ. Biocomputing, EMBL/Max-Delbrueck-Center fur molecular medicin, Berlin-Buch, Germany. huynen@cmbi.kun.nl Much has been learned about the cellular pathology of Friedreich's ataxia, a recessive neurodegenerative disease resulting from insufficient expression of the mitochondrial protein frataxin. However, the biochemical function of frataxin has remained obscure, hampering attempts at therapeutic intervention. To predict functional interactions of frataxin with other proteins we investigated whether its gene specifically co-occurs with any other genes in sequenced genomes. In 56 available genomes we identified two genes with identical phylogenetic distributions to the frataxin/cyaY gene: hscA and hscB/JAC1. These genes have not only emerged in the same evolutionary lineage as the frataxin gene, they have also been lost at least twice with it, and they have been horizontally transferred with it in the evolution of the mitochondria. The proteins encoded by hscA and hscB, the chaperone HSP66 and the co-chaperone HSP20, have been shown to be required for the synthesis of 2Fe-2S clusters on ferredoxin in proteobacteria. JAC1, an ortholog of hscB, and SSQ1, a paralog of hscA, have been shown to be required for iron-sulfur cluster assembly in mitochondria of Saccharomyces cerevisiae. Combining data on the co-occurrence of genes in genomes with experimental and predicted cellular localization data of their proteins supports the hypothesis that frataxin is directly involved in iron-sulfur cluster protein assembly. They indicate that frataxin is specifically involved in the same sub-process as HSP20/Jac1p. PMID: 11689493 [PubMed - indexed for MEDLINE] 59: Gene 2001 Aug 22;274(1-2):169-77 Erratum in: Gene 2001 Oct 31;278(1-2):265 Identification of interaction partners for two closely-related members of the ETS protein family, FLI and ERG. Deramaudt TB, Remy P, Stiegler P. FRE 2168 du CNRS, Mecanismes Moleculaires de la Division Cellulaire et du Developpement, Institut de Physiologie et de Chimie Biologique, 21 rue Rene Descartes, 67084 Strasbourg Cedex, France. Fli and erg are two members of the ETS gene family that encodes transcription factors related to the c-ets-1 proto-oncogene. The products of the ETS genes act as transcriptional effectors in cell proliferation, differentiation, and oncogenic transformation. FLI and ERG, two closely-related proteins, bind, as do all the ETS proteins characterized so far, to DNA sequences with an invariable central GGA core flanked by preferred nucleotides. Nevertheless, promoter-specific responses to FLI or ERG may be driven by mechanisms involving multicomponent complexes. Using a yeast two-hybrid screen, we have identified several proteins that physically interact with either FLI or ERG proteins used as bait. The Xenopus developmentally implicated Xvent-2 and Xvent-2B proteins, and the Xenopus splicing factor RNP-C/U1C physically interact with Xl-FLI and Xl-ERG, both in the yeast two-hybrid system and in vitro. We also report the potential interaction of FLI and ERG with Sox-D, a stabilizing protein that may modulate their transcriptional activity. Furthermore, the possible involvement of the transcriptional effectors FLI and ERG in mRNA processing, hematopoiesis or in the control of angiogenesis is suggested through possible interactions with, respectively, RNA binding proteins and hnRNPs, a repressor of the hematopoietic pathway (SAP18), and the HAF protein. PMID: 11675009 [PubMed - indexed for MEDLINE] 60: Biochemistry 2001 Oct 30;40(43):13088-96 Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase. Bollenbach TJ, Nowak T. Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 USA. Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+). The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme. PMID: 11669647 [PubMed - indexed for MEDLINE] 61: J Biol Chem 2002 Feb 15;277(7):5290-8 Unusual binding properties of the SH3 domain of the yeast actin-binding protein Abp1: structural and functional analysis. Fazi B, Cope MJ, Douangamath A, Ferracuti S, Schirwitz K, Zucconi A, Drubin DG, Wilmanns M, Cesareni G, Castagnoli L. Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133 Roma, Italy. Abp1p is an actin-binding protein that plays a central role in the organization of Saccharomyces cerevisiae actin cytoskeleton. By a combination of two-hybrid and phage-display approaches, we have identified six new ligands of the Abp1-SH3 domain. None of these SH3-mediated novel interactions was detected in recent all genome high throughput protein interaction projects. Here we show that the SH3-mediated association of Abp1p with the Ser/Thr kinases Prk1p and Ark1p is essential for their localization to actin cortical patches. The Abp1-SH3 domain has a rather unusual binding specificity, because its target peptides contain the tetrapentapeptide +XXXPXXPX+PXXL with positive charges flanking the polyproline core on both sides. Here we present the structure of the Abp1-SH3 domain solved at 1.3-A resolution. The peptide-binding pockets in the SH3 domain are flanked by two acidic residues that are uncommon at those positions in the SH3 domain family. We have shown by site-directed mutagenesis that one of these negatively charged side chains may be the key determinant for the preference for non-classical ligands. PMID: 11668184 [PubMed - indexed for MEDLINE] 62: Inorg Chem 1996 Mar 13;35(6):1692-1700 New Type 2 Copper-Cysteinate Proteins. Copper Site Histidine-to-Cysteine Mutants of Yeast Copper-Zinc Superoxide Dismutase. Lu Y, Roe JA, Bender CJ, Peisach J, Banci L, Bertini I, Gralla EB, Valentine JS. Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095, Department of Molecular Pharmacology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, Department of Chemistry and Biochemistry, Loyola Marymount University, Los Angeles, California 90045, and Department of Chemistry, University of Florence, Florence, Italy. Preparation and characterization of two new site-directed mutant copper-zinc superoxide dismutase proteins from Saccharomyces cerevisiae, i.e., His46Cys (H46C) and His120Cys (H120C), in which individual histidyl ligands in the copper-binding site were replaced by cysteine, are reported here. These two mutant CuZnSOD proteins may be described as type 2 (or normal) rather than type 1 (or blue) copper-cysteinate proteins and are characterized by their yellow rather than blue color, resulting from intense copper-to-sulfur charge transfer bands around 400 nm, their type 2 EPR spectra, with large rather than small nuclear hyperfine interactions, and their characteristic type 2 d-d electronic absorption spectra. An interesting difference between these two copper site His-to-Cys mutations is that the imidazolate bridge between the two metal sites that is characteristic of the wild-type protein remains intact in the case of the H46C mutant but is not present in the case of the H120C mutant. PMID: 11666393 [PubMed - as supplied by publisher] 63: J Biol Chem 2001 Dec 7;276(49):46225-9 Asymmetric recognition of DNA local distortion. Structure-based functional studies of eukaryotic Msh2-Msh6. Drotschmann K, Yang W, Brownewell FE, Kool ET, Kunkel TA. Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. Crystal structures of bacterial MutS homodimers bound to mismatched DNA reveal asymmetric interactions of the two subunits with DNA. A phenylalanine and glutamate of one subunit make mismatched base-specific interactions, and residues of both subunits contact the DNA backbone surrounding the mismatched base, but asymmetrically. A number of amino acids in MutS that contact the DNA are conserved in the eukaryotic Msh2-Msh6 heterodimer. We report here that yeast strains with amino acids substituted for residues inferred to interact with the DNA backbone or mismatched base have elevated spontaneous mutation rates consistent with defective mismatch repair. Purified Msh2-Msh6 with substitutions in the conserved Phe(337) and Glu(339) in Msh6 thought to stack or hydrogen bond, respectively, with the mismatched base do have reduced DNA binding affinity but normal ATPase activity. Moreover, wild-type Msh2-Msh6 binds with lower affinity to mismatches with thymine replaced by difluorotoluene, which lacks the ability to hydrogen bond. The results suggest that yeast Msh2-Msh6 interacts asymmetrically with the DNA through base-specific stacking and hydrogen bonding interactions and backbone contacts. The importance of these contacts decreases with increasing distance from the mismatch, implying that interactions at and near the mismatch are important for binding in a kinked DNA conformation. PMID: 11641390 [PubMed - indexed for MEDLINE] 64: Genetics 2001 Oct;159(2):487-97 Genetic interactions of Spt4-Spt5 and TFIIS with the RNA polymerase II CTD and CTD modifying enzymes in Saccharomyces cerevisiae. Lindstrom DL, Hartzog GA. Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, 95064, USA. Genetic and biochemical studies have identified many factors thought to be important for transcription elongation. We investigated relationships between three classes of these factors: (1) transcription elongation factors Spt4-Spt5, TFIIS, and Spt16; (2) the C-terminal heptapeptide repeat domain (CTD) of RNA polymerase II; and (3) protein kinases that phosphorylate the CTD and a phosphatase that dephosphorylates it. We observe that spt4 and spt5 mutations cause strong synthetic phenotypes in combination with mutations that shorten or alter the composition of the CTD; affect the Kin28, Bur1, or Ctk1 CTD kinases; and affect the CTD phosphatase Fcp1. We show that Spt5 co-immunoprecipitates with RNA polymerase II that has either a hyper- or a hypophosphorylated CTD. Furthermore, mutation of the CTD or of CTD modifying enzymes does not affect the ability of Spt5 to bind RNA polymerase II. We find a similar set of genetic interactions between the CTD, CTD modifying enzymes, and TFIIS. In contrast, an spt16 mutation did not show these interactions. These results suggest that the CTD plays a key role in modulating elongation in vivo and that at least a subset of elongation factors are dependent upon the CTD for their normal function. PMID: 11606527 [PubMed - indexed for MEDLINE] 65: Biochemistry 2001 Oct 23;40(42):12704-11 MDP-1 is a new and distinct member of the haloacid dehalogenase family of aspartate-dependent phosphohydrolases. Selengut JD. Laboratory of Biochemistry, National Heart, Lung and Blood Institute, Building 50-2347, National Institutes of Health, 50 South Drive, Bethesda, Maryland 20892-8012, USA. selengut@nih.gov MDP-1 is a eukaryotic magnesium-dependent acid phosphatase with little sequence homology to previously characterized phosphatases. The presence of a conserved motif (Asp-X-Asp-X-Thr) in the N terminus of MDP-1 suggested a relationship to the haloacid dehalogenase (HAD) superfamily, which contains a number of magnesium-dependent acid phosphatases. These phosphatases utilize an aspartate nucleophile and contain a number of conserved active-site residues and hydrophobic patches, which can be plausibly aligned with conserved residues in MDP-1. Seven site-specific point mutants of MDP-1 were produced by modifying the catalytic aspartate, serine, and lysine residues to asparagine or glutamate, alanine, and arginine, respectively. The activity of these mutants confirms the assignment of MDP-1 as a member of the HAD superfamily. Detailed comparison of the sequence of the 15 MDP-1 sequences from various organisms with other HAD superfamily sequences suggests that MDP-1 is not closely related to any particular member of the superfamily. The crystal structures of several HAD family enzymes identify a domain proximal to the active site responsible for important interactions with low molecular weight substrates. The absence of this domain or any other that might perform the same function in MDP-1 suggests an "open" active site capable of interactions with large substrates such as proteins. This suggestion was experimentally confirmed by demonstration that MDP-1 is competent to catalyze the dephosphorylation of tyrosine-phosphorylated proteins. PMID: 11601995 [PubMed - indexed for MEDLINE] 66: EMBO J 2001 Oct 15;20(20):5626-35 Apocytochrome c requires the TOM complex for translocation across the mitochondrial outer membrane. Diekert K, de Kroon AI, Ahting U, Niggemeyer B, Neupert W, de Kruijff B, Lill R. Institut fur Zytobiologie und Zytopathologie der Philipps-Universitat Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany. The import of proteins into the mitochondrial intermembrane space differs in various aspects from the classical import pathway into the matrix. Apocytochrome c defines one of several pathways known to reach the intermembrane space, yet the components and pathways involved in outer membrane translocation are poorly defined. Here, we report the reconstitution of the apocytochrome c import reaction using proteoliposomes harbouring purified components. Import specifically requires the protease-resistant part of the TOM complex and is driven by interactions of the apoprotein with internal parts of the complex (involving Tom40) and the 'trans-side receptor' cytochrome c haem lyase. Despite the necessity of TOM complex function, the translocation pathway of apocytochrome c does not overlap with that of presequence-containing preproteins. We conclude that the TOM complex is a universal preprotein translocase that mediates membrane passage of apocytochrome c and other preproteins along distinct pathways. Apocytochrome c may provide a paradigm for the import of other small proteins into the intermembrane space such as factors used in apoptosis and protection from stress. PMID: 11598006 [PubMed - indexed for MEDLINE] 67: Physiol Genomics 2001 Oct 10;7(1):27-34 Two-hybrid analysis of the Saccharomyces cerevisiae 26S proteasome. Cagney G, Uetz P, Fields S. Departments of Genetics and Medicine, Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195-7360, USA. A two-hybrid screen against an activation domain array of Saccharomyces cerevisiae proteins was carried out for 31 yeast proteasome proteins. Fifty-five putative interactions were identified: 21 between components of the proteasome complex and 34 between proteasome proteins and other proteins. Many of these latter interactions involved either proteins of the ubiquitin pathway, cell cycle proteins, protein kinases or a translation initiation factor subunit. The role of eleven proteins associated with proteasome function by these screens was analyzed by examining the corresponding deletion strains for temperature sensitivity and canavanine sensitivity and for the stability of a ubiquitin-beta-galactosidase fusion protein. These assays additionally implicated three proteins, Bim1, Ump1, and YKL171W, in proteasome function. This study demonstrates the utility of genome-wide two-hybrid assays as an entry point for the further analysis of a large protein complex. PMID: 11595789 [PubMed - indexed for MEDLINE] 68: J Biol Chem 2001 Dec 14;276(50):46745-50 Cdc42 interacts with the exocyst and regulates polarized secretion. Zhang X, Bi E, Novick P, Du L, Kozminski KG, Lipschutz JH, Guo W. Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. Polarized delivery and incorporation of proteins and lipids to specific domains of the plasma membrane is fundamental to a wide range of biological processes such as neuronal synaptogenesis and epithelial cell polarization. The exocyst complex is specifically localized to sites of active exocytosis and plays essential roles in secretory vesicle targeting and docking at the plasma membrane. Sec3p, a component of the exocyst, is thought to be a spatial landmark for polarized exocytosis. In a search for proteins that regulate the localization of the exocyst in the budding yeast Saccharomyces cerevisiae, we found that certain cdc42 mutants affect the polarized localization of the exocyst proteins. In addition, we found that these mutant cells have a randomized protein secretion pattern on the cell surface. Biochemical experiments indicated that Sec3p directly interacts with Cdc42 in its GTP-bound form. Genetic studies demonstrated synthetically lethal interactions between cdc42 and several exocyst mutants. These results have revealed a role for Cdc42 in exocytosis. We propose that Cdc42 coordinates the vesicle docking machinery and the actin cytoskeleton for polarized secretion. PMID: 11595741 [PubMed - indexed for MEDLINE] 69: Gene 2001 Aug 8;273(2):207-14 Molecular cloning and characterization of a steroid receptor-binding regulator of G-protein signaling protein cDNA. Ikeda M, Hirokawa M, Satani N, Kinoshita T, Watanabe Y, Inoue H, Tone S, Ishikawa T, Minatogawa Y. Department of Biochemistry, Kawasaki Medical School, 577 Matsushima Kurashiki, 701-0192, Okayama, Japan. ikeda@bcc.kawasaki-m.ac.jp Steroid hormone receptors are composed of six major functional domains, i.e. the A/B domains as the activation function 1 domain (AF-1), domain C as the DNA-binding domain, domain D as a hinge domain and domain E/F as the ligand-dependent transcriptional domain (AF-2). They regulate gene transcription through interactions with various nuclear factors of their domains, such as AF-1 and AF-2. We have insufficient knowledge of the function of the DNA-binding domain (domain C) except for its DNA-binding function or the hinge domain (domain D). Therefore, we attempted to identify factors interacting with the domains by using a yeast two-hybrid system. Domains C and D of estrogen receptor alpha were used as a bait to isolate cDNA clones from a rat ovary cDNA library. We isolated the cDNA clone of a novel steroid receptor-binding protein bearing the regulator of G-protein signaling (RGS) designated as SRB-RGS. The protein repressed the transcriptional activity of estrogen receptor alpha, suggesting cross-talk of steroid hormones and peptide hormones (or growth factors) for signal transductions mediated by SRB-RGS. PMID: 11595167 [PubMed - indexed for MEDLINE] 70: Structure (Camb) 2001 Oct;9(10):897-904 Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail. Hamilton KS, Ellison MJ, Barber KR, Williams RS, Huzil JT, McKenna S, Ptak C, Glover M, Shaw GS. Department of Biochemistry, The University of Alberta, Edmonton, Alberta T6G 2H7, Canada. BACKGROUND: Ubiquitin-conjugating enzymes (E2s) are central enzymes involved in ubiquitin-mediated protein degradation. During this process, ubiquitin (Ub) and the E2 protein form an unstable E2-Ub thiolester intermediate prior to the transfer of ubiquitin to an E3-ligase protein and the labeling of a substrate for degradation. A series of complex interactions occur among the target substrate, ubiquitin, E2, and E3 in order to efficiently facilitate the transfer of the ubiquitin molecule. However, due to the inherent instability of the E2-Ub thiolester, the structural details of this complex intermediate are not known. RESULTS: A three-dimensional model of the E2-Ub thiolester intermediate has been determined for the catalytic domain of the E2 protein Ubc1 (Ubc1(Delta450)) and ubiquitin from S. cerevisiae. The interface of the E2-Ub intermediate was determined by kinetically monitoring thiolester formation by 1H-(15)N HSQC spectra by using combinations of 15N-labeled and unlabeled Ubc1(Delta450) and Ub proteins. By using the surface interface as a guide and the X-ray structures of Ub and the 1.9 A structure of Ubc1(Delta450) determined here, docking simulations followed by energy minimization were used to produce the first model of a E2-Ub thiolester intermediate. CONCLUSIONS: Complementary surfaces were found on the E2 and Ub proteins whereby the C terminus of Ub wraps around the E2 protein terminating in the thiolester between C88 (Ubc1(Delta450)) and G76 (Ub). The model supports in vivo and in vitro experiments of E2 derivatives carrying surface residue substitutions. Furthermore, the model provides insights into the arrangement of Ub, E2, and E3 within a ternary targeting complex. PMID: 11591345 [PubMed - indexed for MEDLINE] 71: Mol Cell Neurosci 2001 Sep;18(3):307-19 Doublecortin interacts with mu subunits of clathrin adaptor complexes in the developing nervous system. Friocourt G, Chafey P, Billuart P, Koulakoff A, Vinet MC, Schaar BT, McConnell SK, Francis F, Chelly J. Laboratoire de Genetique et Physiopathologie des retards mentaux, ICGM, INSERM, CHU, Cochin, 24, rue du Faubourg Saint Jacques, Paris, 75014, France. Doublecortin is a microtubule-associated protein required for normal corticogenesis in the developing brain. We carried out a yeast two-hybrid screen to identify interacting proteins. One of the isolated clones encodes the mu1 subunit of the adaptor complex AP-1 involved in clathrin-dependent protein sorting. We found that Doublecortin also interacts in yeast with mu2 from the AP-2 complex. Mutagenesis and pull-down experiments showed that these interactions were mediated through a tyrosine-based sorting signal (YLPL) in the C-terminal part of Doublecortin. The functional relevance of these interactions was suggested by the coimmunoprecipitation of Doublecortin with AP-1 and AP-2 from mouse brain extracts. This interaction was further supported by RNA in situ hybridization and immunofluorescence studies. Taken together these data indicate that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in vivo and are consistent with a potential involvement of Doublecortin in protein sorting or vesicular trafficking. Copyright 2001 Academic Press. PMID: 11591131 [PubMed - indexed for MEDLINE] 72: Int J Food Microbiol 2001 Sep 19;69(1-2):101-11 Saccharomyces cerevisiae as a starter culture in Mycella. Hansen TK, Tempel TV, Cantor MD, Jakobsen M. Department of Dairy and Food Science, Food Microbiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark. tkh@kvl.dk The potential use of Saccharomyces cerevisiae FB7 as an additional starter culture for the production of Mycella, a Danish Gorgonzola type cheese, was investigated. Two dairy productions of Mycella, each containing batches of experimental cheeses with S. cerevisiae added and reference cheeses without yeast added were carried out. For both experimental and reference cheeses, chemical analysis (pH, a(w), NaCl, water and fat content) were carried out during the ripening period, but no significant differences were found. The evolution of lactic acid bacteria was almost identical in both the experimental and reference cheeses and similar results were found for the number of yeast. S. cerevisiae FB7 was found to be predominant in the core of the experimental cheeses throughout the ripening period, while Debaryomyces hansenii dominated in the reference cheese and on the surface of the experimental cheeses. In the cheeses with S. cerevisiae FB7, an earlier sporulation and an improved growth of Penicillium roqueforti was observed compared to the reference cheeses. Furthermore, in the experimental cheese, synergistic interactions were also found in the aroma analysis, the degradation of casein and by the sensory analysis. The observed differences indicate a positive contribution to the overall quality of Mycella by S. cerevisiae FB7. PMID: 11589548 [PubMed - indexed for MEDLINE] 73: Europ J Paediatr Neurol 2001;5 Suppl A:89-93 Analysis of CLN3-protein interactions using the yeast two-hybrid system. Leung KY, Greene ND, Munroe PB, Mole SE. Heart Science Centre, Harefield Hospital, Middlesex, UK. kit-yi.leung@harefield.nthames.nhs.uk Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a childhood neurodegenerative disease that is caused by mutations in the CLN3 gene. The protein encoded by CLN3 has no homology with any proteins of known function and its cellular role remains elusive. In order to investigate the role played by the CLN3 protein we aimed to identify interacting proteins. Here, we describe the yeast two-hybrid system as the approach taken to investigate such protein-protein interactions. CLN3 was expressed as a fusion protein with a DNA-binding domain and used to screen a library of human fetal brain cDNAs fused to a transcriptional activation domain. Owing to low level expression of the full length CLN3 fusion protein, truncated regions corresponding to the predicted hydrophilic regions were also tested. No proteins that interact with CLN3 were detected, nor was there any evidence for CLN3-CLN3 interactions. Potential interaction of CLN3 with subunit c of mitochondrial ATP synthase, the major component of the storage material that accumulates in Batten disease patients, was also tested. No interaction was detected suggesting that the accumulation of subunit c does not result from loss of a process that requires a direct interaction with CLN3. We conclude that either CLN3 does not interact with other proteins or such interactions cannot be detected using the two-hybrid system. PMID: 11589015 [PubMed - indexed for MEDLINE] 74: Biochem Biophys Res Commun 2001 Oct 12;287(5):1083-7 The PUB domain: a putative protein-protein interaction domain implicated in the ubiquitin-proteasome pathway. Suzuki T, Park H, Till EA, Lennarz WJ. Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York, 11794-5215, USA. Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which may be involved in the proteasome-dependent pathway for degradation of misfolded glycoproteins formed in the endoplasmic reticulum (ER) that are exported into the cytoplasm. A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al. J. Cell Biol. 149, 1039-1051, 2000). The recently uncovered complete genome sequence of Arabidopsis thaliana prompted us to search for the protein homologue of Png1p in this organism. Interestingly, when the mouse Png1p homologue sequence was used as a query, not only a Png1p homologue containing a transglutaminase-like domain that is believed to contain a catalytic triad for PNGase activity, but also four proteins which had a domain of 46 amino acids in length that exhibited significant similarity to the N-terminus of mouse Png1p were identified. Moreover, three of these homologous proteins were also found to possess a UBA or UBX domain, which are found in various proteins involved in the ubiquitin-related pathway. We name this newly found homologous region the PUB (Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that this domain may mediate protein-protein interactions. Copyright 2001 Academic Press. PMID: 11587532 [PubMed - indexed for MEDLINE] 75: Genome Biol 2001;2(9):RESEARCH0039 Abundant protein domains occur in proportion to proteome size. Malek JA. Agencourt Bioscience Corporation, 100 Cummings Center, Suite 107J, Beverly, MA 01915, USA. jamalek@agencourt.com BACKGROUND: Conserved domains in proteins have crucial roles in protein interactions, DNA binding, enzyme activity and other important cellular processes. It will be of interest to determine the proportions of genes containing such domains in the proteomes of different eukaryotes. RESULTS: The average proportion of conserved domains in each of five eukaryote genomes was calculated. In pairwise genome comparisons, the ratio of genes containing a given conserved domain in the two genomes on average reflected the ratio of the predicted total gene numbers of the two genomes. These ratios have been verified using a repository of databases and one of its subdivisions of conserved domains. CONCLUSIONS: Many conserved domains occur as a constant proportion of proteome size across the five sequenced eukaryotic genomes. This raises the possibility that this proportion is maintained because of functional constraints on interacting domains. The universality of the ratio in the five eukaryotic genomes attests to its potential importance. PMID: 11574058 [PubMed - indexed for MEDLINE] 76: Cell 2001 Sep 21;106(6):723-33 A plant viral "reinitiation" factor interacts with the host translational machinery. Park HS, Himmelbach A, Browning KS, Hohn T, Ryabova LA. Friedrich Miescher-Institute, P.O. Box 2543, CH-4002, Basel, Switzerland. The cauliflower mosaic virus transactivator, TAV, controls translation reinitiation of major open reading frames on polycistronic RNA. We show here that TAV function depends on its association with polysomes and eukaryotic initiation factor eIF3 in vitro and in vivo. TAV physically interacts with eIF3 and the 60S ribosomal subunit. Two proteins mediating these interactions were identified: eIF3g and 60S ribosomal protein L24. Transient expression of eIF3g and L24 in plant protoplasts strongly affects TAV-mediated reinitiation activity. We demonstrate that TAV/eIF3/40S and eIF3/TAV/60S ternary complexes form in vitro, and propose that TAV mediates efficient recruitment of eIF3 to polysomes, allowing translation of polycistronic mRNAs by reinitiation, overcoming the normal cell barriers to this process. PMID: 11572778 [PubMed - indexed for MEDLINE] 77: EMBO J 2001 Sep 17;20(18):5290-301 The structure of an AspRS-tRNA(Asp) complex reveals a tRNA-dependent control mechanism. Moulinier L, Eiler S, Eriani G, Gangloff J, Thierry JC, Gabriel K, McClain WH, Moras D. UPR 9004, Laboratoire de Biologie et Genomique Structurales, Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP 163, 67404 Illkirch Cedex, France. The 2.6 A resolution crystal structure of an inactive complex between yeast tRNA(Asp) and Escherichia coli aspartyl-tRNA synthetase reveals the molecular details of a tRNA-induced mechanism that controls the specificity of the reaction. The dimer is asymmetric, with only one of the two bound tRNAs entering the active site cleft of its subunit. However, the flipping loop, which controls the proper positioning of the amino acid substrate, acts as a lid and prevents the correct positioning of the terminal adenosine. The structure suggests that the acceptor stem regulates the loop movement through sugar phosphate backbone- protein interactions. Solution and cellular studies on mutant tRNAs confirm the crucial role of the tRNA three-dimensional structure versus a specific recognition of bases in the control mechanism. PMID: 11566892 [PubMed - indexed for MEDLINE] 78: EMBO J 2001 Sep 17;20(18):5207-18 Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions. White CL, Suto RK, Luger K. Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA. Chromatin is composed of nucleosomes, the universally repeating protein-DNA complex in eukaryotic cells. The crystal structure of the nucleosome core particle from Saccharomyces cerevisiae reveals that the structure and function of this fundamental complex is conserved between single-cell organisms and metazoans. Our results show that yeast nucleosomes are likely to be subtly destabilized as compared with nucleosomes from higher eukaryotes, consistent with the idea that much of the yeast genome remains constitutively open during much of its life cycle. Importantly, minor sequence variations lead to dramatic changes in the way in which nucleosomes pack against each other within the crystal lattice. This has important implications for our understanding of the formation of higher order chromatin structure and its modulation by post-translational modifications. Finally, the yeast nucleosome core particle provides a structural context by which to interpret genetic data obtained from yeast. Coordinates have been deposited with the Protein Data Bank under accession number 1ID3. PMID: 11566884 [PubMed - indexed for MEDLINE] 79: Mol Cell Biol 2001 Oct;21(20):6782-95 Human STAGA complex is a chromatin-acetylating transcription coactivator that interacts with pre-mRNA splicing and DNA damage-binding factors in vivo. Martinez E, Palhan VB, Tjernberg A, Lymar ES, Gamper AM, Kundu TK, Chait BT, Roeder RG. Laboratories of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021, USA. GCN5 is a histone acetyltransferase (HAT) originally identified in Saccharomyces cerevisiae and required for transcription of specific genes within chromatin as part of the SAGA (SPT-ADA-GCN5 acetylase) coactivator complex. Mammalian cells have two distinct GCN5 homologs (PCAF and GCN5L) that have been found in three different SAGA-like complexes (PCAF complex, TFTC [TATA-binding-protein-free TAF(II)-containing complex], and STAGA [SPT3-TAF(II)31-GCN5L acetylase]). The composition and roles of these mammalian HAT complexes are still poorly characterized. Here, we present the purification and characterization of the human STAGA complex. We show that STAGA contains homologs of most yeast SAGA components, including two novel human proteins with histone-like folds and sequence relationships to yeast SPT7 and ADA1. Furthermore, we demonstrate that STAGA has acetyl coenzyme A-dependent transcriptional coactivator functions from a chromatin-assembled template in vitro and associates in HeLa cells with spliceosome-associated protein 130 (SAP130) and DDB1, two structurally related proteins. SAP130 is a component of the splicing factor SF3b that associates with U2 snRNP and is recruited to prespliceosomal complexes. DDB1 (p127) is a UV-damaged-DNA-binding protein that is involved, as part of a complex with DDB2 (p48), in nucleotide excision repair and the hereditary disease xeroderma pigmentosum. Our results thus suggest cellular roles of STAGA in chromatin modification, transcription, and transcription-coupled processes through direct physical interactions with sequence-specific transcription activators and with components of the splicing and DNA repair machineries. PMID: 11564863 [PubMed - indexed for MEDLINE] 80: Genes Dev 2001 Sep 15;15(18):2445-56 Mechanisms controlling differential promoter-occupancy by the yeast forkhead proteins Fkh1p and Fkh2p: implications for regulating the cell cycle and differentiation. Hollenhorst PC, Pietz G, Fox CA. Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA. The roles of DNA and Mcm1p interactions in determining the overlapping and distinct functions of the yeast cell cycle regulatory transcription factors Fkh1p and Fkh2p were examined. Full-length recombinant Fkh1p and Fkh2p were purified and their binding to bona fide promoters examined in vitro. Each protein bound a variety of target promoters with similar specificity in vitro, consistent with the observation that these proteins bind common promoters in vivo. However, in vivo, the Fkh1p and Fkh2p occupied different target promoters to different extents, suggesting that each was primarily responsible for controlling a different set of genes. Additional in vitro studies provided a mechanistic explanation for this differential promoter-occupancy. Specifically, the Fkh2p, but not the Fkh1p, was capable of binding cooperatively with Mcm1p. The Mcm1p-Fkh2p cooperative binding was enhanced by, but did not require, the presence of a Mcm1p-binding site within a target promoter. Consistent with these data, Mcm1p was present at Fkh-controlled promoters in vivo regardless of whether they contained Mcm1p-binding sites, suggesting a role for Mcm1p at promoters not thought previously to be under Mcm1p control. Analysis of Fkh1p and Fkh2p binding to promoter targets in vivo by use of mutant strains indicated that the two proteins compete for promoter-occupancy at a number of target promoters. We postulate that Fkh1p and a stable Fkh2p/Mcm1p complex compete for binding to target promoters and that the levels and/or binding activity of Fkh1p, but not Fkh2p, are most limiting for promoter-occupancy in vivo. Interestingly, the in vitro DNA-binding assays, using a variety of promoter targets, revealed that bona fide Fkh target promoters contained two or more Fkh-binding sites that allowed the Fkh1p and Fkh2p proteins to form multiple protein-DNA complexes in vitro. Multiple Fkh-binding sites may be a distinguishing feature of bona fide Fkh promoters in yeast and other organisms. PMID: 11562353 [PubMed - indexed for MEDLINE] 81: Genetics 2001 Sep;159(1):91-105 Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe. Tsutsui Y, Khasanov FK, Shinagawa H, Iwasaki H, Bashkirov VI. Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan. Schizosaccharomyces pombe Rhp55 and Rhp57 are RecA-like proteins involved in double-strand break (DSB) repair. Here we demonstrate that Rhp55 and Rhp57 proteins strongly interact in vivo, similar to Saccharomyces cerevisiae Rad55p and Rad57p. Mutations in the conserved ATP-binding/hydrolysis folds of both the Rhp55 and Rhp57 proteins impaired their function in DNA repair but not in cell proliferation. However, when combined, ATPase fold mutations in Rhp55p and Rhp57p resulted in severe defects of both functions, characteristic of the deletion mutants. Yeast two-hybrid analysis also revealed other multiple in vivo interactions among S. pombe proteins involved in recombinational DNA repair. Similar to S. cerevisiae Rad51p-Rad54p, S. pombe Rhp51p and Rhp54p were found to interact. Both putative Rad52 homologs in S. pombe, Rad22p and Rti1p, were found to interact with the C-terminal region of Rhp51 protein. Moreover, Rad22p and Rti1p exhibited mutual, as well as self-, interactions. In contrast to the S. cerevisiae interacting pair Rad51p-Rad55p, S. pombe Rhp51 protein strongly interacted with Rhp57 but not with Rhp55 protein. In addition, the Rti1 and Rad22 proteins were found to form a complex with the large subunit of S. pombe RPA. Our data provide compelling evidence that most, but not all, of the protein-protein interactions found in S. cerevisiae DSB repair are evolutionarily conserved. PMID: 11560889 [PubMed - indexed for MEDLINE] 82: Genetics 2001 Sep;159(1):77-89 The Ras/PKA signaling pathway of Saccharomyces cerevisiae exhibits a functional interaction with the Sin4p complex of the RNA polymerase II holoenzyme. Howard SC, Chang YW, Budovskaya YV, Herman PK. Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, USA. Saccharomyces cerevisiae cells enter into the G(0)-like resting state, stationary phase, in response to specific types of nutrient limitation. We have initiated a genetic analysis of this resting state and have identified a collection of rye mutants that exhibit a defective transcriptional response to nutrient deprivation. These transcriptional defects appear to disrupt the control of normal growth because the rye mutants are unable to enter into a normal stationary phase upon nutrient deprivation. In this study, we examined the mutants in the rye1 complementation group and found that rye1 mutants were also defective for stationary phase entry. Interestingly, the RYE1 gene was found to be identical to SIN4, a gene that encodes a component of the yeast Mediator complex within the RNA polymerase II holoenzyme. Moreover, mutations that affected proteins within the Sin4p module of the Mediator exhibited specific genetic interactions with the Ras protein signaling pathway. For example, mutations that elevated the levels of Ras signaling, like RAS2(val19), were synthetic lethal with sin4. In all, our data suggest that specific proteins within the RNA polymerase II holoenzyme might be targets of signal transduction pathways that are responsible for coordinating gene expression with cell growth. PMID: 11560888 [PubMed - indexed for MEDLINE] 83: J Virol 2001 Oct;75(20):9613-22 Functional interaction map of lyssavirus phosphoprotein: identification of the minimal transcription domains. Jacob Y, Real E, Tordo N. Laboratoire des Lyssavirus, Institut Pasteur, 75724 Paris Cedex 15, France. yjacob@pasteur.fr Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P and N) which participate in the viral ribonucleoprotein (RNP) transcription-replication complex. This strategy combined two-hybrid and green fluorescent protein-reverse two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa) 176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal portion of P (aa 52 to 189) also contains a homomultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained between proteins of the different genotypes. A minimal transcriptional module of the P protein was obtained by fusing the first 60 N-terminal aa containing the L protein binding site to the C-terminal strong N-BD. Random mutation of the strong N-BD on P protein identified three highly conserved K residues crucial for N-P interaction. Their mutagenesis in full-length P induced a transcriptionally defective RNP. The analysis of homologous interactive domains presented here and previously reported dissections of the P protein allowed us to propose a model of the functional interaction network of the lyssavirus P protein. This model underscores the central role of P at the interface between L protein and N-RNA template. PMID: 11559793 [PubMed - indexed for MEDLINE] 84: J Biol Chem 2001 Nov 9;276(45):42003-10 The structural and functional organization of the yeast mediator complex. Kang JS, Kim SH, Hwang MS, Han SJ, Lee YC, Kim YJ. National Creative Research Center for Genome Regulation, Department of Biochemistry, Yonsei University, Seoul 120-749, Korea. The Mediator complex of Saccharomyces cerevisiae is required for diverse aspects of transcription by RNA polymerase II (pol II). Mediator is composed of two functionally distinct subcomplexes, Rgr1 and Srb4. To identify the structures and functions of each subcomplex, we expressed recombinant proteins for each subunit and assayed their interactions with each other and with basal transcription proteins. The Rgr1 subcomplex is composed of the Gal11 module, which binds activators, and the Med9/10 module. The Med9/10 module is required for both transcriptional activation and repression, and these activities appear to be carried out by two submodules. Proteins in the Med9 submodule interact physically and genetically with Srb10/11, suggesting that the Med9 submodule mediates the repression of pol II. Purified recombinant Srb4 subcomplex stimulated basal transcription of pol II but had little effect on activated transcription and phosphorylation of the C-terminal domain of the Rpb1 subunit of pol II. Both subcomplexes of Mediator interacted with a distinct set of basal transcription factors and pol II. The modular organization of Mediator and the associated functions suggest that the Mediator complex may recruit and/or stabilize the preinitiation complex through several points of contact with transcriptional regulators and basal transcription factors. PMID: 11555651 [PubMed - indexed for MEDLINE] 85: Mol Biol Cell 2001 Sep;12(9):2870-80 Control of microtubule dynamics by Stu2p is essential for spindle orientation and metaphase chromosome alignment in yeast. Kosco KA, Pearson CG, Maddox PS, Wang PJ, Adams IR, Salmon ED, Bloom K, Huffaker TC. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA. Stu2p is a member of a conserved family of microtubule-binding proteins and an essential protein in yeast. Here, we report the first in vivo analysis of microtubule dynamics in cells lacking a member of this protein family. For these studies, we have used a conditional Stu2p depletion strain expressing alpha-tubulin fused to green fluorescent protein. Depletion of Stu2p leads to fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase cells. The reduction in cytoplasmic microtubule dynamics is due primarily to decreases in both the catastrophe and rescue frequencies and an increase in the fraction of time microtubules spend pausing. These changes have significant consequences for the cell because they impede the ability of cytoplasmic microtubules to orient the spindle. In addition, recovery of fluorescence after photobleaching indicates that kinetochore microtubules are no longer dynamic in the absence of Stu2p. This deficiency is correlated with a failure to properly align chromosomes at metaphase. Overall, we provide evidence that Stu2p promotes the dynamics of microtubule plus-ends in vivo and that these dynamics are critical for microtubule interactions with kinetochores and cortical sites in the cytoplasm. PMID: 11553724 [PubMed - indexed for MEDLINE] 86: Mol Biol Cell 2001 Sep;12(9):2756-66 The GTPase effector domain sequence of the Dnm1p GTPase regulates self-assembly and controls a rate-limiting step in mitochondrial fission. Fukushima NH, Brisch E, Keegan BR, Bleazard W, Shaw JM. Department of Biology, University of Utah, Salt Lake City, UT 84112, USA. Dnm1p belongs to a family of dynamin-related GTPases required to remodel different cellular membranes. In budding yeast, Dnm1p-containing complexes assemble on the cytoplasmic surface of the outer mitochondrial membrane at sites where mitochondrial tubules divide. Our previous genetic studies suggested that Dnm1p's GTPase activity was required for mitochondrial fission and that Dnm1p interacted with itself. In this study, we show that bacterially expressed Dnm1p can bind and hydrolyze GTP in vitro. Coimmunoprecipitation studies and yeast two-hybrid analysis suggest that Dnm1p oligomerizes in vivo. With the use of the yeast two-hybrid system, we show that this Dnm1p oligomerization is mediated, in part, by a C-terminal sequence related to the GTPase effector domain (GED) in dynamin. The Dnm1p interactions characterized here are similar to those reported for dynamin and dynamin-related proteins that form higher order structures in vivo, suggesting that Dnm1p assembles to form rings or collars that surround mitochondrial tubules. Based on previous findings, a K705A mutation in the Dnm1p GED is predicted to interfere with GTP hydrolysis, stabilize active Dnm1p-GTP, and stimulate a rate-limiting step in fission. Here we show that expression of the Dnm1 K705A protein in yeast enhances mitochondrial fission. Our results provide evidence that the GED region of a dynamin-related protein modulates a rate-limiting step in membrane fission. PMID: 11553714 [PubMed - indexed for MEDLINE] 87: Mol Biol Cell 2001 Sep;12(9):2601-13 Dad1p, third component of the Duo1p/Dam1p complex involved in kinetochore function and mitotic spindle integrity. Enquist-Newman M, Cheeseman IM, Van Goor D, Drubin DG, Meluh PB, Barnes G. Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA. We showed recently that a complex between Duo1p and Dam1p is required for both spindle integrity and kinetochore function in the budding yeast Saccharomyces cerevisiae. To extend our understanding of the functions and interactions of the Duo1p/Dam1p complex, we analyzed the novel gene product Dad1p (for Duo1 and Dam1 interacting). Dad1p physically associates with Duo1p by two-hybrid analysis, coimmunoprecipitates with Duo1p and Dam1p out of yeast protein extracts, and shows interdependent localization with Duo1p and Dam1p to the mitotic spindle. These results indicate that Dad1p functions as a component of the Duo1p/Dam1p complex. Like Duo1p and Dam1p, Dad1p also localizes to kinetochore regions in chromosomes spreads. Here, we also demonstrate by chromatin immunoprecipitation that Duo1p, Dam1p, and Dad1p associate specifically with centromeric DNA in a manner that is dependent upon Ndc10 and partially dependent upon the presence of microtubules. To explore the functions of Dad1p in vivo, we generated a temperature-sensitive allele, dad1-1. This allele shows spindle defects and a mitotic arrest phenotype that is dependent upon the spindle assembly checkpoint. In addition, dad1-1 mutants undergo chromosome mis-segregation at the restrictive temperature, resulting in a dramatic decrease in viability. PMID: 11553702 [PubMed - indexed for MEDLINE] 88: J Clin Endocrinol Metab 2001 Sep;86(9):4416-23 Pitfalls in characterizing P450c17 mutations associated with isolated 17,20-lyase deficiency. Gupta MK, Geller DH, Auchus RJ. Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857, USA. The cytochrome P450c17 enzyme system performs both the 17alpha-hydroxylase and 17,20-lyase reactions in the human adrenal glands and gonads. This 17,20-lyase activity is required for the biosynthesis of dehydroepiandrosterone, the C(19) precursor of sex steroids. Considerable evidence supports the idea that the 17,20-lyase activity of this system is particularly sensitive to alterations in the interactions between P450c17 and its cofactor proteins P450-oxidoreductase and cytochrome b(5). We have described two patients with the clinical phenotype of isolated 17,20-lyase deficiency in whom single amino acid replacement mutations in the redox partner binding site of P450c17 (R347H and R358Q) selectively ablate 17,20-lyase activity while preserving most 17alpha-hydroxylase activity. We have shown by computer modeling and detailed biochemical studies that mutations R347H and R358Q impair the interactions of P450c17 with P450-oxidoreductase and cytochrome b(5) (redox partners). Another mutation reported to cause isolated 17,20-lyase deficiency (F417C) does not map within the redox partner binding site, but might nonetheless alter the interaction of the mutant protein with redox partners. To study the interaction of the F417C mutation with P450 oxidoreductase and cytochrome b(5), we expressed the cDNA for this protein in yeast microsomes, a heterologous expression system in which the composition of redox partner proteins can be varied systematically. Although the full-length protein was expressed in quantities comparable to those of wild-type P450c17 in this system, the F417C mutation did not form a classical P450 difference spectrum and was devoid of both 17alpha-hydroxylase and 17,20-lyase activities. To ensure that this result was not unique to the yeast expression system, we also expressed wild-type P450c17 and the F417C mutation in COS-7 cells, and we again found that the F417C mutation was expressed, but was not active. To conclusively demonstrate that a particular mutation in P450c17 causes isolated 17,20-lyase deficiency, accurate enzymatic studies of the mutant protein must reproducibly show activities consistent with the diagnosis. Mutations R347H and R358Q are the only two such mutations found in humans proven to cause isolated 17,20-lyase deficiency. PMID: 11549685 [PubMed - indexed for MEDLINE] 89: Eur Biophys J 2001 Aug;30(4):273-83 Fluoroalcohol-induced structural changes of proteins: some aspects of cosolvent-protein interactions. Gast K, Siemer A, Zirwer D, Damaschun G. Max-Delbruck-Centrum fur Molekulare Medizin Berlin-Buch, Germany. gast@mdc-berlin.de The conformational transitions of bovine beta-lactoglobulin A and phosphoglycerate kinase from yeast induced by hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) have been studied by dynamic light scattering and circular dichroism spectroscopy in order to elucidate the potential of fluoroalcohols to bring about structural changes of proteins. Moreover, pure fluoroalcohol-water mixed solvents were investigated to prove the relation between cluster formation and the effects on proteins. The results demonstrate that cluster formation is mostly an accompanying phenomenon because important structural changes of the proteins occur well below the critical concentration of fluoroalcohol at which the formation of clusters sets in. According to our light scattering experiments, the remarkable potential of HFIP is a consequence of extensive preferential binding. Surprisingly, preferential binding seems to play a vanishing role in the case of TFE. However, the comparable Stokes radii of both proteins in the highly helical state induced by either HFIP or TFE point to a similar degree of solvation in both mixed solvents. This shows that direct binding or an indirect mechanism must be equally taken into consideration to explain the effects of alcohols on proteins. The existence of a compact helical intermediate with non-native secondary structure on the transition of beta-lactoglobulin A from the native to the highly helical state is clearly demonstrated. PMID: 11548130 [PubMed - indexed for MEDLINE] 90: Oncogene 2001 Aug 30;20(38):5279-90 Toxicity of human adenovirus E4orf4 protein in Saccharomyces cerevisiae results from interactions with the Cdc55 regulatory B subunit of PP2A. Roopchand DE, Lee JM, Shahinian S, Paquette D, Bussey H, Branton PE. Department of Biochemistry, McGill University, McIntyre Medical Building, Montreal, Quebec, Canada, H3G 1Y6. The E4orf4 protein of human adenovirus induces p53-independent apoptosis, a process that may promote cell death and viral spread. When expressed alone, E4orf4 kills transformed cells but not normal human cells. The only clear target of E4orf4 in mammalian cells is the Balpha (B55) subunit of protein phosphatase 2A (PP2A), a member of one of three classes of regulatory B subunits. Here we report the effects of E4orf4 in Saccharomyces cerevisiae, which encodes two PP2A regulatory B subunits, CDC55 and RTS1, that share homology with mammalian B and B' subunits, respectively. E4orf4 expression was found to be toxic in yeast, resulting in the accumulation of cells in G2/M phase that failed to grow upon removal of E4orf4. E4orf4-expressing yeast also displayed an elongated cell morphology similar to cdc55 deletion strains. E4orf4 required CDC55 to elicit its effect, whereas RTS1 was dispensable. The recruitment of the PP2A holoenzyme by E4orf4 was entirely dependent on Cdc55. These studies indicate that E4orf4-induced apoptosis in mammalian cells and cell death in yeast require functional interactions with B-type subunits of PP2A. However, some inhibition of growth by E4orf4 was observed in the cdc55 strain and with an E4orf4 mutant that fails to interact with Cdc55, indicating that E4orf4 may possess a second Cdc55-independent function affecting cell growth. PMID: 11536041 [PubMed - indexed for MEDLINE] 91: Biochem J 2001 Sep 15;358(Pt 3):727-35 Binding of the merlin-I product of the neurofibromatosis type 2 tumour suppressor gene to a novel site in beta-fodrin is regulated by association between merlin domains. Neill GW, Crompton MR. Centre for Cutaneous Research, St Bartholomew's and the Royal London, Queen Mary and Westfield College, 2 Newark Street, London E1 2AT, UK. The mechanism underlying the tumour-suppressor activity of the neurofibromatosis type 2 (NF2) gene product, merlin, is largely undefined but there is evidence that the biological function of the protein might be mediated partly through interactions with the cytoskeleton. Merlin is expressed predominantly as two isoforms that differ at their C-termini owing to alternative splicing of exon 16. By expressing merlin isoform I as bait in a yeast two-hybrid screen, we isolated a clone encoding a region of the cytoskeletal protein beta-fodrin. Confirmation of the merlin-fodrin interaction was provided by using the mammalian two-hybrid system and binding assays in vitro. In addition, these assays and co-immunoprecipitation from mammalian cells revealed that the binding site for fodrin is located in the C-terminal half of merlin at a site that is masked in the native protein. Co-expression of the N-terminus of merlin decreased the interaction of its C-terminus with fodrin, implicating homophilic interactions of merlin isoform I in masking the fodrin-binding site. The effect of three disease-associated mutations on the merlin-fodrin interaction and merlin dimerization was also investigated. The mutation L535P, but not L360P or K413E, significantly decreased the merlin-fodrin interaction but not dimerization, indicating that the tumour suppressor ability of merlin might reside partly in its ability to interact with the cytoskeleton via fodrin. PMID: 11535133 [PubMed - indexed for MEDLINE] 92: Mol Cell Biol 2001 Oct;21(19):6606-14 Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by interactions between integrase and Sir4p. Xie W, Gai X, Zhu Y, Zappulla DC, Sternglanz R, Voytas DF. Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011-3260, USA. The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity. PMID: 11533248 [PubMed - indexed for MEDLINE] 93: Mol Cell Biol 2001 Oct;21(19):6429-39 A novel upstream RNA polymerase III promoter element becomes essential when the chromatin structure of the yeast U6 RNA gene is altered. Martin MP, Gerlach VL, Brow DA. Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706-1532, USA. The Saccharomyces cerevisiae U6 RNA gene, SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription of SNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position -30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele of SNR6 with decreased spacing between the A and B blocks, snr6-Delta42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-Delta42 is much more sensitive to mutations in a (dT-dA)(7) tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Delta42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription of SNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)(7) tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo. PMID: 11533232 [PubMed - indexed for MEDLINE] 94: EMBO J 2001 Sep 3;20(17):4935-43 The chromatin remodelling factor Brg-1 interacts with beta-catenin to promote target gene activation. Barker N, Hurlstone A, Musisi H, Miles A, Bienz M, Clevers H. Department of Immunology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Wnt-induced formation of nuclear Tcf-beta-catenin complexes promotes transcriptional activation of target genes involved in cell fate decisions. Inappropriate expression of Tcf target genes resulting from mutational activation of this pathway is also implicated in tumorigenesis. The C-terminus of beta-catenin is indispensable for the transactivation function, which probably reflects the presence of binding sites for essential transcriptional coactivators such as p300/CBP. However, the precise mechanism of transactivation remains unclear. Here we demonstrate an interaction between beta-catenin and Brg-1, a component of mammalian SWI/SNF and Rsc chromatin-remodelling complexes. A functional consequence of reintroduction of Brg-1 into Brg-1-deficient cells is enhanced activity of a Tcf-responsive reporter gene. Consistent with this, stable expression of inactive forms of Brg-1 in colon carcinoma cell lines specifically inhibits expression of endogenous Tcf target genes. In addition, we observe genetic interactions between the Brg-1 and beta-catenin homologues in flies. We conclude that beta-catenin recruits Brg-1 to Tcf target gene promoters, facilitating chromatin remodelling as a prerequisite for transcriptional activation. PMID: 11532957 [PubMed - indexed for MEDLINE] 95: EMBO J 2001 Sep 3;20(17):4684-93 Ski7p G protein interacts with the exosome and the Ski complex for 3'-to-5' mRNA decay in yeast. Araki Y, Takahashi S, Kobayashi T, Kajiho H, Hoshino S, Katada T. Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan. Two cytoplasmic mRNA-decay pathways have been characterized in yeast, and both are initiated by shortening of the 3'-poly(A) tail. In the major 5'-to-3' decay pathway, the deadenylation triggers removal of the 5'-cap, exposing the transcript body for 5'-to-3' degradation. An alternative 3'-to-5' decay pathway also follows the deadenylation and requires two multi-complexes: the exosome containing various 3'-exonucleases and the Ski complex consisting of the RNA helicase Ski2p, Ski3p and Ski8p. In addition, Ski7p, which has an N-terminal domain and a C-terminal elongation factor 1alpha-like GTP-binding domain, is involved in the 3'-to-5' decay. However, physical interaction between the exosome and the Ski complex, together with the function of Ski7p, has remained unknown. Here we report that the N domain of Ski7p is required and sufficient for the 3'-to-5' decay. Furthermore, the exosome and the Ski complex interact with the different regions of Ski7p N domain, and both interactions are required for the 3'-to-5' decay. Thus, Ski7p G protein appears to function as a signal-coupling factor between the two multi-complexes operating in the 3'-to-5' mRNA-decay pathway. PMID: 11532933 [PubMed - indexed for MEDLINE] 96: Virology 2001 Sep 1;287(2):266-74 Hepatitis B virus X protein interferes with cell viability through interaction with the p127-kDa UV-damaged DNA-binding protein. Lin-Marq N, Bontron S, Leupin O, Strubin M. Department of Genetics and Microbiology, University Medical Centre, Rue Michel-Servet 1, Geneva 4, 1211, Switzerland. The hepatitis B virus X protein (HBx) is essential for establishing natural viral infection and has been implicated in the development of liver cancer associated with chronic infection. The basis for HBx function in either process is not understood. In cell culture, HBx exhibits pleiotropic activities affecting transcription, DNA repair, cell growth, and apoptotic cell death. Numerous cellular proteins including the p127-kDa subunit of UV-damaged DNA-binding activity have been reported to interact with HBx but the functional significance of these interactions remains unclear. Here we show that the binding of HBx to p127 interferes with cell viability. Mutational analysis reveals that HBx contacts p127 via a region to which no function has been assigned previously. An HBx variant bearing a single-charge reversal substitution within this region loses p127 binding and concomitant cytotoxicity. This mutant regains activity when directly fused to p127. These studies confirm that p127 is an important cellular target of HBx, and they indicate that HBx does not exert its effect by sequestering p127, and thereby preventing its normal function, but instead by conferring to p127 a deleterious activity. Copyright 2001 Academic Press. PMID: 11531405 [PubMed - indexed for MEDLINE] 97: Methods Mol Biol 2001;177:319-28 Membrane recruitment systems for analysis of protein-protein interactions. Aronheim A. B. Rappaport Faculty of Medicine, Israel Institute of Technology, Haifa, Israel. PMID: 11530615 [PubMed - indexed for MEDLINE] 98: Methods Mol Biol 2001;177:261-70 The split-hybrid system. Uncoding multiprotein networks and defining mutations that affect protein interactions. Goldman PS, DeMaggio AJ, Goodman RH, Hoekstra MF. Icos Corporation, Bothell, WA, USA. PMID: 11530611 [PubMed - indexed for MEDLINE] 99: Methods Mol Biol 2001;177:241-59 One-hybrid systems for detecting protein-DNA interactions. Alexander MK, Bourns BD, Zakian VA. Lewis Thomas Lab, Princeton University, Princeton, NJ, USA. PMID: 11530610 [PubMed - indexed for MEDLINE] 100: Methods Mol Biol 2001;177:179-98 Protein interactions important in eukaryotic translation initiation. Asano K, Hinnebusch AG. Laboratory of Gene Regulation and Development, National Institute of Child Health and Development, Bethesda, MD, USA. PMID: 11530606 [PubMed - indexed for MEDLINE] 101: Methods Mol Biol 2001;177:151-9 Two-hybrid interactions confirmed by coimmunoprecipitation of epitope-tagged clones. Naumovski L. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA. PMID: 11530604 [PubMed - indexed for MEDLINE] 102: Methods Mol Biol 2001;177:135-50 Confirming yeast two-hybrid protein interactions using in vitro glutathione-S-transferase pulldowns. Kraichely DM, MacDonald PN. Proctor and Gamble, Cincinnati, OH, USA. PMID: 11530602 [PubMed - indexed for MEDLINE] 103: Nucleic Acids Res 2001 Sep 1;29(17):3566-75 DNA-binding activity and subunit interaction of the mariner transposase. Zhang L, Dawson A, Finnegan DJ. Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, UK. Mos1 is a member of the mariner/Tc1 family of transposable elements originally identified in Drosophila mauritiana. It has 28 bp terminal inverted repeats and like other elements of this type it transposes by a cut and paste mechanism, inserts at TA dinucleotides and codes for a transposase. This is the only protein required for transposition in vitro. We have investigated the DNA binding properties of Mos1 transposase and the role of transposase-transposase interactions in transposition. Purified transposase recognises the terminal inverted repeats of Mos1 due to a DNA-binding domain in the N-terminal 120 amino acids. This requires a putative helix-turn-helix motif between residues 88 and 108. Binding is preferentially to the right hand end, which differs at four positions from the repeat at the left end. Cleavage of Mos1 by transposase is also preferentially at the right hand end. Wild-type transposase monomers interact with each other in a yeast two-hybrid assay and we have used this to isolate mutations resulting in reduced interaction. These mutations lie along the length of the protein, indicating that transposase-transposase interactions are not due to a single interaction domain. One such mutation which retains both DNA-binding and catalytic activity has greatly reduced ability to excise Mos1 from plasmid DNA through coordinate cleavage of the two ends and transposition in vitro is lowered to a level 20-fold below that of the wild-type. This suggests that transposase-transposase interaction is required to form a synaptic complex necessary for coordinate cleavage at the ends of Mos1 during transposition. This mutant enzyme allows insertion at dinucleotides other than TA, including sequences with GC base pairs. This is the first example of a mariner/Tc1 transposase with altered target specificity. PMID: 11522826 [PubMed - indexed for MEDLINE] 104: Nucleic Acids Res 2001 Sep 1;29(17):3513-9 A relationship between gene expression and protein interactions on the proteome scale: analysis of the bacteriophage T7 and the yeast Saccharomyces cerevisiae. Grigoriev A. GPC Biotech, Fraunhoferstrasse 20, Martinsried 82152, Germany. andrei.grigoriev@gpc-biotech.com The relationship between the similarity of expression patterns for a pair of genes and interaction of the proteins they encode is demonstrated both for the simple genome of the bacteriophage T7 and the considerably more complex genome of the yeast Saccharomyces cerevisiae. Statistical analysis of large-scale gene expression and protein interaction data shows that protein pairs encoded by co-expressed genes interact with each other more frequently than with random proteins. Furthermore, the mean similarity of expression profiles is significantly higher for respective interacting protein pairs than for random ones. Such coupled analysis of gene expression and protein interaction data may allow evaluation of the results of large-scale gene expression and protein interaction screens as demonstrated for several publicly available datasets. The role of this link between expression and interaction in the evolution from monomeric to oligomeric protein structures is also discussed. PMID: 11522820 [PubMed - indexed for MEDLINE] 105: J Biol Chem 2001 Oct 26;276(43):39945-9 An accessible hydrophobic surface is a key element of the molecular chaperone action of Atp11p. Sheluho D, Ackerman SH. Department of Surgery, Wayne State University School of Medicine, 1225 Ellman Bldg., 421 E. Canfield Ave., Detroit, MI 48201, USA. Atp11p is a soluble protein of mitochondria that binds unassembled beta subunits of the F(1)-ATPase and prevents them from aggregating in the matrix. In this report, we show that Atp11p protects the insulin B chain from aggregating in vitro and therefore acts as a molecular chaperone. The chaperone action of Atp11p is mediated by hydrophobic interactions. An accessible hydrophobic surface in Atp11p was identified with the environment-sensitive fluorescent probe 1,1'-bis(4-anilino-5-napththalenesulfonic acid (bis-ANS). The spectral changes of bis-ANS in the presence of Atp11p indicate that the probe binds to a nonpolar region of the protein. Furthermore, the dye quenches the fluorescence of Atp11p tryptophan residues in a concentration-dependent manner. Although up to three molecules of bis-ANS can bind cooperatively to Atp11p, the binding of only one dye molecule is sufficient to virtually eliminate the chaperone activity of the protein. PMID: 11522798 [PubMed - indexed for MEDLINE] 106: Biochim Biophys Acta 2001 Aug 17;1506(2):89-102 Analysis of suppressor mutation reveals long distance interactions in the bc(1) complex of Saccharomyces cerevisiae. Brasseur G, Di Rago JP, Slonimski PP, Lemesle-Meunier D. Laboratoire de Bioenergetique et Ingenierie des Proteines, CNRS, Marseilles, France. brasseur@ibsm.cnrs-mrs.fr Four totally conserved glycines are involved in the packing of the two cytochrome b hemes, b(L) and b(H), of the bc(1) complex. The conserved glycine 131 is involved in the packing of heme b(L) and is separated by only 3 A from this heme in the bc(1) complex structure. The cytochrome b respiratory deficient mutant G131S is affected in the assembly of the bc(1) complex. An intragenic suppressor mutation was obtained at position 260, in the ef loop, where a glycine was replaced by an alanine. This respiratory competent revertant exhibited a low bc(1) complex activity and was affected in the electron transfer at the Q(P) site. The k(min) for the substrate DBH(2) was diminished by an order of magnitude and EPR spectra showed a partially empty Q(P) site. However, the binding of the Q(P) site inhibitors stigmatellin and myxothiazol remained unchanged in the suppressor strain. Optical spectroscopy revealed that heme b(L) is red shifted by 0.8 nm and that the E(m) of heme b(L) was slightly increased (+20 mV) in the revertant strain as compared to wild type strain values. Addition of a methyl group at position 260 is thus sufficient to allow the assembly of the bc(1) complex and the insertion of heme b(L) despite the presence of the serine at position 131. Surprisingly, reversion at position 260 was located 13 A away from the original mutation and revealed a long distance interaction in the yeast bc(1) complex. PMID: 11522251 [PubMed - indexed for MEDLINE] 107: J Biol Chem 2001 Oct 19;276(42):38394-9 Differential utilization of enzyme-substrate interactions for acylation but not deacylation during the catalytic cycle of Kex2 protease. Rockwell NC, Fuller RS. Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA. Kex2 protease from Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases belonging to the subtilase superfamily of serine proteases. Kex2 can be distinguished from degradative subtilisins on the basis of stringent substrate specificity and distinct pre-steady-state behavior. To better understand these mechanistic differences, we have examined the effects of substrate residues at P(1) and P(4) on individual steps in the Kex2 catalytic cycle with a systematic series of isosteric peptidyl amide and ester substrates. The results demonstrate that substrates based on known, physiological cleavage sites exhibit high acylation rates (> or =550 s(-1)) with Kex2. Substitution of Lys for the physiologically correct Arg at P(1) resulted in a > or =200-fold drop in acylation rate with almost no apparent effect on binding or deacylation. In contrast, substitution of the physiologically incorrect Ala for Nle at P(4) resulted in a much smaller defect in acylation and a modest but significant effect on binding with Lys at P(1). This substitution also had no effect on deacylation. These results demonstrate that Kex2 utilizes enzyme-substrate interactions in different ways at different steps in the catalytic cycle, with the S(1)-P(1) contact providing a key specificity determinant at the acylation step. PMID: 11514565 [PubMed - indexed for MEDLINE] 108: FEBS Lett 2001 Aug 17;503(2-3):196-200 The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors. Erskine PT, Coates L, Newbold R, Brindley AA, Stauffer F, Wood SP, Warren MJ, Cooper JB, Shoolingin-Jordan PM, Neier R. Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, UK. The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed. PMID: 11513881 [PubMed - indexed for MEDLINE] 109: Mol Cell Biol 2001 Sep;21(18):6270-9 H2A.Z is required for global chromatin integrity and for recruitment of RNA polymerase II under specific conditions. Adam M, Robert F, Larochelle M, Gaudreau L. Departement de Biologie, Universite de Sherbrooke, Sherbrooke, Quebec J1K 2R1, Canada. Evolutionarily conserved variant histone H2A.Z has been recently shown to regulate gene transcription in Saccharomyces cerevisiae. Here we show that loss of H2A.Z in this organism negatively affects the induction of GAL genes. Importantly, fusion of the H2A.Z C-terminal region to S phase H2A without its corresponding C-terminal region can mediate the variant histone's specialized function in GAL1-10 gene induction, and it restores the slow-growth phenotype of cells with a deletion of HTZ1. Furthermore, we show that the C-terminal region of H2A.Z can interact with some components of the transcriptional apparatus. In cells lacking H2A.Z, recruitment of RNA polymerase II and TATA-binding protein to the GAL1-10 promoters is significantly diminished under inducing conditions. Unexpectedly, we also find that H2A.Z is required to globally maintain chromatin integrity under GAL gene-inducing conditions. We hypothesize that H2A.Z can positively regulate gene transcription, at least in part, by modulating interactions with RNA polymerase II-associated factors at certain genes under specific cell growth conditions. PMID: 11509669 [PubMed - indexed for MEDLINE] 110: J Biol Chem 2001 Oct 26;276(43):40254-62 The DNA-binding domain of yeast heat shock transcription factor independently regulates both the N- and C-terminal activation domains. Bulman AL, Hubl ST, Nelson HC. Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 422 Curie Blvd., Philadelphia, PA 19104, USA. The expression of heat shock proteins in response to cellular stresses is dependent on the activity of the heat shock transcription factor (HSF). In yeast, HSF is constitutively bound to DNA; however, the mitigation of negative regulation in response to stress dramatically increases transcriptional activity. Through alanine-scanning mutagenesis of the surface residues of the DNA-binding domain, we have identified a large number of mutants with increased transcriptional activity. Six of the strongest mutations were selected for detailed study. Our studies suggest that the DNA-binding domain is involved in the negative regulation of both the N-terminal and C-terminal activation domains of HSF. These mutations do not significantly affect DNA binding. Circular dichroism analysis suggests that a subset of the mutants may have altered secondary structure, whereas a different subset has decreased thermal stability. Our findings suggest that the regulation of HSF transcriptional activity (under both constitutive and stressed conditions) may be partially dependent on the local topology of the DNA-binding domain. In addition, the DNA-binding domain may mediate key interactions with ancillary factors and/or other intramolecular regulatory regions in order to modulate the complex regulation of HSF's transcriptional activity. PMID: 11509572 [PubMed - indexed for MEDLINE] 111: J Biol Chem 2001 Nov 2;276(44):41205-12 Molecular interactions of the Gbeta binding domain of the Ste20p/PAK family of protein kinases. An isolated but fully functional Gbeta binding domain from Ste20p is only partially folded as shown by heteronuclear NMR spectroscopy. Song J, Chen Z, Xu P, Gingras R, Ng A, Leberer E, Thomas DY, Ni F. Biomolecular NMR Laboratory and the Montreal Joint Centre for Structural Biology, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada. The transmission of the mating signal of the budding yeast Saccharomyces cerevisiae requires Ste20p, a member of the serine/threonine protein kinases of the Ste20p/PAK family, to link the Gbeta subunit of the heterotrimeric G protein to the mitogen-activated protein kinase cascades. The binding site of Ste20p to the Gbeta subunit was mapped to a consensus sequence of SSLphiPLI/VXphiphibeta (X for any residue; phi for A, I, L, S or T; beta for basic residues), which was shown to be a novel Gbeta binding (GBB) motif present only in the noncatalytic C-terminal domains of the Ste20p/PAK family of protein kinases (Leeuw, T., Wu, C., Schrag, J. D., Whiteway, M., Thomas, D. Y., and Leberer, E. (1998) Nature 391, 191-195; Leberer, E., Dignard, D., Thomas, D. Y., and Leeuw, T. (2000) Biol. Chem. 381, 427-431). Here, we report the results of an NMR study on two GBB motif peptides and the entire C-terminal domain derived from Ste20p. The NMR data show that the two peptide fragments are not uniquely structured in aqueous solution, but in the presence of 40% trifluoroethanol, the longer 37-residue peptide exhibited two well defined, but flexibly linked helical structure elements. Heteronuclear NMR data indicate that the fully functional 86-residue C-terminal domain of Ste20p is again unfolded in aqueous solution but has helical secondary structure preferences similar to those of the two peptide fragments. The NMR results on the two GBB peptides and the entire GBB domain all indicate that the two important binding residues, Ser(879) and Ser(880), are located at the junction between two helical segments. These experimental observations with the prototype GBB domain of a novel family of Gbeta-controlled effectors may have important implications in understanding the molecular mechanisms of the signal transduction from the heterotrimeric G protein to the mitogen-activated protein kinase cascade. PMID: 11509560 [PubMed - indexed for MEDLINE] 112: Biometals 2001 Jun;14(2):99-112 Old iron, young copper: from Mars to Venus. Crichton RR, Pierre JL. Unite de Biochimie, Universite Catholique de Louvain, Belgium. Crichton@bioc.ucl.ac.be Iron and copper are metals which play an important role in the living world. From a brief consideration of their chemistry and biochemistry we conclude that the early chemistry of life used water soluble ferrous iron while copper was in the water-insoluble Cu(I) state as highly insoluble sulphides. The advent of oxygen was a catastrophic event for most living organisms, and can be considered to be the first general irreversible pollution of the earth. In contrast to the oxidation of iron and its loss of bioavailability as insoluble Fe(III), the oxidation of insoluble Cu(I) led to soluble Cu(II). A new iron biochemistry became possible after the advent of oxygen, with the development of chelators of Fe(III), which rendered iron once again accessible, and with the control of the potential toxicity of iron by its storage in a water soluble, non-toxic, bio-available storage protein (ferritin). Biology also discovered that whereas enzymes involved in anaerobic metabolism were designed to operate in the lower portion of the redox spectrum, the arrival of dioxygen created the need for a new redox active metal which could attain higher redox potentials. Copper, now bioavailable, was ideally suited to exploit the oxidizing power of dioxygen. The arrival of copper also coincided with the development of multicellular organisms which had extracellular cross-linked matrices capable of resisting attack by oxygen free radicals. After the initial 'iron age' subsequent evolution moved, not towards a 'copper age', but rather to an 'iron-copper' age. In the second part of the review, this symbiosis of iron and copper is examined in yeast. We then briefly consider iron and copper metabolism in mammals, before looking at iron-copper interactions in mammals, particularly man, and conclude with the reflection that, as in Greek and Roman mythology, a better understanding of the potentially positive interactions between Mars (iron) and Venus (copper) can only be to the advantage of our species. Publication Types: Review Review, Tutorial PMID: 11508852 [PubMed - indexed for MEDLINE] 113: EMBO J 2001 Aug 15;20(16):4577-87 Mechanistic aspects of DnaA-RepA interaction as revealed by yeast forward and reverse two-hybrid analysis. Sharma R, Kachroo A, Bastia D. Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA. Using yeast forward and reverse two-hybrid analysis and biochemical techniques, we present novel and definitive in vivo and in vitro evidence that both the N-terminal domain I and C-terminal domain IV of the host-encoded DnaA initiator protein of Escherichia coli interact physically with plasmid-encoded RepA initiator of pSC101. The N-terminal, but not the C-terminal, region of RepA interacted with DnaA in vitro. These protein-protein interactions are critical for two very early steps of replication initiation, namely origin unwinding and helicase loading. Neither domain I nor IV of DnaA could individually collaborate with RepA to promote pSC101 replication. However, when the two domains are co-expressed within a common cell milieu and allowed to associate non-covalently with each other via a pair of leucine zippers, replication of the plasmid was supported in vivo. Thus, the result shows that physical tethering, either non-covalent or covalent, of domain I and IV of DnaA and interaction of both domains with RepA, are critical for replication initiation. The results also provide the molecular basis for a novel, potential, replication-based bacterial two-hybrid system. PMID: 11500384 [PubMed - indexed for MEDLINE] 114: RNA 2001 Aug;7(8):1084-96 Ribosomal protein L5 helps anchor peptidyl-tRNA to the P-site in Saccharomyces cerevisiae. Meskauskas A, Dinman JD. Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854, USA. Our previous demonstration that mutants of 5S rRNA called mof9 can specifically alter efficiencies of programmed ribosomal frameshifting (PRF) suggested a role for this ubiquitous molecule in the maintenance of translational reading frame, though the repetitive nature of the 5S rDNA gene (>100 copies/cell) inhibited more detailed analyses. However, given the known interactions between 5S rRNA and ribosomal protein L5 (previously called L1 or YL3) encoded by an essential, single-copy gene, we monitored the effects of a series of well-defined rpl5 mutants on PRF and virus propagation. Consistent with the mof9 results, we find that the rpl5 mutants promoted increased frameshifting efficiencies in both the -1 and +1 directions, and conferred defects in the ability of cells to propagate two endogenous viruses. Biochemical analyses demonstrated that mutant ribosomes had decreased affinities for peptidyl-tRNA. Pharmacological studies showed that sparsomycin, a peptidyltransferase inhibitor that specifically increases the binding of peptidyl-tRNA with ribosomes, was antagonistic to the frameshifting defects of the most severe mutant, and the extent of sparsomycin resistance correlated with the severity of the frameshifting defects in all of the mutants. These results provide biochemical and physiological evidence that one function of L5 is to anchor peptidyl-tRNA to the P-site. A model is presented describing how decreased affinity of ribosomes for peptidyl-tRNA can affect both -1 and +1 frameshifting, and for the effects of sparsomycin. PMID: 11497428 [PubMed - indexed for MEDLINE] 115: Microbiology 2001 Aug;147(Pt 8):2007-19 A GAS-like gene family in the pathogenic fungus Candida glabrata. Weig M, Haynes K, Rogers TR, Kurzai O, Frosch M, Muhlschlegel FA. Institut fur Hygiene und Mikrobiologie, Universitat Wurzburg, Josef-Schneider-Str. 2, 97080 Wurzburg, Germany. In fungi, the cell wall plays a major role in host-pathogen interactions. Despite this, little is known about the molecular basis of cell wall assembly in Candida glabrata, which has emerged as the second most common cause of systemic candidosis. A C. glabrata gene family, CgGAS1-3, that shares significant homologies with both the GAS1 gene of Saccharomyces cerevisiae, which is necessary for cell wall assembly, and the pH-regulated genes PHR1 and PHR2 of Candida albicans, which are involved in cell wall assembly and required for virulence, has been cloned. Among the members of this family, CgGAS1-3 display a unique expression pattern. Both CgGAS1 and CgGAS2 are constitutively expressed. In contrast, CgGAS3 transcript was not detectable under any of the assayed conditions. The C. glabrata actin gene, CgACT1, has also been cloned to be used as a meaningful loading control in Northern blots. CgGAS1 and CgGAS2 were deleted by two different methodological approaches. A rapid PCR-based strategy by which gene disruption was achieved with short regions of homology (50 bp) was applied successfully to C. glabrata. DeltaCggas1 or DeltaCggas2 cells demonstrated similar aberrant morphologies, displaying an altered bud morphology and forming floccose aggregates. These phenotypes suggest a role for CgGAS1 and CgGAS2 in cell wall biosynthesis. Further evidence for this hypothesis was obtained by successful functional complementation of a gas1 null mutation in S. cerevisiae with the C. glabrata CgGAS1 or CgGAS2 gene. PMID: 11495979 [PubMed - indexed for MEDLINE] 116: Oncogene 2001 Jul 5;20(30):3995-4006 Multiple signaling interactions of Abl and Arg kinases with the EphB2 receptor. Yu HH, Zisch AH, Dodelet VC, Pasquale EB. The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, California, CA 92037, USA. The Eph family of receptor tyrosine kinases and the Abl family of non-receptor tyrosine kinases have both been implicated in tissue morphogenesis. They regulate the organization of the actin cytoskeleton in the developing nervous system and participate in signaling pathways involved in axon growth. Both Eph receptors and Abl are localized in the neuronal growth cone, suggesting that they play a role in axon pathfinding. Two-hybrid screens identified regions of Abl and Arg that bind to the EphB2 and EphA4 receptors, suggesting a novel signaling connection involving the two kinase families. The association of full-length Abl and Arg with EphB2 was confirmed by co-immunoprecipitation and found to involve several distinct protein interactions. The SH2 domains of Abl and Arg bind to tyrosine-phosphorylated motifs in the juxtamembrane region of EphB2. A second, phosphorylation-independent interaction with EphB2 involves non-conserved sequences in the C-terminal tails of Abl and Arg. A third interaction between Abl and EphB2 is probably mediated by an intermediary protein because it requires tyrosine phosphorylation of EphB2, but not the binding sites for the Abl SH2 domain. The connection between EphB2 and Abl/Arg appears to be reciprocal. Activated EphB2 causes tyrosine phosphorylation of Abl and Arg, and vice versa. Interestingly, treatment of COS cells and B35 neuronal-like cells with ephrin-B1 to activate endogenous EphB2 decreased the kinase activity of endogenous Abl. These data are consistent with the opposite effects that Eph receptors and Abl have on neurite ougrowth and suggest that Eph receptors and Abl family kinases have shared signaling activities. PMID: 11494128 [PubMed - indexed for MEDLINE] 117: Proc Natl Acad Sci U S A 2001 Aug 14;98(17):9648-53 The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells. Matern HT, Yeaman C, Nelson WJ, Scheller RH. Genentech, Inc., Department of Richard Scheller, South San Francisco, CA 94080-4990, USA. The yeast exocyst complex (also called Sec6/8 complex in higher eukaryotes) is a multiprotein complex essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. It is composed of eight proteins (Sec3, -5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weights ranging from 70 to 144 kDa. Mammalian orthologues for seven of these proteins have been described and here we report the cloning and initial characterization of the remaining subunit, Sec3. Human Sec3 (hSec3) shares 17% sequence identity with yeast Sec3p, interacts in the two-hybrid system with other subunits of the complex (Sec5 and Sec8), and is expressed in almost all tissues tested. In yeast, Sec3p has been proposed to be a spatial landmark for polarized secretion (1), and its localization depends on its interaction with Rho1p (2). We demonstrate here that hSec3 lacks the potential Rho1-binding site and GFP-fusions of hSec3 are cytosolic. Green fluorescent protein (GFP)-fusions of nearly every subunit of the mammalian Sec6/8 complex were expressed in Madin-Darby canine kidney (MDCK) cells, but they failed to assemble into a complex with endogenous proteins and localized in the cytosol. Of the subunits tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact when expressed in MDCK cells. Cells overexpressing GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70 targeting interaction is critical for normal development of polarized epithelial cells. PMID: 11493706 [PubMed - indexed for MEDLINE] 118: J Cell Biol 2001 Aug 6;154(3):549-71 A protein interaction map for cell polarity development. Drees BL, Sundin B, Brazeau E, Caviston JP, Chen GC, Guo W, Kozminski KG, Lau MW, Moskow JJ, Tong A, Schenkman LR, McKenzie A 3rd, Brennwald P, Longtine M, Bi E, Chan C, Novick P, Boone C, Pringle JR, Davis TN, Fields S, Drubin DG. Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed. PMID: 11489916 [PubMed - indexed for MEDLINE] 119: J Biol Chem 2001 Oct 19;276(42):38820-9 Functional analysis of the hydrophobic patch on nuclear transport factor 2 involved in interactions with the nuclear pore in vivo. Quimby BB, Leung SW, Bayliss R, Harreman MT, Thirumala G, Stewart M, Corbett AH. Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA. Nuclear transport factor 2 (NTF2) is a small homodimeric protein that interacts simultaneously with both RanGDP and FxFG nucleoporins. The interaction between NTF2 and Ran is essential for the import of Ran into the nucleus. Here we use mutational analysis to dissect the in vivo role of the interaction between NTF2 and nucleoporins. We identify a series of surface residues that form a hydrophobic patch on NTF2, which when mutated disrupt the NTF2-nucleoporin interaction. Analysis of these mutants in vivo demonstrates that the strength of this interaction can be significantly reduced without affecting cell viability. However, cells cease to be viable if the interaction between NTF2 and nucleoporins is abolished completely, indicating that this interaction is essential for the function of NTF2 in vivo. In addition, we have isolated a dominant negative mutant of NTF2, N77Y, which has increased affinity for nucleoporins. Overexpression of the N77Y protein blocks nuclear protein import and concentrates Ran at the nuclear rim. These data support a mechanism in which NTF2 interacts transiently with FxFG nucleoporins to translocate through the pore and import RanGDP into the nucleus. PMID: 11489893 [PubMed - indexed for MEDLINE] 120: Traffic 2001 Aug;2(8):565-76 Clathrin interactions with C-terminal regions of the yeast AP-1 beta and gamma subunits are important for AP-1 association with clathrin coats. Yeung BG, Payne GS. Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90095-1737, USA. Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo recruitment and clathrin assembly during clathrin-coated vesicle biogenesis. We have identified, and characterized the physiological significance of clathrin-binding activities in the two large subunits of the AP-1 complex in Saccharomyces cerevisiae. Using GST-fusion chromatography, two clathrin-binding sites were defined in the beta1 subunit that match consensus clathrin-binding sequences in other mammalian and yeast clathrin-binding proteins. Clathrin interactions were also identified with the C-terminal region of the gamma subunit. When introduced into chromosomal genes, point mutations in the beta1 clathrin-binding motifs, or deletion of the gamma C-terminal region, reduced association of AP-1 with clathrin in coimmunoprecipitation assays. The beta1 mutations or the gamma truncation individually produced minor effects on AP-1 distribution by subcellular fractionation. However, when beta1 and gamma mutations were combined, severe defects were observed in AP-1 association with membranes and incorporation into clathrin-coated vesicles. The combination of subunit mutations accentuated growth and alpha-factor pheromone maturation defects in chc1-ts cells, though not to the extent caused by complete loss of AP-1 activity. Our results suggest that both the beta1 and gamma subunits contribute interactions with clathrin that are important for stable assembly of AP-1 complexes into clathrin coats in vivo. PMID: 11489214 [PubMed - indexed for MEDLINE] 121: J Autoimmun 2001 Aug;17(1):51-61 Expression of protein tyrosine phosphatase-like molecule ICA512/IA-2 induces growth arrest in yeast cells and transfected mammalian cell lines. Papakonstantinou T, Myers MA, Jois J, Roucou X, Prescott M, Rowley MJ, Mackay IR. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168, Australia. The ICA512/IA-2 molecule, a protein with similarity to receptor-type protein tyrosine phosphatases, was discovered during studies to identify autoantigens in Type 1 diabetes. The biological function of ICA512/IA-2 is unknown. We describe striking effects of ICA512/IA-2 on viability and growth of both yeast cells and cultured mammalian cells. In transformed yeast Saccharomyces cerevisiae cells, expression of ICA512/IA-2 induced growth retardation as judged by measurements of optical density and counts of colony-forming units. In contrast, expression of the intracellular domain (amino acids 600-979) of ICA512/IA-2 in yeast or mammalian cells had no such effects. In investigations on apoptosis, expression of ICA512/IA-2 in yeast cells caused loss of plasma membrane asymmetry, but not release of cytochrome c from mitochondria which did occur in a control system after expression of the pro-apoptotic molecule Bax. Possible interactions between ICA512/IA-2 and components of the cytoskeleton were not supported by studies on staining of fixed yeast cells with phalloidin-Texas Red. With transfected mammalian cell lines COS-7 and NIH3T3, expression of ICA512/IA-2 likewise induced growth arrest, with some of the morphological features of apoptosis. Thus obligatory expression of ICA512/IA-2 in eukaryotic cells causes disruption of cellular activities, with growth arrest in yeast and nuclear pycnosis/fragmentation in mammalian cells. A possible explanation is that growth inhibition reflects a part of the presently unknown function of ICA512/IA-2. Copyright 2001 Academic Press. PMID: 11488637 [PubMed - indexed for MEDLINE] 122: J Biol Chem 2001 Sep 21;276(38):35644-51 Interactions in the error-prone postreplication repair proteins hREV1, hREV3, and hREV7. Murakumo Y, Ogura Y, Ishii H, Numata S, Ichihara M, Croce CM, Fishel R, Takahashi M. Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. murakumo@med.nagoya-u.ac.jp Most mutations after DNA damage in yeast Saccharomyces cerevisiae are induced by error-prone translesion DNA synthesis employing scRev1 and DNA polymerase zeta that consists of scRev3 and scRev7 proteins. Recently, the human REV1 (hREV1) and REV3 (hREV3) genes were identified, and their products were revealed to be involved in UV-induced mutagenesis, as observed for their yeast counterparts. Human REV7 (hREV7) was also cloned, and its product was found to interact with hREV3, but the biological function of hREV7 remained unknown. We report here the analyses of precise interactions in the human REV proteins. The interaction between hREV1 and hREV7 was identified by the yeast two-hybrid library screening using a bait of hREV7, which was confirmed by in vitro and in vivo binding assays. The homodimerization of hREV7 was also detected in the two-hybrid analysis. In addition, the precise domains for interaction between hREV7 and hREV1 or hREV3 and for hREV7 homodimerization were determined. Although hREV7 interacts with both hREV1 and hREV3, a stable complex formation of the three proteins was undetectable in vitro. These findings suggest the possibility that hREV7 might play an important role in regulating the enzymatic activities of hREV1 and hREV3 for mutagenesis in response to DNA damage. PMID: 11485998 [PubMed - indexed for MEDLINE] 123: Genes Dev 2001 Aug 1;15(15):1957-70 Characterization of U4 and U6 interactions with the 5' splice site using a S. cerevisiae in vitro trans-splicing system. Johnson TL, Abelson J. Division of Biology, California Institute of Technology, Pasadena, California 91125, USA. Spliceosome assembly has been characterized as the ordered association of the snRNP particles U1, U2, and U4/U6.U5 onto pre-mRNA. We have used an in vitro trans-splicing/cross-linking system in Saccharomyces cerevisiae nuclear extracts to examine the first step of this process, 5' splice site recognition. This trans-splicing reaction has ATP, Mg(2+), and splice-site sequence requirements similar to those of cis-splicing reactions. Using this system, we identified and characterized a novel U4-5' splice site interaction that is ATP-dependent, but does not require the branch point, the 3' splice site, or the 5' end of the U1 snRNA. Additionally, we identified several ATP-dependent U6 cross-links at the 5' splice site, indicating that different regions of U6 sample it before a U6-5' splice site interaction is stabilized that persists through the first step of splicing. This work provides evidence for ATP-dependent U4/U6 association with the 5' splice site independent of ATP-mediated U2 association with the branch point. Furthermore, it defines specific nucleotides in U4 and U6 that interact with the 5' splice site at this early stage, even in the absence of base-pairing with the U1 snRNA. PMID: 11485990 [PubMed - indexed for MEDLINE] 124: Biochem J 2001 Aug 15;358(Pt 1):7-16 A large family of endosome-localized proteins related to sorting nexin 1. Teasdale RD, Loci D, Houghton F, Karlsson L, Gleeson PA. R.W. Johnson Pharmaceutical Research Institute, 3210 Merryfield Row, San Diego, CA 92121, USA. r.teasdale@imb.uq.edu.au Sorting nexin 1 (SNX1), a peripheral membrane protein, has previously been shown to regulate the cell-surface expression of the human epidermal growth factor receptor [Kurten, Cadena and Gill (1996) Science 272, 1008-1010]. Searches of human expressed sequence tag databases with SNX1 revealed eleven related human cDNA sequences, termed SNX2 to SNX12, eight of them novel. Analysis of SNX1-related sequences in the Saccharomyces cerevisiae genome clearly shows a greatly expanded SNX family in humans in comparison with yeast. On the basis of the predicted protein sequences, all members of this family of hydrophilic molecules contain a conserved 70-110-residue Phox homology (PX) domain, referred to as the SNX-PX domain. Within the SNX family, subgroups were identified on the basis of the sequence similarities of the SNX-PX domain and the overall domain structure of each protein. The members of one subgroup, which includes human SNX1, SNX2, SNX4, SNX5 and SNX6 and the yeast Vps5p and YJL036W, all contain coiled-coil regions within their large C-terminal domains and are found distributed in both membrane and cytosolic fractions, typical of hydrophilic peripheral membrane proteins. Localization of the human SNX1 subgroup members in HeLa cells transfected with the full-length cDNA species revealed a similar intracellular distribution that in all cases overlapped substantially with the early endosome marker, early endosome autoantigen 1. The intracellular localization of deletion mutants and fusions with green fluorescent protein showed that the C-terminal regions of SNX1 and SNX5 are responsible for their endosomal localization. On the basis of these results, the functions of these SNX molecules are likely to be unique to endosomes, mediated in part by interactions with SNX-specific C-terminal sequences and membrane-associated determinants. PMID: 11485546 [PubMed - indexed for MEDLINE] 125: EMBO J 2001 Aug 1;20(15):4041-54 Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes. Hoogenraad CC, Akhmanova A, Howell SA, Dortland BR, De Zeeuw CI, Willemsen R, Visser P, Grosveld F, Galjart N. MGC Department of Cell Biology, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands. Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein- dynactin interaction on the surface of membranous organelles, by associating with these complexes. PMID: 11483508 [PubMed - indexed for MEDLINE] 126: EMBO J 2001 Aug 1;20(15):4035-40 Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion. Kato M, Wickner W. Department of Biochemistry, Dartmouth Medical School, Vail Building, Hanover, NH 03755-3844, USA. In vitro homotypic fusion of yeast vacuoles occurs in three stages: priming, the Sec18 (NSF)-mediated changes that precede vacuole association; docking, the Ypt7 and SNARE-mediated pairing of vacuoles; and fusion, mediated by calmodulin/V0/t-SNARE interactions. Defects in catalysts of each stage result in fragmented (unfused) vacuoles. Strains with deletions in any of ERG genes 3-6, lacking normal ergosterol biosynthesis, have fragmented vacuoles. The ergosterol ligands filipin, nystatin and amphotericin B block the in vitro fusion of vacuoles from wild-type cells. Each of these inhibitors acts at the priming stage to inhibit Sec17p release from vacuoles. A reversible delay in Sec18p action prevents vacuoles from acquiring resistance to any of these three drugs, confirming that their action is on the normal fusion pathway. Ergosterol or cholesterol delivery to wild-type vacuoles stimulates their in vitro fusion, and the in vitro fusion of ergDelta vacuoles requires added sterol. The need for ergosterol for vacuole priming underscores the role of lipids in organizing the membrane elements of this complex reaction. PMID: 11483507 [PubMed - indexed for MEDLINE] 127: EMBO J 2001 Aug 1;20(15):3938-46 Novel modular domain PB1 recognizes PC motif to mediate functional protein-protein interactions. Ito T, Matsui Y, Ago T, Ota K, Sumimoto H. Division of Genome Biology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan. titolab@kenroku.kanazawa-u.ac.jp Modular domains mediating specific protein-protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67(phox), which activates the microbicidal phagocyte NADPH oxidase. Each of these specifically recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40(phox) (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67(phox), which abolishes its interaction with Cdc24p, confers on cells temperature- sensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif-containing region of p40(phox), which restores the interaction with the altered Bem1p. This domain-swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules. PMID: 11483497 [PubMed - indexed for MEDLINE] 128: Proc Natl Acad Sci U S A 2001 Aug 14;98(17):9581-6 Kinetic trapping of DNA by transcription factor IIIB. Cloutier TE, Librizzi MD, Mollah AK, Brenowitz M, Willis IM. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA. High levels of RNA polymerase III gene transcription are achieved by facilitated recycling of the polymerase on transcription factor IIIB (TFIIIB)-DNA complexes that are stable through multiple rounds of initiation. TFIIIB-DNA complexes in yeast comprise the TATA-binding protein (TBP), the TFIIB-related factor TFIIIB70, and TFIIIB90. The high stability of the TFIIIB-DNA complex is conferred by TFIIIB90 binding to TFIIIB70-TBP-DNA complexes. This stability is thought to result from compound bends introduced in the DNA by TBP and TFIIIB90 and by protein-protein interactions that obstruct DNA dissociation. Here we present biochemical evidence that the high stability of TFIIIB-DNA complexes results from kinetic trapping of the DNA. Thermodynamic analysis shows that the free energies of formation of TFIIIB70-TBP-DNA (DeltaG degrees = -12.10 +/- 0.12 kcal/mol) and TFIIIB-DNA (DeltaG degrees = -11.90 +/- 0.14 kcal/mol) complexes are equivalent whereas a kinetic analysis shows that the half-lives of these complexes (46 +/- 3 min and 95 +/- 6 min, respectively) differ significantly. The differential stability of these isoenergetic complexes demonstrates that TFIIIB90 binding energy is used to drive conformational changes and increase the barrier to complex dissociation. PMID: 11481428 [PubMed - indexed for MEDLINE] 129: Proc Natl Acad Sci U S A 2001 Aug 14;98(17):9760-5 Interactions of Exo1p with components of MutLalpha in Saccharomyces cerevisiae. Tran PT, Simon JA, Liskay RM. Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland 97201, USA. Previously, we reported evidence suggesting that Saccharomyces cerevisiae MutLalpha, composed of Mlh1p and Pms1p, was a functional member of the gyrase b/Hsp90/MutL (GHL) dimeric ATPase superfamily characterized by highly conserved ATPase domains. Similar to other GHL ATPases, these putative ATPase domains of MutLalpha may be important for the recruitment and/or activation of downstream effectors. One downstream effector candidate is Exo1p, a 5'-3' double stranded DNA exonuclease that has previously been implicated in DNA mismatch repair (MMR). Here we report yeast two-hybrid results suggesting that Exo1p can interact physically with MutLalpha through the Mlh1p subunit. We also report epistasis analysis involving MutLalpha ATPase mutations combined with exo1Delta. One interpretation of our genetic results is that MutLalpha ATPase domains function to direct Exo1p and other functionally redundant exonucleases during MMR. Finally, our results show that much of the increase in spontaneous mutation observed in an exo1Delta strain is REV3-dependent, in turn suggesting that Exo1p is also involved in one or more MMR-independent mutation avoidance pathways. PMID: 11481425 [PubMed - indexed for MEDLINE] 130: Nat Genet 2001 Aug;28(4):303-4 Comment on: Nat Genet. 2001 Aug;28(4):327-34. To bind or not to bind. Biggin MD. Gene expression is regulated by transcription factors binding selectively to particular portions of the genome. To what extent are these protein-DNA interactions influenced by the intrinsic sequence-specific recognition properties at each protein, and to what extent are they affected by other factors, such as chromatin structure or cooperative interactions with other proteins. Genome-wide surveys of DNA binding by transcription factors in vivo are beginning to provide some answers. Publication Types: Comment News PMID: 11479583 [PubMed - indexed for MEDLINE] 131: J Biol Chem 2001 Sep 28;276(39):36295-302 Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell nuclear antigen (PCNA) is independent of its in vitro interaction via a consensus PCNA binding region. Frank G, Qiu J, Zheng L, Shen B. Department of Cell and Tumor Biology, City of Hope National Medical Center, Duarte, California 91010, USA. Interaction between human flap endonuclease-1 (hFEN-1) and proliferating cell nuclear antigen (PCNA) represents a good model for interactions between multiple functional proteins involved in DNA metabolic pathways. A region of 9 conserved amino acid residues (residues Gln-337 through Lys-345) in the C terminus of human FEN-1 (hFEN-1) was shown to be responsible for the interaction with PCNA. Our current study indicates that 4 amino acid residues in hFEN-1 (Leu-340, Asp-341, Phe-343, and Phe-344) are critical for human PCNA (hPCNA) interaction. A conserved PCNA interaction motif in various proteins from assorted species has been defined as Q(1)X(2)X(3)(L/I)(4)X(5)X(6)F(7)(F/Y)(8), although our results fail to implicate Q(1) (Gln-337 in hFEN-1) as a crucial residue. Surprisingly, all hFEN-1 mutants, including L340A, D341A, F343A, and F344A, retained hPCNA-mediated stimulation of both exo- and flap endonuclease activities. Furthermore, our in vitro assay showed that hPCNA failed to bind to the scRad27 (yeast homolog of FEN-1) nuclease. However, its nuclease activities were significantly enhanced in the presence of hPCNA. Four additional Saccharomyces cerevisiae scRad27 mutants, including multiple alanine mutants and a deletion mutant of the entire PCNA binding region, were constructed to confirm this result. All of these mutants retained PCNA-driven nuclease activity stimulation. We therefore conclude that stimulation of eukaryotic hFEN-1 nuclease activities by PCNA is independent of its in vitro interaction via the PCNA binding region. PMID: 11477073 [PubMed - indexed for MEDLINE] 132: Science 2001 Sep 14;293(5537):2101-5 Global analysis of protein activities using proteome chips. Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, Bertone P, Lan N, Jansen R, Bidlingmaier S, Houfek T, Mitchell T, Miller P, Dean RA, Gerstein M, Snyder M. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA. To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications. PMID: 11474067 [PubMed - indexed for MEDLINE] 133: Nat Struct Biol 2001 Aug;8(8):695-700 Erratum in: Nat Struct Biol 2002 Mar;9(3):231 A histone fold TAF octamer within the yeast TFIID transcriptional coactivator. Selleck W, Howley R, Fang Q, Podolny V, Fried MG, Buratowski S, Tan S. Center for Gene Regulation, Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802-1014, USA. Gene activity in a eukaryotic cell is regulated by accessory factors to RNA polymerase II, which include the general transcription factor complex TFIID, composed of TBP and TBP-associated factors (TAFs). Three TAFs that contain histone fold motifs (yTAF17, yTAF60 and yTAF61) are critical for transcriptional regulation in the yeast Saccharomyces cerevisiae and are found in both TFIID and SAGA, a multicomponent histone acetyltransferase transcriptional coactivator. Although these three TAFs were proposed to assemble into a pseudooctamer complex, we find instead that yTAF17, yTAF60 and yTAF61 form a specific TAF octamer complex with a fourth TAF found in TFIID, yTAF48. We have reconstituted this complex in vitro and established that it is an octamer containing two copies each of the four components. Point mutations within the histone folds disrupt the octamer in vitro, and temperature-sensitive mutations in the histone folds can be specifically suppressed by overexpressing the other TAF octamer components in vivo. Our results indicate that the TAF octamer is similar both in stoichiometry and histone fold interactions to the histone octamer component of chromatin. PMID: 11473260 [PubMed - indexed for MEDLINE] 134: Biochim Biophys Acta 2001 Jun 29;1532(3):234-47 A genetic screen for ethanolamine auxotrophs in Saccharomyces cerevisiae identifies a novel mutation in Mcd4p, a protein implicated in glycosylphosphatidylinositol anchor synthesis. Storey MK, Wu WI, Voelker DR. Department of Medicine, Program in Cell Biology, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA. A genetic screen for ethanolamine auxotrophs has identified a novel mutant allele of the morphogenesis checkpoint dependent (MCD)-4 gene, designated mcd4-P301L. In the presence of a null allele for the phosphatidylserine (PtdSer) decarboxylase 1 gene (psd1 Delta), the mcd4-P301L mutation confers temperature sensitivity for growth on minimal medium. This growth defect is reversed by either ethanolamine or choline supplementation. Incubation of mutant cells with [(3)H]serine followed by analysis of the aminoglycerophospholipids demonstrated a 60% decrease in phosphatidylethanolamine (PtdEtn) formation compared to parental cells. Chemical analysis of phospholipid content after culture under non-permissive conditions also demonstrated a 60% decrease in the PtdEtn pool compared to the parental strain. Although the morphogenesis checkpoint dependent (MCD)-4 gene and its homologues have been shown to play a role in glycosylphosphatidylinositol (GPI) anchor synthesis, the mcd4-P301L strain displayed normal incorporation of [(3)H]inositol into both proteins and lipids. Thus, a defect in GPI anchor synthesis does not explain either the ethanolamine auxotrophy or biochemical phenotype of this mutant. We also examined the growth characteristics and PtdSer metabolism of a previously described mcd4-174 mutant strain, with defects in GPI anchor synthesis, protein modification and cell wall maintenance. The mcd4-174, psd1 Delta strain is a temperature sensitive ethanolamine auxotroph that requires osmotic support for growth, and displays normal PtdEtn formation compared to parental cells. These results reveal important genetic interactions between PSD1 and MCD4 genes, and provide evidence that Mcd4p can modulate aminoglycerophospholipid metabolism, in a way independent of its role in GPI anchor synthesis. PMID: 11470244 [PubMed - indexed for MEDLINE] 135: Biochemistry 2001 Jul 31;40(30):9049-58 Temperature-induced denaturation and renaturation of triosephosphate isomerase from Saccharomyces cerevisiae: evidence of dimerization coupled to refolding of the thermally unfolded protein. Benitez-Cardoza CG, Rojo-Dominguez A, Hernandez-Arana A. Area de Biofisicoquimica, Departamento de Quimica, Universidad Autonoma Metropolitana-Iztapalapa, Apartado Postal 55-534, Iztapalapa D.F. 09340, Mexico. The thermal denaturation of the dimeric enzyme triosephosphate isomerase (TIM) from Saccharomyces cerevisiae was studied by spectroscopic and calorimetric methods. At low protein concentration the structural transition proved to be reversible in thermal scannings conducted at a rate greater than 1.0 degrees C min(-1). Under these conditions, however, the denaturation-renaturation cycle exhibited marked hysteresis. The use of lower scanning rates lead to pronounced irreversibility. Kinetic studies indicated that denaturation of the enzyme likely consists of an initial first-order reaction that forms thermally unfolded (U) TIM, followed by irreversibility-inducing reactions which are probably linked to aggregation of the unfolded protein. As judged from CD measurements, U possesses residual secondary structure but lacks most of the tertiary interactions present in native TIM. Furthermore, the large increment in heat capacity upon denaturation suggests that extensive exposure of surface area occurs when U is formed. Above 63 degrees C, reactions leading to irreversibility were much slower than the unfolding process; as a result, U was sufficiently long-lived as to allow an investigation of its refolding kinetics. We found that U transforms into nativelike TIM through a second-order reaction in which association is coupled to the regain of secondary structure. The rate constants for unfolding and refolding of TIM displayed temperature dependences resembling those reported for monomeric proteins but with considerably larger activation enthalpies. Such large temperature dependences seem to be determinant for the occurrence of kinetically controlled transitions and thus constitute a simple explanation for the hysteresis observed in thermal scannings. PMID: 11467968 [PubMed - indexed for MEDLINE] 136: J Bacteriol 2001 Aug;183(16):4761-70 Domain interactions in the yeast ATP binding cassette transporter Ycf1p: intragenic suppressor analysis of mutations in the nucleotide binding domains. Falcon-Perez JM, Martinez-Burgos M, Molano J, Mazon MJ, Eraso P. Instituto de Investigaciones Biomedicas "Alberto Sols," CSIC-UAM, Madrid, Spain. The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V(max) of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane. PMID: 11466279 [PubMed - indexed for MEDLINE] 137: Mol Cell Biol 2001 Aug;21(16):5541-53 Yeast RNA polymerase I enhancer is dispensable for transcription of the chromosomal rRNA gene and cell growth, and its apparent transcription enhancement from ectopic promoters requires Fob1 protein. Wai H, Johzuka K, Vu L, Eliason K, Kobayashi T, Horiuchi T, Nomura M. Department of Biological Chemistry, University of California-Irvine, Irvine, California 92697-1700, USA. At the end of the 35S rRNA gene within ribosomal DNA (rDNA) repeats in Saccharomyces cerevisiae lies an enhancer that has been shown to greatly stimulate rDNA transcription in ectopic reporter systems. We found, however, that the enhancer is not necessary for normal levels of rRNA synthesis from chromosomal rDNA or for cell growth. Yeast strains which have the entire enhancer from rDNA deleted did not show any defects in growth or rRNA synthesis. We found that the stimulatory activity of the enhancer for ectopic reporters is not observed in cells with disrupted nucleolar structures, suggesting that reporter genes are in general poorly accessible to RNA polymerase I (Pol I) machinery in the nucleolus and that the enhancer improves accessibility. We also found that a fob1 mutation abolishes transcription from the enhancer-dependent rDNA promoter integrated at the HIS4 locus without any effect on transcription from chromosomal rDNA. FOB1 is required for recombination hot spot (HOT1) activity, which also requires the enhancer region, and for recombination within rDNA repeats. We suggest that Fob1 protein stimulates interactions between rDNA repeats through the enhancer region, thus helping ectopic rDNA promoters to recruit the Pol I machinery normally present in the nucleolus. PMID: 11463836 [PubMed - indexed for MEDLINE] 138: Proc Natl Acad Sci U S A 2001 Jul 17;98(15):8447-53 Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchange. Solinger JA, Heyer WD. Division of Biological Sciences, Section of Microbiology, University of California, Davis, CA 95616, USA. Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. As previously shown, Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Here we demonstrate that Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depended on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins. PMID: 11459988 [PubMed - indexed for MEDLINE] 139: Mol Genet Genomics 2001 Jun;265(4):705-10 Genetic interactions within TFIIIC, the promoter-binding factor of yeast RNA polymerase III. Rozenfeld S, Thuriaux P. Service de Biochimie et Genetique Moleculaire, CEA/Saclay, Gif-su-Yvette, France. TFIIIC is a heteromultimeric protein, made of six distinct subunits in Saccharomyces cerevisiae, that binds to RNA polymerase III promoters and triggers the assembly of the transcription complex. The largest yeast subunit tau138, encoded by TFC3, binds to the B-box promoter element. This binding is defective in the temperature-sensitive mutant tfc3-G349E; the mutation responsible is located in one of two conserved motifs shared with the B-binding component of human TFIIIC. Rare dominant gain-of-function mutations that restore growth at high temperature were obtained following ultraviolet mutagenesis of tfc3-G349E. All of them resulted from single amino acid substitutions that alter the structure of TFIIIC. Three were due to reversion or intragenic suppression (TFC3-K754E and TFC3-L804H) events. Three were identical isolates of TFC6-E330K, a previously described mutation of the tau91 subunit. The remaining suppressors mapped in TFC4, and resulted in amino acid replacements in the second largest subunit of TFIIIC (tau131). With the exception TFC4-E711K, these affect positions that are invariant between the S. cerevisiae and Homo sapiens proteins, and are localised in conserved tetratricopeptide motifs. These findings demonstrate a close functional interaction between the two largest subunits of TFIIIC and underscore the importance of the tetratricopeptide motif of tau131. PMID: 11459191 [PubMed - indexed for MEDLINE] 140: J Biol Chem 2001 Sep 14;276(37):34832-9 Modulation of myosin function by isoform-specific properties of Saccharomyces cerevisiae and muscle tropomyosins. Strand J, Nili M, Homsher E, Tobacman LS. Departments of Internal Medicine and Biochemistry, the University of Iowa, Iowa City, Iowa 52242, USA. Tropomyosin is an extended coiled-coil protein that influences actin function by binding longitudinally along thin filaments. The present work compares cardiac tropomyosin and the two tropomyosins from Saccharomyces cerevisiae, TPM1 and TPM2, that are much shorter than vertebrate tropomyosins. Unlike cardiac tropomyosin, the phase of the coiled-coil-forming heptad repeat of TPM2 is discontinuous; it is interrupted by a 4-residue deletion. TPM1 has two such deletions, which flank the 38-residue partial gene duplication that causes TPM1 to span five actins instead of the four of TPM2. Each of the three tropomyosin isoforms modulates actin-myosin interactions, with isoform-specific effects on cooperativity and strength of myosin binding. These different properties can be explained by a model that combines opposite effects, steric hindrance between myosin and tropomyosin when the latter is bound to a subset of its sites on actin, and also indirect, favorable interactions between tropomyosin and myosin, mediated by mutually promoted changes in actin. Both of these effects are influenced by which tropomyosin isoform is present. Finally, the tropomyosins have isoform-specific effects on in vitro sliding speed and on the myosin concentration dependence of this movement, suggesting that non-muscle tropomyosin isoforms exist, at least in part, to modulate myosin function. PMID: 11457840 [PubMed - indexed for MEDLINE] 141: J Biol Chem 2001 Sep 14;276(37):34948-57 Saccharomyces cerevisiae protein Pci8p and human protein eIF3e/Int-6 interact with the eIF3 core complex by binding to cognate eIF3b subunits. Shalev A, Valasek L, Pise-Masison CA, Radonovich M, Phan L, Clayton J, He H, Brady JN, Hinnebusch AG, Asano K. Laboratory of Gene Regulation and Development, NICHD, and the Basic Research Laboratory, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. Mammalian, plant, and Schizosaccharomyces pombe eukaryotic initiation factor-3 (eIF3) contains a protein homologous to the product of int-6 (eIF3e), a frequent integration site of mouse mammary tumor viruses. By contrast, Saccharomyces cerevisiae does not encode a protein closely related to eIF3e/Int-6. Here, we characterize a novel S. cerevisiae protein (Pci8p, Yil071cp) that contains a PCI (proteasome-COP9 signalosome-eIF3) domain conserved in eIF3e/Int-6. We show that both Pci8p and human eIF3e/Int-6 expressed in budding yeast interact with the yeast eIF3 complex in vivo and in vitro by binding to a discrete segment of its eIF3b subunit Prt1p and that human eIF3e/Int-6 interacts with the human eIF3b segment homologous to the Pci8p-binding site of yeast Prt1p. These results refine our understanding of subunit interactions in the eIF3 complex and suggest structural similarity between human eIF3e/Int-6 and yeast Pci8p. However, deletion of PCI8 had no discernible effect on cell growth or translation initiation as judged by polysome analysis, suggesting that Pci8p is not required for the essential function of eIF3 in translation initiation. Motivated by the involvement of Int-6 in transcriptional control, we investigated the effects of deleting PCI8 on the total mRNA expression profile by oligonucleotide microarray analysis and found reduced mRNA levels for a subset of heat shock proteins in the pci8Delta mutant. We discuss possible dual functions of Pci8p and Int-6 in transcriptional and translational control. PMID: 11457827 [PubMed - indexed for MEDLINE] 142: Genetics 2001 Jul;158(3):989-97 The defect in transcription-coupled repair displayed by a Saccharomyces cerevisiae rad26 mutant is dependent on carbon source and is not associated with a lack of transcription. Bucheli M, Lommel L, Sweder K. Laboratory for Cancer Research, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854-8020, USA. Nucleotide excision repair (NER) is an evolutionarily conserved pathway that removes DNA damage induced by ultraviolet irradiation and various chemical agents that cause bulky adducts. Two subpathways within NER remove damage from the genome overall or the transcribed strands of transcribing genes (TCR). TCR is a faster repair process than overall genomic repair and has been thought to require the RAD26 gene in Saccharomyces cerevisiae. Rad26 is a member of the SWI/SNF family of proteins that either disrupt chromatin or facilitate interactions between the RNA Pol II and transcription activators. SWI/SNF proteins are required for the expression or repression of a diverse set of genes, many of which are differentially transcribed in response to particular carbon sources. The remodeling of chromatin by Rad26 could affect transcription and/or TCR following formation of DNA damage and other stress-inducing conditions. We speculate that another factor(s) can substitute for Rad26 under particular growth conditions. We therefore measured the level of repair and transcription in two different carbon sources and found that the defect in the rad26 mutant for TCR was dependent on the type of carbon source. Furthermore, TCR did not correlate with transcription rate, suggesting that disruption of RAD26 leads to a specific defect in DNA repair and not transcription. PMID: 11454749 [PubMed - indexed for MEDLINE] 143: Curr Opin Cell Biol 2001 Aug;13(4):399-404 Ion homeostasis during salt stress in plants. Serrano R, Rodriguez-Navarro A. Instituto de Biologia Molecular y Celular de Plantas Universidad Politecnica de Valencia-C.S.I.C., Camino de Vera, 46022, Valencia, Spain. serrano@ibmcp.upv.es Recent progress has been made in the characterization of cation transporters that maintain ion homeostasis during salt stress in plants. Sodium-proton antiporters at the vacuolar (NHX1) and plasma membrane (SOS1) have been identified in Arabidopsis. SOS1 is regulated by the calcium-activated protein kinase complex SOS2-SOS3. In yeast, a transcription repressor, Sko1, mediates regulation of the sodium-pump ENA1 gene by the Hog1 MAP kinase. The recent visualization at the atomic level of the inhibitory site of sodium in the known target Hal2 has helped identify the interactions determining Na(+) toxicity. Publication Types: Review Review Literature PMID: 11454443 [PubMed - indexed for MEDLINE] 144: Curr Genet 2001 Jun;39(4):205-9 The Schizosaccharomyces pombe Cdc42p GTPase signals through Pak2p and the Mkh1p-Pek1p-Spm1p MAP kinase pathway. Merla A, Johnson DI. Department of Microbiology and Molecular Genetics, University of Vermont, Burlington 05405, USA. The Cdc42p GTPase is involved in many aspects of growth and cell-cycle regulation, including actin cytoskeletal rearrangements and activation of signal transduction pathways. To further investigate these functions, genetic interactions were examined between Schizosaccharomyces pombe Cdc42p, its effectors Pak1p and Pak2p, and the Mkh1p-Pek1p-Spm1p signal transduction pathway, which functions in cytokinesis and cell division. Expression of a truncated version of Pak2p lacking its N-terminal autoinhibitory domain led to a growth defect that was suppressed by deltamkh1 and deltaspm1 null mutations and an elongated cell phenotype indicative of a cell division defect that was suppressed by the deltamkh1 mutation. In addition, expression of the constitutively activated cdc42G12V mutant allele led to a growth defect that was rescued by the deltapak2 and deltamkh1 mutations. The deltapak2 mutation did not suppress the growth defect conferred by plasmid expression of Mkh1p, suggesting that Pak2p functions upstream of Mkh1p in this pathway. A two-hybrid protein interaction was observed between Pak2p and Mkh1p, but not between Pak1p and Mkh1p. These results are consistent with Cdc42p interacting with Pak2p to signal through the Mkh1p-Pek1p-Spm1p pathway. PMID: 11453249 [PubMed - indexed for MEDLINE] 145: Nucleic Acids Res 2001 Jul 15;29(14):3069-79 Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease. Bae SH, Kim JA, Choi E, Lee KH, Kang HY, Kim HD, Kim JH, Bae KH, Cho Y, Park C, Seo YS. National Creative Research Initiative Center for Cell Cycle Control, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchun-Dong, Changan-Ku, Suwon, Kyunggi-Do 440-746, Korea. In order to gain insights into the structural basis of the multifunctional Dna2 enzyme involved in Okazaki fragment processing, we performed biochemical, biophysical and genetic studies to dissect the domain structure of Dna2. Proteolytic digestion of Dna2 using subtilisin produced a 127 kDa polypeptide that lacked the 45 kDa N-terminal region of Dna2. Further digestion generated two subtilisin-resistant core fragments of approximately equal size, 58 and 60 kDa. Surprisingly, digestion resulted in a significant (3- to 8-fold) increase in both ATPase and endonuclease activities compared to the intact enzyme. However, cells with a mutant DNA2 allele lacking the corresponding N-terminal region were severely impaired in growth, being unable to grow at 37 degrees C, indicating that the N-terminal region contains a domain critical for a cellular function(s) of Dna2. Analyses of the hydrodynamic properties of and in vivo complex formation by wild-type and/or mutant Dna2 lacking the N-terminal 45 kDa domain revealed that Dna2 is active as the monomer and thus the defect in the mutant Dna2 protein is not due to its inability to multimerize. In addition, we found that the N-terminal 45 kDa domain interacts physically with a central region located between the two catalytic domains. Our results suggest that the N-terminal 45 kDa domain of Dna2 plays a critical role in regulation of the enzymatic activities of Dna2 by serving as a site for intra- and intermolecular interactions essential for optimal function of Dna2 in Okazaki fragment processing. The possible mode of regulation of Dna2 is discussed based upon our recent finding that replication protein A interacts functionally and physically with Dna2 during Okazaki fragment processing. PMID: 11452032 [PubMed - indexed for MEDLINE] 146: Bioinformatics 2001 Jul;17(7):669-71 A Java applet for visualizing protein-protein interaction. Mrowka R. Johannes-Muller-Institut fur Physiologie, Charite, Humboldt-Universitat zu Berlin, Tucholsky Str. 2, D-10117 Berlin, Germany. Mrowka@rz.hu-berlin.de A web applet for browsing protein-protein interactions was implemented. It enables the display of interaction relationships, based upon neighboring distance and biological function. AVAILABILITY: The Java applet is available at http://www.charite.de/bioinformatics PMID: 11448890 [PubMed - indexed for MEDLINE] 147: Bioinformatics 2001 Jul;17(7):608-21 Identifying target sites for cooperatively binding factors. GuhaThakurta D, Stormo GD. Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue, Campus Box: 8232, St Louis, MO 63110, USA. dg@genetics.wustl.edu MOTIVATION: Transcriptional activation in eukaryotic organisms normally requires combinatorial interactions of multiple transcription factors. Though several methods exist for identification of individual protein binding site patterns in DNA sequences, there are few methods for discovery of binding site patterns for cooperatively acting factors. Here we present an algorithm, Co-Bind (for COperative BINDing), for discovering DNA target sites for cooperatively acting transcription factors. The method utilizes a Gibbs sampling strategy to model the cooperativity between two transcription factors and defines position weight matrices for the binding sites. Sequences from both the training set and the entire genome are taken into account, in order to discriminate against commonly occurring patterns in the genome, and produce patterns which are significant only in the training set. RESULTS: We have tested Co-Bind on semi-synthetic and real data sets to show it can efficiently identify DNA target site patterns for cooperatively binding transcription factors. In cases where binding site patterns are weak and cannot be identified by other available methods, Co-Bind, by virtue of modeling the cooperativity between factors, can identify those sites efficiently. Though developed to model protein-DNA interactions, the scope of Co-Bind may be extended to combinatorial, sequence specific, interactions in other macromolecules. AVAILABILITY: The program is available upon request from the authors or may be downloaded from http://ural.wustl.edu. PMID: 11448879 [PubMed - indexed for MEDLINE] 148: Virology 2001 Jul 20;286(1):216-24 Ty1 retrotransposition and programmed +1 ribosomal frameshifting require the integrity of the protein synthetic translocation step. Harger JW, Meskauskas A, Nielsen J, Justice MC, Dinman JD. Department of Molecular Genetics and Microbiology, Graduate Program in Molecular Biosciences at UMDNJ/Rutgers Universities, The Cancer Institute of New Jersey, Piscataway, New Jersey 08854, USA. Programmed ribosomal frameshifting is utilized by a number of RNA viruses to ensure the correct ratio of viral structural to enzymatic proteins for viral particle assembly. Altering frameshifting efficiencies upsets this ratio, inhibiting virus propagation. Two yeast viruses that induce host cell ribosomes to shift translational reading frame were used as tools to explore the interactions between viruses and host cellular protein synthetic machinery. Previous studies showed that the ribosome-inactivating protein pokeweed antiviral protein specifically inhibited propagation of the Ty1 retrotransposable element of yeast as a consequence of inhibition of programmed +1 ribosomal frameshifting. Here, complementary genetic and pharmacological approaches were employed to test whether inhibition of Ty1 retrotransposition is a general feature of alterations in the translocation step of elongation and +1 frameshifting. The results demonstrate that cells harboring a variety of mutant alleles of two host-encoded proteins that are involved in translocation, eukaryotic elongation factor-2 and the ribosome-associated protein RPP0, have Ty1 propagation defects. We also show that sordarin, a fungus-specific inhibitor of eEF-2 function, specifically inhibits programmed +1 ribosomal frameshifting and Ty1 retrotransposition. These findings serve to link inhibition of Ty1 retrotransposition and +1 frameshifting to changes in the translocation step of elongation. Copyright 2001 Academic Press. PMID: 11448174 [PubMed - indexed for MEDLINE] 149: Yeast 2001 Jul;18(10):943-51 Yeast Lrg1p acts as a specialized RhoGAP regulating 1,3-beta-glucan synthesis. Watanabe D, Abe M, Ohya Y. Department of Integrated Biosciences, Graduate School of Frontier Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Selection of an extragenic suppressor of fks1-1154 Deltafks2, mutations in the catalytic subunits of yeast 1,3-beta-glucan synthase (GS) conferring temperature-sensitivity, led to the LRG1 gene, which was originally identified as a LIM-RhoGAP homologous gene. Mutations in the LRG1 gene restore impaired 1,3-beta-glucan synthesis in the fks1-1154 Deltafks2 mutant as well as that in rho1-2, a temperature-sensitive mutant of Rho-type GTPase that functions as a regulatory subunit of GS. Two-hybrid analyses of Lrg1p, which contains a sequence conserved among Rho GTPase-activating proteins (GAPs), revealed its specific interactions with the active form of Rho1p. Among eight potential yeast RhoGAPs, Lrg1p is the only member that negatively regulates GS activity: mutations in the rest of GAPs, including bem2, Deltabem3, Deltasac7, Deltabag7, Deltarga1, Deltarga2 and Deltargd1, do not suppress impairment of 1,3-beta-glucan synthesis. Analyses of Mpk1p phosphorylation revealed the inability of Lrg1p to regulate the Pkc1p-MAP kinase cascade, a distinct Rho1p-regulating signalling pathway known to be affected by the GAPs, Bem2p and Sac7p. Thus, different groups of Rho1p GAPs control the activity of different Rho1p-effector proteins. Copyright 2001 John Wiley & Sons, Ltd. PMID: 11447600 [PubMed - indexed for MEDLINE] 150: J Biol Chem 2001 Sep 14;276(37):34537-44 Genetic interactions with the yeast Q-SNARE VTI1 reveal novel functions for the R-SNARE YKT6. Dilcher M, Kohler B, von Mollard GF. Zentrum Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universitat Gottingen, 37073 Gottingen, Germany. SNARE proteins are required for fusion of transport vesicles with target membranes. Previously, we found that the yeast Q-SNARE Vti1p is involved in transport to the cis-Golgi, to the prevacuole/late endosome, and to the vacuole. Here we identified a previously uncharacterized gene, VTS1, and the R-SNARE YKT6 both as multicopy and as low copy suppressors of the growth and vacuolar transport defect in vti1-2 cells. Ykt6p was known to function in retrograde traffic to the cis-Golgi and homotypic vacuolar fusion. We found that VTI1 and YKT6 also interacted in traffic to the prevacuole and vacuole, indicating that these SNARE complexes contain Ykt6p, Vti1p, plus Pep12p and Ykt6p, Vti1p, Vam3p, plus Vam7p, respectively. As Ykt6p was required for several transport steps, R-SNAREs cannot be the sole determinants of specificity. To study the role of the 0 layer in the SNARE motif, we introduced the mutations vti1-Q158R and ykt6-R165Q. SNARE complexes to which Ykt6p contributed a fourth glutamine residue in the 0 layer were nonfunctional, suggesting an essential function for arginine in the 0 layer of these complexes. vti1-Q158R cells had severe defects in several transport steps, indicating that the second arginine in the 0 layer interfered with function. PMID: 11445562 [PubMed - indexed for MEDLINE] 151: J Biol Chem 2001 Sep 7;276(36):33689-96 Coordinated ATP hydrolysis by the Hsp90 dimer. Richter K, Muschler P, Hainzl O, Buchner J. Institut fur Organische Chemie und Biochemie, Technische Universitat Munchen, Lichtenbergstr. 4, Garching 85747, Germany. The Hsp90 dimer is a molecular chaperone with an unusual N-terminal ATP binding site. The structure of the ATP binding site makes it a member of a new class of ATP-hydrolyzing enzymes, known as the GHKL family. While for some of the family members structural data on conformational changes occurring after ATP binding are available, these are still lacking for Hsp90. Here we set out to investigate the correlation between dimerization and ATP hydrolysis by Hsp90. The dimerization constant of wild type (WT) Hsp90 was determined to be 60 nm. Heterodimers of WT Hsp90 with fragments lacking the ATP binding domain form readily and exhibit dimerization constants similar to full-length Hsp90. However, the ATPase activity of these heterodimers was significantly lower than that of the wild type protein, indicating cooperative interactions in the N-terminal part of the protein that lead to the activation of the ATPase activity. To further address the contribution of the N-terminal domains to the ATPase activity, we used an Hsp90 point mutant that is unable to bind ATP. Since heterodimers between the WT protein and this mutant showed WT ATPase activity, this mutant, although unable to bind ATP, still has the ability to stimulate the activity in its WT partner domain. Thus, contact formation between the N-terminal domains might not depend on ATP bound to both domains. Together, these results suggest a mechanism for coupling the hydrolysis of ATP to the opening-closing movement of the Hsp90 molecular chaperone. PMID: 11441008 [PubMed - indexed for MEDLINE] 152: Mol Cell Biol 2001 Aug;21(15):5142-55 exo1-Dependent mutator mutations: model system for studying functional interactions in mismatch repair. Amin NS, Nguyen MN, Oh S, Kolodner RD. Ludwig Institute for Cancer Research, University of California, San Diego School of Medicine, La Jolla, California 92093-0660, USA. EXO1 interacts with MSH2 and MLH1 and has been proposed to be a redundant exonuclease that functions in mismatch repair (MMR). To better understand the role of EXO1 in mismatch repair, a genetic screen was performed to identify mutations that increase the mutation rates caused by weak mutator mutations such as exo1Delta and pms1-A130V mutations. In a screen starting with an exo1 mutation, exo1-dependent mutator mutations were obtained in MLH1, PMS1, MSH2, MSH3, POL30 (PCNA), POL32, and RNR1, whereas starting with the weak pms1 allele pms1-A130V, pms1-dependent mutator mutations were identified in MLH1, MSH2, MSH3, MSH6, and EXO1. These mutations only cause weak MMR defects as single mutants but cause strong MMR defects when combined with each other. Most of the mutations obtained caused amino acid substitutions in MLH1 or PMS1, and these clustered in either the ATP-binding region or the MLH1-PMS1 interaction regions of these proteins. The mutations showed two other types of interactions: specific pairs of mutations showed unlinked noncomplementation in diploid strains, and the defect caused by pairs of mutations could be suppressed by high-copy-number expression of a third gene, an effect that showed allele and overexpressed gene specificity. These results support a model in which EXO1 plays a structural role in MMR and stabilizes multiprotein complexes containing a number of MMR proteins. A similar role is proposed for PCNA based on the data presented. PMID: 11438669 [PubMed - indexed for MEDLINE] 153: J Clin Lab Anal 2001;15(4):223-9 Direct measurement of HDL cholesterol: method eliminating apolipoprotein E-rich particles. Okada M, Matsui H, Ito Y, Fujiwara A. Department of Laboratory Medicine, Niigata University School of Medicine, Niigata City, Japan. okadar@med.niigata-u.ac.jp It has been reported that the existing direct method of high density lipoprotein (HDL) cholesterol measures particles enriched with apolipoprotein E (apoE). The aim of our study was to investigate a new analytical protocol to directly measure HDL cholesterol that eliminates apoE-rich particles. The interactions of four lipoproteins (HDL(3), HDL(2), LDL, and VLDL + chylomicron) with surfactants, divalent cations, sugars, and lectins were investigated. By analyzing sera, HDL(3), and HDL(2), we examined the relationships among the measurements obtained by our protocol, a precipitation method using heparin-MnCl(2), and a commercially available kit for this direct method. A significant difference was found between the direct method and the heparin-MnCl(2) method, but not between our protocol and the heparin-MnCl(2) method. Multiple regression analysis showed that the difference between the direct method and the heparin MnCl(2) method is dependent on sources of apoE-rich HDL. In conclusion, our protocol enables a direct measurement of HDL cholesterol that eliminates apoE-rich particles. Copyright 2001 Wiley-Liss, Inc. PMID: 11436206 [PubMed - indexed for MEDLINE] 154: J Biol Chem 2001 Aug 31;276(35):33093-100 Schwannomin isoform-1 interacts with syntenin via PDZ domains. Jannatipour M, Dion P, Khan S, Jindal H, Fan X, Laganiere J, Chishti AH, Rouleau GA. Center for Research in Neuroscience, McGill University and the Montreal General Hospital, 1650 Cedar Avenue, Montreal, Quebec, Canada. The neurofibromatosis type 2 gene (NF2) is involved in the pathogenesis of benign tumors of the human nervous system. The NF2 protein, called schwannomin or merlin, is inactivated in virtually all schwannomas and meningiomas. The molecular mechanisms by which schwannomin functions as a tumor suppressor is unknown but believed to involve plasma membrane-cytoskeletal interactions. Two major alternatively spliced isoforms of schwannomin differing in their C termini have been reported. Using the yeast two-hybrid system, we have identified syntenin as a binding partner for schwannomin isoform-1 (sch-1). Syntenin is an adapter protein that couples transmembrane proteoglycans to cytoskeletal components and is involved in intracellular vesicle transport. The C terminus 25 amino acids of sch-1 and the two PDZ domains of syntenin mediate their binding, and mutations introduced within the VAFFEEL region of sch-1 defined a sequence crucial for syntenin recognition. We have showed that the two proteins interacted in vitro and in vivo and localized underneath the plasma membrane. Fibroblast cells expressing heterologous antisense syntenin display alterations in the subcellular distribution of sch-1. Together, these results provide the first functional clue to the existence of schwannomin isoforms and could unravel novel pathways for the transport and subcellular localization of schwannomin in vivo. PMID: 11432873 [PubMed - indexed for MEDLINE] 155: EMBO J 2001 Jul 2;20(13):3506-17 Spt16-Pob3 and the HMG protein Nhp6 combine to form the nucleosome-binding factor SPN. Formosa T, Eriksson P, Wittmeyer J, Ginn J, Yu Y, Stillman DJ. Department of Biochemistry, University of Utah School of Medicine, 50 N. Medical Drive Rm 211, Salt Lake City, UT 84132, USA. Tim.Formosa@hsc.utah.edu Yeast Spt16/Cdc68 and Pob3 form a heterodimer that acts in both DNA replication and transcription. This is supported by studies of new alleles of SPT16 described here. We show that Spt16-Pob3 enhances HO transcription through a mechanism that is affected by chromatin modification, since some of the defects caused by mutations can be suppressed by deleting the histone deacetylase Rpd3. While otherwise conserved among many eukaryotes, Pob3 lacks the HMG1 DNA-binding motif found in similar proteins such as the SSRP1 subunit of human FACT. SPT16 and POB3 display strong genetic interactions with NHP6A/B, which encodes an HMG1 motif, suggesting that these gene products function coordinately in vivo. While Spt16-Pob3 and Nhp6 do not appear to form stable heterotrimers, Nhp6 binds to nucleosomes and these Nhp6-nucleosomes can recruit Spt16-Pob3 to form SPN-nucleosomes. These complexes have altered electrophoretic mobility and a distinct pattern of enhanced sensitivity to DNase I. These results suggest that Spt16-Pob3 and Nhp6 cooperate to function as a novel nucleosome reorganizing factor. PMID: 11432837 [PubMed - indexed for MEDLINE] 156: Eur J Biochem 2001 Jul;268(13):3674-84 Mechanism of activation of the double-stranded-RNA-dependent protein kinase, PKR: role of dimerization and cellular localization in the stimulation of PKR phosphorylation of eukaryotic initiation factor-2 (eIF2). Vattem KM, Staschke KA, Wek RC. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, USA. An important defense against viral infection involves inhibition of translation by PKR phosphorylation of the alpha subunit of eIF2. Binding of viral dsRNAs to two dsRNA-binding domains (dsRBDs) in PKR leads to relief of an inhibitory region and activation of eIF2 kinase activity. Interestingly, while deletion of the regulatory region of PKR significantly induces activity in vitro, the truncated kinase does not inhibit translation in vivo, suggesting that these sequences carry out additional functions required for PKR control. To delineate these functions and determine the order of events leading to activation of PKR, we fused truncated PKR to domains of known function and assayed the chimeras for in vivo activity. We found that fusion of a heterologous dimerization domain with the PKR catalytic domain enhanced autophosphorylation and eIF2 kinase function in vivo. The dsRBDs also mediate ribosome association and we proposed that such targeting increases the localized concentration of PKR, enhancing interaction between PKR molecules. We addressed this premise by linking the truncated PKR to RAS sequences mediating farnesylation and membrane localization and found that the fusion protein was functional in vivo. These results indicate that cellular localization along with oligomerization enhances interaction between PKR molecules. Alanine substitution for the phosphorylation site, threonine 446, impeded in vivo and in vitro activity of the PKR fusion proteins, while aspartate or glutamate substitutions partially restored the function of the truncated kinase. These results indicate that both dimerization and cellular localization play a role in transient protein-protein interactions and that trans-autophosphorylation is the final step in the mechanism of activation of PKR. PMID: 11432733 [PubMed - indexed for MEDLINE] 157: J Biol Chem 2001 Aug 31;276(35):32474-9 Asymmetric interactions between the acidic P1 and P2 proteins in the Saccharomyces cerevisiae ribosomal stalk. Guarinos E, Remacha M, Ballesta JP. Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas and Universidad Autonoma de Madrid, Canto Blanco, 28049 Madrid, Spain. The Saccharomyces cerevisiae ribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1alpha, P1beta, P2alpha, and P2beta. The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure. Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1alpha, P0-1beta, P0-2alpha, and P0-2beta. The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth. In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition. An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein. The following associations were found: P0-1alpha.P2beta, P0-1beta.P2alpha, P0-2alpha.P1beta, and P0-2beta.P1alpha. These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1alpha.P2beta and P1beta.P2alpha, which have different structural and functional roles. PMID: 11431471 [PubMed - indexed for MEDLINE] 158: Nature 2001 Jun 28;411(6841):1073-6 Multiple pathways cooperate in the suppression of genome instability in Saccharomyces cerevisiae. Myung K, Chen C, Kolodner RD. Ludwig Institute for Cancer Research, University of California San Diego, 92093, USA. Gross chromosome rearrangements (GCRs), such as translocations, deletion of a chromosome arm, interstitial deletions and inversions, are often observed in cancer cells. Spontaneous GCRs are rare in Saccharomyces cerevisiae; however, the existence of mutator mutants with increased genome instability suggests that GCRs are actively suppressed. Here we show by genetic analysis that these genome rearrangements probably result from DNA replication errors and are suppressed by at least three interacting pathways or groups of proteins: S-phase checkpoint functions, recombination proteins and proteins that prevent de novo addition of telomeres at double-strand breaks (DSBs). Mutations that inactivate these pathways cause high rates of GCRs and show synergistic interactions, indicating that the pathways that suppress GCRs all compete for the same DNA substrates. PMID: 11429610 [PubMed - indexed for MEDLINE] 159: Mutat Res 2001 Jul 12;486(2):137-46 Deletion of the SRS2 gene suppresses elevated recombination and DNA damage sensitivity in rad5 and rad18 mutants of Saccharomyces cerevisiae. Friedl AA, Liefshitz B, Steinlauf R, Kupiec M. Institute of Radiation Biology, GSF-National Research Center for Environment and Health, P.O. Box 1149, 85758, Oberschleissheim, Germany. anna.friedl@lrz.uni-muenchen.de The Saccharomyces cerevisiae genes RAD5, RAD18, and SRS2 are proposed to act in post-replicational repair of DNA damage. We have investigated the genetic interactions between mutations in these genes with respect to cell survival and ectopic gene conversion following treatment of logarithmic and early stationary cells with UV- and gamma-rays. We find that the genetic interaction between the rad5 and rad18 mutations depends on DNA damage type and position in the cell cycle at the time of treatment. Inactivation of SRS2 suppresses damage sensitivity both in rad5 and rad18 mutants, but only when treated in logarithmic phase. When irradiated in stationary phase, the srs2 mutation enhances the sensitivity of rad5 mutants, whereas it has no effect on rad18 mutants. Irrespective of the growth phase, the srs2 mutation reduces the frequency of damage-induced ectopic gene conversion in rad5 and rad18 mutants. In addition, we find that srs2 mutants exhibit reduced spontaneous and UV-induced sister chromatid recombination (SCR), whereas rad5 and rad18 mutants are proficient for SCR. We propose a model in which the Srs2 protein has pro-recombinogenic or anti-recombinogenic activity, depending on the context of the DNA damage. PMID: 11425518 [PubMed - indexed for MEDLINE] 160: RNA 2001 Jun;7(6):833-45 A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture. Newby MI, Greenbaum NL. Institute of Molecular Biophysics, Florida State University, Tallahassee 32306-4380, USA. The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 2'OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (psi) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of psi at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of psi also stabilized the branch-site interaction, contributing -0.7 kcal/mol to duplex deltaG degrees 37. These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing. PMID: 11424937 [PubMed - indexed for MEDLINE] 161: Traffic 2001 Jul;2(7):476-86 The class C Vps complex functions at multiple stages of the vacuolar transport pathway. Peterson MR, Emr SD. Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0668, USA. The Class C Vps complex, consisting of Vps11, Vps16, Vps18, and Vps33, is required for SNARE-mediated membrane fusion at the lysosome-like yeast vacuole. However, Class C vps mutants display more severe and pleiotropic phenotypes than mutants specifically defective in endosome-to-vacuole transport, suggesting that there are additional functions for the Class C Vps complex. A SNARE double mutant which is defective for both Golgi-to-endosome and endosome-to-vacuole trafficking replicates many of the phenotypes observed in Class C vps mutants. We show that genetic interactions exist between Class C vps alleles and alleles of the Class D vps group, which are defective in the docking and fusion of Golgi-derived vesicles at the endosome. Moreover, the Class D protein Vac1 was found to physically bind to the Class C Vps complex through a direct association with Vps11. Finally, using a random mutagenic screen, a temperature-conditional allele which shares many of the phenotypes of mutants which are selectively defective in Golgi-to-endosome trafficking was isolated (vps11-3ts). Collectively, these results indicate that the Class C Vps complex plays essential roles in the processes of membrane docking and fusion at both the Golgi-to-endosome and endosome-to-vacuole stages of transport. PMID: 11422941 [PubMed - indexed for MEDLINE] 162: Mol Biol Evol 2001 Jul;18(7):1283-92 The yeast protein interaction network evolves rapidly and contains few redundant duplicate genes. Wagner A. Department of Biology, University of New Mexico, Albequerque, New Mexico 87131-1091, USA. wagnera@unm.edu In this paper, the structure and evolution of the protein interaction network of the yeast Saccharomyces cerevisiae is analyzed. The network is viewed as a graph whose nodes correspond to proteins. Two proteins are connected by an edge if they interact. The network resembles a random graph in that it consists of many small subnets (groups of proteins that interact with each other but do not interact with any other protein) and one large connected subnet comprising more than half of all interacting proteins. The number of interactions per protein appears to follow a power law distribution. Within approximately 200 Myr after a duplication, the products of duplicate genes become almost equally likely to (1) have common protein interaction partners and (2) be part of the same subnetwork as two proteins chosen at random from within the network. This indicates that the persistence of redundant interaction partners is the exception rather than the rule. After gene duplication, the likelihood that an interaction gets lost exceeds 2.2 x 10(-3)/Myr. New interactions are estimated to evolve at a rate that is approximately three orders of magnitude smaller. Every 300 Myr, as many as half of all interactions may be replaced by new interactions. PMID: 11420367 [PubMed - indexed for MEDLINE] 163: Mol Cell Biol 2001 Jul;21(14):4568-78 GCN5 dependence of chromatin remodeling and transcriptional activation by the GAL4 and VP16 activation domains in budding yeast. Stafford GA, Morse RH. Department of Biomedical Sciences, State University of New York at Albany School of Public Health, Albany, New York 12201-2002, USA. Chromatin-modifying enzymes such as the histone acetyltransferase GCN5 can contribute to transcriptional activation at steps subsequent to the initial binding of transcriptional activators. However, few studies have directly examined dependence of chromatin remodeling in vivo on GCN5 or other acetyltransferases, and none have examined remodeling via nucleosomal activator binding sites. In this study, we have monitored chromatin perturbation via nucleosomal binding sites in the yeast episome TALS by GAL4 derivatives in GCN5(+) and gcn5Delta yeast cells. The strong activator GAL4 shows no dependence on GCN5 for remodeling TALS chromatin, whereas GAL4-estrogen receptor-VP16 shows substantial, albeit not complete, GCN5 dependence. Mini-GAL4 derivatives having weakened interactions with TATA-binding protein and TFIIB exhibit a strong dependence on GCN5 for both transcriptional activation and TALS remodeling not seen for native GAL4. These results indicate that GCN5 can contribute to chromatin remodeling at activator binding sites and that dependence on coactivator function for a given activator can vary according to the type and strength of contacts that it makes with other factors. We also found a weaker dependence for chromatin remodeling on SPT7 than on GCN5, indicating that GCN5 can function via pathways independent of the SAGA complex. Finally, we examine dependence on GCN5 and SWI-SNF at two model promoters and find that although these two chromatin-remodeling and/or modification activities may sometimes work together, in other instances they act in complementary fashion. PMID: 11416135 [PubMed - indexed for MEDLINE] 164: Biochem J 2001 Jul 1;357(Pt 1):83-95 Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding. Kramer B, Ferrari DM, Klappa P, Pohlmann N, Soling HD. Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D37077 Gottingen, Germany. The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b' domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as 'scrambled' ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b' domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may promote oxidative folding by different kinetic pathways. PMID: 11415439 [PubMed - indexed for MEDLINE] 165: Biochem Biophys Res Commun 2001 Jun 22;284(4):1083-9 Similar subunit interactions contribute to assembly of clathrin adaptor complexes and COPI complex: analysis using yeast three-hybrid system. Takatsu H, Futatsumori M, Yoshino K, Yoshida Y, Shin HW, Nakayama K. Institute of Biological Sciences and Gene Experiment Center, University of Tsukuba, Ibaraki, Tsukuba Science City, 305-8572, Japan. Clathrin adaptor protein (AP) complexes are heterotetramers composed of two large, one medium, and one small subunits. By exploiting the yeast three-hybrid system, we have found that an interaction between the two large subunits of the AP-1 complex, gamma-adaptin and beta1-adaptin, is markedly enhanced in the presence of the small subunit, sigma1. Similarly, two large subunits of the AP-4 complex, epsilon-adaptin and beta4-adaptin, are found to interact with each other only in the presence of the small subunit, sigma4. Furthermore, we have found that an interaction between two large subunits of the COPI F subcomplex, gamma-COP and beta-COP, is detectable only in the presence of zeta-COP. Because these COPI subunits have common ancestral origins to the corresponding AP subunits, these three-hybrid data, taken together with the previous two-hybrid data, suggest that the AP complexes and the COPI F subcomplex assemble by virtue of similar subunit interactions. Copyright 2001 Academic Press. PMID: 11409905 [PubMed - indexed for MEDLINE] 166: FEBS Lett 2001 Jun 8;498(2-3):150-6 Nuclear export of mRNA. Zenklusen D, Stutz F. Institute of Microbiology, Centre Hospitalier Universitaire Vaudois, 44, rue du Bugnon, 1011, Lausanne, Switzerland. Export of mRNA through nuclear pore complexes (NPC) is preceded by multiple and well coordinated processing steps, resulting in the formation of an export competent ribonucleoprotein complex (mRNP). Numerous factors involved in the translocation of the mRNP through the NPC and its release into the cytoplasm have been isolated mainly through genetic approaches in yeast, and putative functional homologues have been identified in metazoan systems. Understanding the mechanism of mRNA export relies, in part, on the functional characterization of these factors and the establishment of a complete network of molecular interactions. Here we summarize recent progress in the characterization of yeast and mammalian components implicated in the export of an mRNA from the nucleus to the cytoplasm. Publication Types: Review Review, Tutorial PMID: 11412847 [PubMed - indexed for MEDLINE] 167: Genes Dev 2001 Jun 15;15(12):1528-39 The 19S complex of the proteasome regulates nucleotide excision repair in yeast. Gillette TG, Huang W, Russell SJ, Reed SH, Johnston SA, Friedberg EC. Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9072, USA. Previous studies suggest that the amino-terminal ubiquitin-like (ubl) domain of Rad23 protein can recruit the proteasome for a stimulatory role during nucleotide excision repair in the yeast Saccharomyces cerevisiae. In this report, we show that the 19S regulatory complex of the yeast proteasome can affect nucleotide excision repair independently of Rad23 protein. Strains with mutations in 19S regulatory subunits (but not 20S subunits) of the proteasome promote partial recovery of nucleotide excision repair in vivo in rad23 deletion mutants, but not in other nucleotide excision repair-defective strains tested. In addition, a strain that expresses a temperature-degradable ATPase subunit of the 19S regulatory complex manifests a dramatically increased rate of nucleotide excision repair in vivo. These data indicate that the 19S regulatory complex of the 26S proteasome can negatively regulate the rate of nucleotide excision repair in yeast and suggest that Rad23 protein not only recruits the 19S regulatory complex, but also can mediate functional interactions between the 19S regulatory complex and the nucleotide excision repair machinery. The 19S regulatory complex of the yeast proteasome functions in nucleotide excision repair independent of proteolysis. PMID: 11410533 [PubMed - indexed for MEDLINE] 168: Proc Natl Acad Sci U S A 2001 Jun 19;98(13):7325-30 Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex. Mah AS, Jang J, Deshaies RJ. Division of Biology and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA. Exit from mitosis in budding yeast requires inactivation of cyclin-dependent kinases through mechanisms triggered by the protein phosphatase Cdc14. Cdc14 activity, in turn, is regulated by a group of proteins, the mitotic exit network (MEN), which includes Lte1, Tem1, Cdc5, Cdc15, Dbf2/Dbf20, and Mob1. The direct biochemical interactions between the components of the MEN remain largely unresolved. Here, we investigate the mechanisms that underlie activation of the protein kinase Dbf2. Dbf2 kinase activity depended on Tem1, Cdc15, and Mob1 in vivo. In vitro, recombinant protein kinase Cdc15 activated recombinant Dbf2, but only when Dbf2 was bound to Mob1. Conserved phosphorylation sites Ser-374 and Thr-544 (present in the human, Caenorhabditis elegans, and Drosophila melanogaster relatives of Dbf2) were required for DBF2 function in vivo, and activation of Dbf2-Mob1 by Cdc15 in vitro. Although Cdc15 phosphorylated Dbf2, Dbf2-Mob1, and Dbf2(S374A/T544A)-Mob1, the pattern of phosphate incorporation into Dbf2 was substantially altered by either the S374A T544A mutations or omission of Mob1. Thus, Cdc15 promotes the exit from mitosis by directly switching on the kinase activity of Dbf2. We propose that Mob1 promotes this activation process by enabling Cdc15 to phosphorylate the critical Ser-374 and Thr-544 phosphoacceptor sites of Dbf2. PMID: 11404483 [PubMed - indexed for MEDLINE] 169: Methods 2001 Jul;24(3):297-306 High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. Walhout AJ, Vidal M. Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. Protein-protein interactions play fundamental roles in many biological processes. Hence, protein interaction mapping is becoming a well-established functional genomics approach to generate functional annotations for predicted proteins that so far have remained uncharacterized. The yeast two-hybrid system is currently one of the most standardized protein interaction mapping techniques. Here, we describe the protocols for a semiautomated, high-throughput, Gal4-based yeast two-hybrid system. Copyright 2001 Academic Press. PMID: 11403578 [PubMed - indexed for MEDLINE] 170: Methods 2001 Jul;24(3):201-17 Using the yeast interaction trap and other two-hybrid-based approaches to study protein-protein interactions. Toby GG, Golemis EA. Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. The detection of physical interaction between two or more molecules of interest can be facilitated if the act of association between the interactive partners leads to the production of a readily observed biological or physical readout. Many interacting molecule pairs (X, Y) can be made to induce such a readout if X and Y are each fused to defined protein elements with desired properties. For example, in the yeast forward two-hybrid system, X is synthesized as a translational fusion to a DNA-binding domain (DBD), Y is synthesized as a fusion to a transcriptional activation domain (AD), and coexpression of DBD-X and AD-Y induces transcription of easily scored responsive reporters. Other approaches use paradigms based on the artificial production of two, hybrid, molecules, but substitute a variety of readouts including the repression of transcription, activation of signal transduction pathways, or reconstitution of a disrupted enzymatic activity. In this article, we summarize a number of two-hybrid-based approaches, and detail the use of the forward yeast two-hybrid system in a screen to identify novel interacting partners for a protein of interest. Copyright 2001 Academic Press. Publication Types: Review Review, Tutorial PMID: 11403570 [PubMed - indexed for MEDLINE] 171: J Mol Biol 2001 Jun 22;309(5):1007-15 Sfl1 functions via the co-repressor Ssn6-Tup1 and the cAMP-dependent protein kinase Tpk2. Conlan RS, Tzamarias D. Institute of Molecular Biology & Biotechnology-Foundation of Research & Technology, Vassilika Vouton, Heraklion, Crete, GR-711 10, Greece. r.s.conlan@swan.ac.uk Ssn6 (Cyc8) is a component of the yeast general corepressor Ssn6-Tup1 that inhibits the transcription of many diversely regulated genes. The corepressor does not interact directly with DNA but is recruited to different promoters through interactions with distinct pathway-specific, DNA-binding repressor proteins. Using yeast two-hybrid and GST chromatography interaction experiments, we have determined that Sfl1, a novel repressor protein, interacts directly with Ssn6, and in vivo repression data suggest that Sfl1 inhibits transcription by recruiting Ssn6-Tup1 via a specific domain in the Sfl1 protein. Sin4 and Srb10, components of specific RNA polymerase II sub-complexes that are required for Ssn6-Tup1 repression activity, are found to be required for Sfl1 repression function. These results indicate a possible mechanism for Sfl1-mediated repression via Ssn6-Tup1 and specific subunits of the RNA polymerase II holoenzyme. Electrophoretic mobility shift and chromatin immuno-precipitation assays demonstrate that Sfl1 is present at the promoters of three Ssn6-Tup1-repressible genes; namely, FLO11, HSP26, and SUC2. Sfl1 is known to interact with Tpk2, a cAMP-dependent protein kinase that negatively regulates Sfl1 function. Consistently, we show that phosphorylation by protein kinase A inhibits Sfl1 DNA binding in vitro, and that a tpk2Delta mutation increases the levels of Sfl1 protein associated with specific promoter elements in vivo. These data indicate a possible mechanism for regulating Sfl1-mediated repression through modulation of DNA binding by cAMP-dependent protein kinase-dependent phosphorylation. Taken together with previous data, these new observations suggest a link between cAMP signaling and Ssn6-Tup1-mediated transcriptional repression. Copyright 2001 Academic Press. PMID: 11399075 [PubMed - indexed for MEDLINE] 172: Mol Membr Biol 2001 Jan-Mar;18(1):105-12 Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae. Hauser M, Narita V, Donhardt AM, Naider F, Becker JM. Department of Microbiology and Biochemistry, University of Tennessee, Knoxville, 37996-0845, USA. The model eukaryote Saccharomyces cerevisiae has two distinct peptide transport mechanisms, one for di-/tripeptides (the PTR system) and another for tetra-/pentapeptides (the OPT system). The PTR system consists of three genes, PTR1, PTR2 and PTR3. The transporter (Ptr2p), encoded by the gene PTR2, is a 12 transmembrane domain (TMD) integral membrane protein that translocates di-/tripeptides. Homologues to Ptr2p have been identified in virtually all organisms examined to date and comprise the PTR family of transport proteins. In S. cerevisiae, the expression of PTR2 is highly regulated at the cellular level by complex interactions of many genes, including PTR1, PTR3, CUP9 and SSY1. Oligopeptides, consisting of four to five amino acids, are transported by the 12-14 TMD integral membrane protein Opt1p. Unlike Ptr2p, distribution of this protein appears limited to fungi and plants, and there appears to be three paralogues in S. cerevisiae. This transporter has an affinity for enkephalin, an endogenous mammalian pentapeptide, as well as for glutathione. Although it is known that OPT1 is normally expressed only during sporulation, to date little is known about the genes and proteins involved in the regulation of OPT1 expression. Publication Types: Review Review, Tutorial PMID: 11396605 [PubMed - indexed for MEDLINE] 173: Mol Cell Biol 2001 Jul;21(13):4233-45 New function of CDC13 in positive telomere length regulation. Meier B, Driller L, Jaklin S, Feldmann HM. Institute for Biochemistry, University of Munich (LMU), D-81377 Munich, Germany. Two roles for the Saccharomyces cerevisiae Cdc13 protein at the telomere have previously been characterized: it recruits telomerase to the telomere and protects chromosome ends from degradation. In a synthetic lethality screen with YKU70, the 70-kDa subunit of the telomere-associated Yku heterodimer, we identified a new mutation in CDC13, cdc13-4, that points toward an additional regulatory function of CDC13. Although CDC13 is an essential telomerase component in vivo, no replicative senescence can be observed in cdc13-4 cells. Telomeres of cdc13-4 mutants shorten for about 150 generations until they reach a stable level. Thus, in cdc13-4 mutants, telomerase seems to be inhibited at normal telomere length but fully active at short telomeres. Furthermore, chromosome end structure remains protected in cdc13-4 mutants. Progressive telomere shortening to a steady-state level has also been described for mutants of the positive telomere length regulator TEL1. Strikingly, cdc13-4/tel1Delta double mutants display shorter telomeres than either single mutant after 125 generations and a significant amplification of Y' elements after 225 generations. Therefore CDC13, TEL1, and the Yku heterodimer seem to represent distinct pathways in telomere length maintenance. Whereas several CDC13 mutants have been reported to display elongated telomeres indicating that Cdc13p functions in negative telomere length control, we report a new mutation leading to shortened and eventually stable telomeres. Therefore we discuss a key role of CDC13 not only in telomerase recruitment but also in regulating telomerase access, which might be modulated by protein-protein interactions acting as inhibitors or activators of telomerase activity. PMID: 11390652 [PubMed - indexed for MEDLINE] 174: Mol Cell Biol 2001 Jul;21(13):4089-96 Phosphorylation of the RNA polymerase II carboxy-terminal domain by the Bur1 cyclin-dependent kinase. Murray S, Udupa R, Yao S, Hartzog G, Prelich G. Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York 10461, USA. BUR1, which was previously identified by a selection for mutations that have general effects on transcription in Saccharomyces cerevisiae, encodes a cyclin-dependent kinase that is essential for viability, but none of its substrates have been identified to date. Using an unbiased biochemical approach, we have identified the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II, as a Bur1 substrate. Phosphorylation of Rpb1 by Bur1 is likely to be physiologically relevant, since bur1 mutations interact genetically with rpb1 CTD truncations and with mutations in other genes involved in CTD function. Several genetic interactions are presented, implying a role for Bur1 during transcriptional elongation. These results identify Bur1 as a fourth S. cerevisiae CTD kinase and provide striking functional similarities between Bur1 and metazoan P-TEFb. PMID: 11390638 [PubMed - indexed for MEDLINE] 175: FEBS Lett 2001 Jun 1;498(1):46-51 Shy1p occurs in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast. Nijtmans LG, Artal Sanz M, Bucko M, Farhoud MH, Feenstra M, Hakkaart GA, Zeviani M, Grivell LA. Section for Molecular Biology, Swammerdam Institute of Life Sciences, University of Amsterdam, The Netherlands. nijtmans@science.uva.nl Surf1p is a protein involved in the assembly of mitochondrial respiratory chain complexes. However its exact role in this process remains to be elucidated. We studied SHY1, the yeast homologue of SURF1, with an aim to obtain a better understanding of the molecular pathogenesis of cytochrome c oxidase (COX) deficiency in SURF1 mutant cells from Leigh syndrome patients. Assembly of COX was analysed in a shy1 null mutant strain by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Steady-state levels of the enzyme were found to be strongly reduced, the total amount of assembled complex being approximately 30% of control. The presence of a significant amount of holo-COX in the SHY1-disruptant strain suggests that Shy1p may either facilitate assembly of the enzyme, or increase its stability. However, our observations, based on 2D-PAGE analysis of mitochondria labelled in vitro, now provide the first direct evidence that COX assembly is impaired in a Deltashy1 strain. COX enzyme assembled in the absence of Shy1p appears to be structurally and enzymically normal. The in vitro labelling studies additionally indicate that mitochondrial translation is significantly increased in the shy1 null mutant strain, possibly reflecting a compensatory mechanism for reduced respiratory capacity. Protein interactions of both Shy1p and Surf1p are implied by their appearance in a high molecular weight complex of about 250 kDa, as shown by 2D-PAGE. PMID: 11389896 [PubMed - indexed for MEDLINE] 176: Mol Cell 2001 May;7(5):1013-23 Evolutionarily conserved interaction between CstF-64 and PC4 links transcription, polyadenylation, and termination. Calvo O, Manley JL. Department of Biological Sciences, Columbia University, New York, NY 10027, USA. Tight connections exist between transcription and subsequent processing of mRNA precursors, and interactions between the transcription and polyadenylation machineries seem especially extensive. Using a yeast two-hybrid screen to identify factors that interact with the polyadenylation factor CstF-64, we uncovered an interaction with the transcriptional coactivator PC4. Both human proteins have yeast homologs, Rna15p and Sub1p, respectively, and we show that these two proteins also interact. Given evidence that certain polyadenylation factors, including Rna15p, are necessary for termination in yeast, we show that deletion or overexpression of SUB1 suppresses or enhances, respectively, both growth and termination defects detected in an rna15 mutant strain. Our findings provide an additional, unexpected connection between transcription and polyadenylation and suggest that PC4/Sub1p, via its interaction with CstF-64/Rna15p, possesses an evolutionarily conserved antitermination activity. PMID: 11389848 [PubMed - indexed for MEDLINE] 177: J Biol Chem 2001 Aug 10;276(32):29782-91 Marked stepwise differences within a common kinetic mechanism characterize TATA-binding protein interactions with two consensus promoters. Powell RM, Parkhurst KM, Brenowitz M, Parkhurst LJ. Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA. Binding of the TATA-binding protein (TBP) to promoter DNA bearing the TATA sequence is an obligatory initial step in RNA polymerase II transcription initiation. The interactions of Saccharomyces cerevisiae TBP with the E4 (TATATATA) and adenovirus major late (TATAAAAG) promoters have been modeled via global analysis of kinetic and thermodynamic data obtained using fluorescence resonance energy transfer. A linear two-intermediate kinetic mechanism describes the reaction of both of these consensus strong promoters with TBP. Qualitative features common to both interactions include tightly bound TBP-DNA complexes with similar solution geometries, simultaneous DNA binding and bending, and the presence of intermediate TBP-DNA conformers at high mole fraction throughout most of the reaction and at equilibrium. Despite very similar energetic changes overall, the stepwise entropic and enthalpic compensations along the two pathways differ markedly following the initial binding/bending event. Furthermore, TBP-E4 dissociation ensues from both replacement and displacement processes, in contrast to replacement alone for TBP-adenovirus major late promoter. A model is proposed that explicitly correlates these similarities and differences with the sequence-specific structural properties inherent to each promoter. This detailed mechanistic comparison of two strong promoters interacting with TBP provides a foundation for subsequent comparison between consensus and variant promoter sequences reacting with TBP. PMID: 11387341 [PubMed - indexed for MEDLINE] 178: J Biol Chem 2001 Aug 3;276(31):29268-74 Proteomic analysis of nucleoporin interacting proteins. Allen NP, Huang L, Burlingame A, Rexach M. Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA. The Saccharomyces cerevisiae nuclear pore complex is a supramolecular assembly of 30 nucleoporins that cooperatively facilitate nucleocytoplasmic transport. Thirteen nucleoporins that contain FG peptide repeats (FG Nups) are proposed to function as stepping stones in karyopherin-mediated transport pathways. Here, protein interactions that occur at individual FG Nups were sampled using immobilized nucleoporins and yeast extracts. We find that many proteins bind to FG Nups in highly reproducible patterns. Among 135 proteins identified by mass spectrometry, most were karyopherins and nucleoporins. The PSFG nucleoporin Nup42p and the GLFG nucleoporins Nup49p, Nup57p, Nup100p, and Nup116p exhibited generic interactions with karyopherins; each bound 6--10 different karyopherin betas, including importins as well as exportins. Unexpectedly, the same Nups also captured the hexameric Nup84p complex and Nup2p. In contrast, the FXFG nucleoporins Nup1p, Nup2p, and Nup60p were more selective and captured mostly the Kap95p.Kap60p heterodimer. When the concentration of Gsp1p-GTP was elevated in the extracts to mimic the nucleoplasmic environment, the patterns of interacting proteins changed; exportins exhibited enhanced binding to FG Nups, and importins exhibited reduced binding. The results demonstrate a global role for Gsp1p-GTP on karyopherin-nucleoporin interactions and provide a rudimentary map of the routes that karyopherins take as they cross the nuclear pore complex. PMID: 11387327 [PubMed - indexed for MEDLINE] 179: EMBO J 2001 Jun 1;20(11):2742-56 SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase. Farras R, Ferrando A, Jasik J, Kleinow T, Okresz L, Tiburcio A, Salchert K, del Pozo C, Schell J, Koncz C. Max-Planck Institut fur Zuchtungsforschung, Carl-von-Linne-Weg 10, D-50829 Cologne, Germany. Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD-protein. Here we show that SKP1/ASK1, a conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1-binding proteasomal protein, alpha4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs. SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts. SKP1/ASK1, cullin and proteasomal alpha-subunits show nuclear co-localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division. Detection of SnRK in purified 26S proteasomes and co-purification of epitope- tagged SKP1/ASK1 with SnRK, cullin and proteasomal alpha-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and alpha4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis. PMID: 11387208 [PubMed - indexed for MEDLINE] 180: J Biol Chem 2001 Aug 3;276(31):29393-402 Molecular characterization of mammalian homologues of class C Vps proteins that interact with syntaxin-7. Kim BY, Kramer H, Yamamoto A, Kominami E, Kohsaka S, Akazawa C. Department of Neurochemistry, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan. Vesicle-mediated protein sorting plays an important role in segregation of intracellular molecules into distinct organelles. Extensive genetic studies using yeast have identified more than 40 vacuolar protein sorting (VPS) genes involved in vesicle transport to vacuoles. However, their mammalian counterparts are not fully elucidated. In this study, we identified two human homologues of yeast Class C VPS genes, human VPS11 (hVPS11) and human VPS18 (hVPS18). We also characterized the subcellular localization and interactions of the protein products not only from these genes but also from the other mammalian Class C VPS homologue genes, hVPS16 and rVPS33a. The protein products of hVPS11 (hVps11) and hVPS18 (hVps18) were ubiquitously expressed in peripheral tissues, suggesting that they have a fundamental role in cellular function. Indirect immunofluorescence microscopy revealed that the mammalian Class C Vps proteins are predominantly associated with late endosomes/lysosomes. Immunoprecipitation and gel filtration studies showed that the mammalian Class C Vps proteins constitute a large hetero-oligomeric complex that interacts with syntaxin-7. These results indicate that like their yeast counterparts, mammalian Class C Vps proteins mediate vesicle trafficking steps in the endosome/lysosome pathway. PMID: 11382755 [PubMed - indexed for MEDLINE] 181: Trends Genet 2001 Jun;17(6):346-52 Protein--protein interaction maps: a lead towards cellular functions. Legrain P, Wojcik J, Gauthier JM. Hybrigenics, 180 Avenue Daumesnil, Paris 75012, France. plegrain@hybrigenics.fr The availability of complete genome sequences now permits the development of tools for functional biology on a proteomic scale. Several experimental approaches or in silico algorithms aim at clustering proteins into networks with biological significance. Among those, the yeast two-hybrid system is the technology of choice to detect protein-protein interactions. Recently, optimized versions were applied at a genomic scale, leading to databases on the web. However, as with any other 'genetic' assay, yeast two-hybrid assays are prone to false positives and false negatives. Here we discuss these various technologies, their general limitations and the potential advances they make possible, especially when in combination with other functional genomics or bioinformatics analyses. Publication Types: Review Review, Tutorial PMID: 11377797 [PubMed - indexed for MEDLINE] 182: J Biol Chem 2001 Aug 3;276(31):29382-92 Single amino acid substitutions and deletions that alter the G protein coupling properties of the V2 vasopressin receptor identified in yeast by receptor random mutagenesis. Erlenbach I, Kostenis E, Schmidt C, Serradeil-Le Gal C, Raufaste D, Dumont ME, Pausch MH, Wess J. Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA. To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G(s)-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met(145) in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met(145) led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met(145) had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding. PMID: 11375990 [PubMed - indexed for MEDLINE] 183: Biotechnol Bioeng 2001 Jul 20;74(2):96-107 Prediction of the pilot-scale recovery of a recombinant yeast enzyme using integrated models. Varga EG, Titchener-Hooker NJ, Dunnill P. The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. This article describes the rapid prediction of recovery process performance for a new recombinant enzyme product on the basis of a broad portfolio of computer models and highly targeted experimentation. A process model for the recombinant system was generated by linking unit operation models in an integrated fashion, with required parameter estimation and physical property determination accomplished using data from scale-down studies. This enabled the generic modeling framework established for processing of a natural enzyme from bakers' yeast to be applied. An experimental study of the same operations at the pilot scale showed that the process model gave a conservative prediction of recombinant enzyme recovery. The model successfully captured interactions leading to a low overall product yield and indicated the need for further study of precipitate breakage in the feed zone of a disc stack centrifuge in order to improve performance. The utility of scale-down units as an aid to fast model generation and the advantage of integrating computer modeling and scale-down studies to accelerate bioprocess development are highlighted. Copyright 2001 John Wiley & Sons, Inc. Publication Types: Evaluation Studies PMID: 11369998 [PubMed - indexed for MEDLINE] 184: Protein Sci 2001 Jun;10(6):1113-23 Environmentally induced reversible conformational switching in the yeast cell adhesion protein alpha-agglutinin. Zhao H, Chen MH, Shen ZM, Kahn PC, Lipke PN. Department of Biological Sciences and the Institute for Biomolecular Structure and Function, Hunter College of the City University of New York, New York 10021,USA. The yeast cell adhesion protein alpha-agglutinin is expressed on the surface of a free-living organism and is subjected to a variety of environmental conditions. Circular dichroism (CD) spectroscopy shows that the binding region of alpha-agglutinin has a beta-sheet-rich structure, with only approximately 2% alpha-helix under native conditions (15-40 degrees C at pH 5.5). This region is predicted to fold into three immunoglobulin-like domains, and models are consistent with the CD spectra as well as with peptide mapping and site-specific mutagenesis. However, secondary structure prediction algorithms show that segments comprising approximately 17% of the residues have high alpha-helical and low beta-sheet potential. Two model peptides of such segments had helical tendencies, and one of these peptides showed pH-dependent conformational switching. Similarly, CD spectroscopy of the binding region of alpha-agglutinin showed reversible conversion from beta-rich to mixed alpha/beta structure at elevated temperatures or when the pH was changed. The reversibility of these changes implied that there is a small energy difference between the all-beta and the alpha/beta states. Similar changes followed cleavage of peptide or disulfide bonds. Together, these observations imply that short sequences of high helical propensity are constrained to a beta-rich state by covalent and local charge interactions under native conditions, but form helices under non-native conditions. PMID: 11369849 [PubMed - indexed for MEDLINE] 185: Arch Biochem Biophys 2001 Jan 15;385(2):301-10 Photoaffinity labeling and photoaffinity cross-linking of phosphofructokinase-1 from Saccharomyces cerevisiae by 8-azidoadeninenucleotides. Knoche M, Monnich K, Schafer HJ, Kopperschlager G. Institut fur Biochemie, Fachbereich Chemie und Pharmazie, Johannes Gutenberg-Universitat Mainz, Germany. Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four alpha- and four beta-subunits, each of them carrying catalytic and regulatory bindings sites for MgATP. In this paper, various photoaffinity labels, such as 8-azidoadenosine 5'-triphosphate, 8-azido-1,N6-ethenoadenosine 5'-triphosphate, and 8-N3-3'(2')-O-biotinyl-8-azidoadenosine 5'-triphosphate have been used to study their interaction with the enzyme in the dark and during irradiation. All nucleotidetriphosphates function as phosphate donor forming fructose 1,6-bisphosphate from fructose 6-phosphate. However, the kinetic analysis revealed distinctly differences between them. Photolabeling causes a decrease in enzyme activity to a similar extent, and ATP acts as competitive effector to inactivation. Three bifunctional diazidodiadeninedinucleotides (8-diN3AP4A, monoepsilon-8-diN3AP4A, and diepsilon-8-diN3AP4A) were applied for studying the spatial arrangement of the nucleotide binding sites. No cross-linking of the subunits was obtained by irradiation of the enzyme with 8-diN3AP4A. Photolabeling with diepsilon-8-diN3AP4A resulted in the formation of two alpha-beta cross-links with different mobilities in the SDS-polyacrylamide gel electrophoresis, while monoepsilon-8-diN3AP4A yielded only one alpha-beta cross-link. Because an interfacial location of the catalytic sites between two subunits is less likely, we suggest that the formation of cross-linked subunits may be the result of specific interactions of the bifunctional photolabels with regulatory sites at the interface of both subunits. PMID: 11368011 [PubMed - indexed for MEDLINE] 186: Mol Biol Cell 2001 May;12(5):1381-92 Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export. Azad AK, Stanford DR, Sarkar S, Hopper AK. Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA. Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export. PMID: 11359929 [PubMed - indexed for MEDLINE] 187: Methods Mol Biol 2001;148:431-49 Genetic analysis of DNA-protein interactions using a reporter gene assay in yeast. Setzer DR, Schulman DB, Bumbulis MJ. Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA. PMID: 11357604 [PubMed - indexed for MEDLINE] 188: J Biol Chem 2001 Jul 13;276(28):26715-23 Characterization of DNA damage-stimulated self-interaction of Saccharomyces cerevisiae checkpoint protein Rad17p. Zhang H, Zhu Z, Vidanes G, Mbangkollo D, Liu Y, Siede W. Department of Radiation Oncology and the Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322, USA. Saccharomyces cerevisiae Rad17p is necessary for cell cycle checkpoint arrests in response to DNA damage. Its known interactions with the checkpoint proteins Mec3p and Ddc1p in a PCNA-like complex indicate a sensor role in damage recognition. In a novel application of the yeast two-hybrid system and by immunoprecipitation, we show here that Rad17p is capable of increased self-interaction following DNA damage introduced by 4-nitroquinoline-N-oxide, camptothecin or partial inactivation of DNA ligase I. Despite overlap of regions required for Rad17p interactions with Rad17p or Mec3p, single amino acid substitutions revealed that Rad17p x Rad17p complex formation is independent of Mec3p. E128K (rad17-1) was found to inhibit Rad17p interaction with Mec3p but not with Rad17p. On the other hand, Phe-121 is essential for Rad17p self-interaction, and its function in checkpoint arrest but not for Mec3p interaction. These differential effects indicate that Rad17p-Rad17p interaction plays a role that is independent of the Rad17p x Mec3p x Ddc1p complex, although our results are also compatible with Rad17p-mediated supercomplex formation of the Rad17p x Mec3p x Ddc1p heterotrimer in response to DNA damage. PMID: 11356855 [PubMed - indexed for MEDLINE] 189: Cancer Radiother 2001 Apr;5(2):109-29 [Molecular mechanisms controlling the cell cycle: fundamental aspects and implications for oncology] [Article in French] Viallard JF, Lacombe F, Belloc F, Pellegrin JL, Reiffers J. Service de medecine interne et maladies infectieuses, centre Francois-Magendie, hopital du Haut-Leveque, 5, avenue Magellan, 33604 Pessac, France. jean-francois.viallard@chu-bordeaux.fr INTRODUCTION: Comprehension of cell cycle regulation mechanisms has progressed very quickly these past few years and regulators of the cell cycle have gained widespread importance in cancer. This review first summarizes major advances in the understanding of the control of cell cycle mechanisms. Examples of how this control is altered in tumoral cells are then described. CURRENT KNOWLEDGE AND KEY POINTS: The typical mammalian cell cycle consists of four distinct phases occurring in a well-defined order, each of which should be completed successfully before the next begins. Progression of eukaryotic cells through major cell cycle transitions is mediated by sequential assembly and activation of a family of serine-threonine protein kinases, the cyclin dependent kinases (CDK). The timing of their activation is determined by their post-translational modifications (phosphorylations/dephosphorylations), and by the association of a protein called cyclin, which is the regulatory subunit of the kinase complex. The cyclin family is divided into two main classes. The 'G1 cyclins' include cyclins C, D1-3, and E, and their accumulation is rate-limiting for progression from the G1 to S phase. The 'mitotic or G2 cyclins', which include cyclin A and cyclin B, are involved in the control of G2/M transition and mitosis. The cyclins bind to and activate the CDK, which leads to phosphorylation (and then inhibition) of the tumor suppressor protein, pRb. pRb controls commitment to progress from the G1 to S phase, at least in part by repressing the activity of the E2F transcription factors known to promote cell proliferation. Both the D-type cyclins and their partner kinases CDK4/6 have proto-oncogenic properties, and their activity is carefully regulated at multiple levels including negative control by two families of CDK inhibitors. While members of the INK4 family (p16INK4A, p15INK4B, p18INK4C, p19INK4D) interact specifically with CDK4 and CDK6, the CIP/KIP inhibitors p21CIP1/WAF1, p27KIP1 and p57KIP2 inhibit a broader spectrum of CDK. The interplay between p16INK4A, cyclin D/CDK, and pRb/E2F together constitute a functional unit collectively known as the 'pRb pathway'. Each of the major components of this mechanism may become deregulated in cancer, and accumulating evidence points to the 'pRb pathway' as a candidate obligatory target in multistep oncogenesis of possibly all human tumor types. FUTURE PROSPECTS AND PROJECTS: Major advances in the understanding of cell cycle regulation mechanisms provided a better knowledge of the molecular interactions involved in human cancer. This progress has led to the promotion of new therapeutic agents presently in clinical trials or under development. Moreover, the components of the cell cycle are probably involved in other non-cancerous diseases and their role must be defined. Publication Types: Review Review, Academic PMID: 11355576 [PubMed - indexed for MEDLINE] 190: Biotechniques 2001 May;30(5):984-8 Erratum in: Biotechniques 2001 Sep;31(3):488 Rapid selection against truncation mutants in yeast reverse two-hybrid screens. Puthalakath H, Strasser A, Huang DC. Walter and Eliza Hall Institute, Melbourne, Australia. The yeast reverse two-hybrid system is a powerful technique for isolating mutations in a protein that abolish its interaction with a known partner. Selection is based on abrogation of growth suppression imposed when wild-type interactions confer 5-fluoroorotic acid (5-FOA) sensitivity to yeast cells. A laborious component of this system is to eliminate those mutations that cause protein truncation. By fusing the green fluorescent protein (GFP) to the C-terminus of a protein of interest, dynein light chain (LC8), we were able to rapidly isolate mutations that did not result in protein truncation. Publication Types: Technical Report PMID: 11355361 [PubMed - indexed for MEDLINE] 191: J Biol Chem 2001 Jul 13;276(28):26526-33 Interaction of gamma 1-syntrophin with diacylglycerol kinase-zeta. Regulation of nuclear localization by PDZ interactions. Hogan A, Shepherd L, Chabot J, Quenneville S, Prescott SM, Topham MK, Gee SH. Department of Cellular and Molecular Medicine, Center for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada. Syntrophins are modular adapter proteins that link ion channels and signaling proteins to dystrophin and its homologues. A yeast two-hybrid screen of a human brain cDNA library using the PDZ domain of gamma 1- syntrophin, a recently identified brain-specific isoform, yielded overlapping clones encoding the C terminus of diacylglycerol kinase-zeta (DGK-zeta), an enzyme that converts diacylglycerol into phosphatidic acid. In biochemical assays, the C terminus of DGK-zeta, which contains a consensus PDZ-binding motif, was found to be necessary and sufficient for association with gamma 1-syntrophin. When coexpressed in HeLa cells, DGK-zeta and gamma 1-syntrophin formed a stable complex that partitioned between the cytoplasm and nucleus. DGK-zeta translocates from the cytosol to the nucleus, a process negatively regulated by protein kinase C phosphorylation. We found that DGK-zeta recruits gamma 1-syntrophin into the nucleus and that the PDZ-binding motif is required. Disrupting the interaction altered the intracellular localization of both proteins; DGK-zeta accumulated in the nucleus, whereas gamma 1-syntrophin remained in the cytoplasm. The level of endogenous syntrophins in the nucleus of HeLa cells also reflected the amount of nuclear DGK-zeta. In the brain, DGK-zeta and gamma 1-syntrophin were colocalized in cell bodies and dendrites of cerebellar Purkinjie neurons and other neuronal cell types, suggesting that their interaction is physiologically relevant. Moreover, coimmunoprecipitation and pull-down experiments from brain extracts and cells suggest that DGK-zeta, gamma 1-syntrophin, and dystrophin form a ternary complex. Collectively, our results suggest that gamma 1-syntrophin participates in regulating the subcellular localization of DGK-zeta to ensure correct termination of diacylglycerol signaling. PMID: 11352924 [PubMed - indexed for MEDLINE] 192: RNA 2001 Apr;7(4):565-75 An essential protein-binding domain of nuclear RNase P RNA. Ziehler WA, Morris J, Scott FH, Millikin C, Engelke DR. Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA. Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and protein subunits. The RNA subunits of each enzyme share substantial secondary structural features, and most of the protein subunits are shared between the two. One of the conserved RNA subdomains, designated P3, has previously been shown to be required for nucleolar localization. Phylogenetic sequence analysis suggests that the P3 domain interacts with one of the proteins common to RNase P and RNase MRP, a conclusion strengthened by an earlier observation that the essential domain can be interchanged between the two enzymes. To examine possible functions of the P3 domain, four conserved nucleotides in the P3 domain of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a library of all possible sequence combinations at those positions. Selection of functional genes in vivo identified permissible variations, and viable clones that caused yeast to exhibit conditional growth phenotypes were tested for defects in RNase P RNA and tRNA biosynthesis. Under nonpermissive conditions, the mutants had reduced maturation of the RPR1 RNA precursor, an expected phenotype in cases where RNase P holoenzyme assembly is defective. This loss of RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation characteristic of RNase P defects. To test whether mutations at the conserved positions inhibited interactions with a particular protein, specific binding of the individual protein subunits to the RNA subunit was tested in yeast using the three-hybrid system. Pop1p, the largest subunit shared by RNases P and MRP, bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was eliminated by mutations at the conserved P3 residues. These results indicate that Pop1p interacts with the P3 domain common to RNases P and MRP, and that this interaction is critical in the maturation of RNase P holoenzyme. PMID: 11345435 [PubMed - indexed for MEDLINE] 193: Proc Natl Acad Sci U S A 2001 May 22;98(11):6080-5 Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I. Gross S, Moore C. Department of Molecular Biology and Microbiology, Sackler School of Graduate Biomedical Sciences, Tufts University, Stearns 509, 136 Harrison Avenue, Boston, MA 02111, USA. Cleavage and polyadenylation of mRNA 3' ends in Saccharomyces cerevisiae requires several factors, one of which is cleavage factor I (CF I). Purification of CF I activity from yeast extract has implicated numerous proteins as functioning in both cleavage and/or polyadenylation. Through reconstitution of active CF I from separately expressed and purified proteins, we show that CF I contains five subunits, Rna14, Rna15, Pcf11, Clp1, and Hrp1. These five are necessary and sufficient for reconstitution of cleavage activity in vitro when mixed with CF II, and for specific polyadenylation when mixed with polyadenylation factor I, purified poly(A) polymerase, and poly(A) binding protein. Analysis of the individual protein-protein interactions supports an architectural model for CF I in which Pcf11 simultaneously interacts with Rna14, Rna15, and Clp1, whereas Rna14 bridges Rna15 and Hrp1. PMID: 11344258 [PubMed - indexed for MEDLINE] 194: Science 2001 May 4;292(5518):929-34 Integrated genomic and proteomic analyses of a systematically perturbed metabolic network. Ideker T, Thorsson V, Ranish JA, Christmas R, Buhler J, Eng JK, Bumgarner R, Goodlett DR, Aebersold R, Hood L. The Institute for Systems Biology, 4225 Roosevelt Way NE, Suite 200, Seattle, WA 98105, USA. tideker@systemsbiology.org We demonstrate an integrated approach to build, test, and refine a model of a cellular pathway, in which perturbations to critical pathway components are analyzed using DNA microarrays, quantitative proteomics, and databases of known physical interactions. Using this approach, we identify 997 messenger RNAs responding to 20 systematic perturbations of the yeast galactose-utilization pathway, provide evidence that approximately 15 of 289 detected proteins are regulated posttranscriptionally, and identify explicit physical interactions governing the cellular response to each perturbation. We refine the model through further iterations of perturbation and global measurements, suggesting hypotheses about the regulation of galactose utilization and physical interactions between this and a variety of other metabolic pathways. PMID: 11340206 [PubMed - indexed for MEDLINE] 195: Mol Cell Biol 2001 Jun;21(11):3725-37 Rfc4 interacts with Rpa1 and is required for both DNA replication and DNA damage checkpoints in Saccharomyces cerevisiae. Kim HS, Brill SJ. Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854, USA. The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function. To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast. A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such as rfa1-t11, are lethal in combination with rfc4-2. The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints. RFC4 and the DNA damage checkpoint gene RAD24 were found to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G(1)/S DNA damage checkpoint response and that both the rfc4-2 and rfa1-t11 strains are defective in the G(2)/M DNA damage checkpoint. Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways. Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles. PMID: 11340166 [PubMed - indexed for MEDLINE] 196: Biochem J 2001 May 15;356(Pt 1):207-15 Self-association and precursor protein binding of Saccharomyces cerevisiae Tom40p, the core component of the protein translocation channel of the mitochondrial outer membrane. Gordon DM, Wang J, Amutha B, Pain D. Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, D403 Richards Building, Philadelphia, PA 19104-6085, USA. The precursor protein translocase of the mitochondrial outer membrane (Tom) is a multi-subunit complex containing receptors and a general import channel, of which the core component is Tom40p. Nuclear-encoded mitochondrial precursor proteins are first recognized by surface receptors and then pass through the import channel. The Tom complex has been purified; however, the protein-protein interactions that drive its assembly and maintain its stability have been difficult to study. Here we show that Saccharomyces cerevisiae Tom40p expressed in bacteria and purified to homogeneity associates efficiently with itself. The self-association is very strong and can withstand up to 4 M urea or 1 M salt. The tight self-association does not require the N-terminal segment of Tom40p. Furthermore, purified Tom40p preferentially recognizes the targeting sequence of mitochondrial precursor proteins. Although the binding of the targeting sequence to Tom40p is inhibited by urea concentrations in excess of 1 M, it is moderately resistant to 1 M salt. Simultaneous self-assembly and precursor protein binding suggest that Tom40p contains at least two different domains mediating these processes. The experimental approach described here should be useful for analysing protein-protein interactions involving individual or groups of components of the mitochondrial import machinery. PMID: 11336653 [PubMed - indexed for MEDLINE] 197: FEBS Lett 2001 Apr 27;495(3):148-53 A yeast two-hybrid study of human p97/Gab2 interactions with its SH2 domain-containing binding partners. Crouin C, Arnaud M, Gesbert F, Camonis J, Bertoglio J. Inserm Unit 461, Faculte de Pharmacie Paris-XI, Chatenay-Malabry, france. p97/Gab2 is a recently characterized member of a large family of scaffold proteins that play essential roles in signal transduction. Gab2 becomes tyrosine-phosphorylated in response to a variety of growth factors and forms multimolecular complexes with SH2 domain-containing signaling molecules such as the p85-regulatory subunit of the phosphoinositide-3-kinase (p85-PI3K), the tyrosine phosphatase SHP-2 and the adapter protein CrkL. To characterize the interactions between Gab2 and its SH2-containing binding partners, we designed a modified yeast two-hybrid system in which the Lyn tyrosine kinase is expressed in a regulated manner in yeast. Using this assay, we demonstrated that p97/Gab2 specifically interacts with the SH2 domains of PI3K, SHP-2 and CrkL. Interaction with p85-PI3K is mediated by tyrosine residues Y452, Y476 and Y584 of Gab2, while interaction with SHP-2 depends exclusively on tyrosine Y614. CrkL interaction is mediated by its SH2 domain recognizing Y266 and Y293, despite the latter being in a non-consensus (YTFK) environment. PMID: 11334882 [PubMed - indexed for MEDLINE] 198: Genetics 2001 May;158(1):187-96 Multiple functional interactions between components of the Lsm2-Lsm8 complex, U6 snRNA, and the yeast La protein. Pannone BK, Kim SD, Noe DA, Wolin SL. Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536, USA. The U6 small nuclear ribonucleoprotein is a critical component of the eukaryotic spliceosome. The first protein that binds the U6 snRNA is the La protein, an abundant phosphoprotein that binds the 3' end of many nascent small RNAs. A complex of seven Sm-like proteins, Lsm2-Lsm8, also binds the 3' end of U6 snRNA. A mutation within the Sm motif of Lsm8p causes Saccharomyces cerevisiae cells to require the La protein Lhp1p to stabilize nascent U6 snRNA. Here we describe functional interactions between Lhp1p, the Lsm proteins, and U6 snRNA. LSM2 and LSM4, but not other LSM genes, act as allele-specific, low-copy suppressors of mutations in Lsm8p. Overexpression of LSM2 in the lsm8 mutant strain increases the levels of both Lsm8p and U6 snRNPs. In the presence of extra U6 snRNA genes, LSM8 becomes dispensable for growth, suggesting that the only essential function of LSM8 is in U6 RNA biogenesis or function. Furthermore, deletions of LSM5, LSM6, or LSM7 cause LHP1 to become required for growth. Our experiments are consistent with a model in which Lsm2p and Lsm4p contact Lsm8p in the Lsm2-Lsm8 ring and suggest that Lhp1p acts redundantly with the entire Lsm2-Lsm8 complex to stabilize nascent U6 snRNA. PMID: 11333229 [PubMed - indexed for MEDLINE] 199: Genetics 2001 May;158(1):87-93 Multiple functions of the nonconserved N-terminal domain of yeast TATA-binding protein. Lee M, Struhl K. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. The TATA-binding protein (TBP) is composed of a highly conserved core domain sufficient for TATA-element binding and preinitiation complex formation as well as a highly divergent N-terminal region that is dispensable for yeast cell viability. In vitro, removal of the N-terminal region domain enhances TBP-TATA association and TBP dimerization. Here, we examine the effects of truncation of the N-terminal region in the context of yeast TBP mutants with specific defects in DNA binding and in interactions with various proteins. For a subset of mutations that disrupt DNA binding and the response to transcriptional activators, removal of the N-terminal domain rescues their transcriptional defects. By contrast, deletion of the N-terminal region is lethal in combination with mutations on a limited surface of TBP. Although this surface is important for interactions with TFIIA and Brf1, TBP interactions with these two factors do not appear to be responsible for this dependence on the N-terminal region. Our results suggest that the N-terminal region of TBP has at least two distinct functions in vivo. It inhibits the interaction of TBP with TATA elements, and it acts positively in combination with a specific region of the TBP core domain that presumably interacts with another protein(s). PMID: 11333220 [PubMed - indexed for MEDLINE] 200: EMBO J 2001 May 1;20(9):2326-37 Multiple roles for the C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation. Asano K, Shalev A, Phan L, Nielsen K, Clayton J, Valasek L, Donahue TF, Hinnebusch AG. Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development/NIH, Bethesda, MD 20892, USA. eIF5 stimulates the GTPase activity of eIF2 bound to Met-tRNA(i)(Met), and its C-terminal domain (eIF5-CTD) bridges interaction between eIF2 and eIF3/eIF1 in a multifactor complex containing Met-tRNA(i)(Met). The tif5-7A mutation in eIF5-CTD, which destabilizes the multifactor complex in vivo, reduced the binding of Met-tRNA(i)(Met) and mRNA to 40S subunits in vitro. Interestingly, eIF5-CTD bound simultaneously to the eIF4G subunit of the cap-binding complex and the NIP1 subunit of eIF3. These interactions may enhance association of eIF4G with eIF3 to promote mRNA binding to the ribosome. In vivo, tif5-7A eliminated eIF5 as a stable component of the pre-initiation complex and led to accumulation of 48S complexes containing eIF2; thus, conversion of 48S to 80S complexes is the rate-limiting defect in this mutant. We propose that eIF5-CTD stimulates binding of Met-tRNA(i)(Met) and mRNA to 40S subunits through interactions with eIF2, eIF3 and eIF4G; however, its most important function is to anchor eIF5 to other components of the 48S complex in a manner required to couple GTP hydrolysis to AUG recognition during the scanning phase of initiation. PMID: 11331597 [PubMed - indexed for MEDLINE] 201: EMBO J 2001 May 1;20(9):2111-9 Oligopeptide repeats in the yeast protein Sup35p stabilize intermolecular prion interactions. Parham SN, Resende CG, Tuite MF. Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK. The nuclear-encoded Sup35p protein is responsible for the prion-like [PSI(+)] determinant of yeast, with Sup35p existing largely as a high molecular weight aggregate in [PSI(+)] strains. Here we show that the five oligopeptide repeats present at the N-terminus of Sup35p are responsible for stabilizing aggregation of Sup35p in vivo. Sequential deletion of the oligopeptide repeats prevented the maintenance of [PSI(+)] by the truncated Sup35p, although deletants containing only two repeats could be incorporated into pre-existing aggregates of wild-type Sup35p. The mammalian prion protein PrP also contains similar oligopeptide repeats and we show here that a human PrP repeat (PHGGGWGQ) is able functionally to replace a Sup35p oligopeptide repeat to allow stable [PSI(+)] propagation in vivo. Our data suggest a model in which the oligopeptide repeats in Sup35p stabilize intermolecular interactions between Sup35p proteins that initiate establishment of the aggregated state. Modulating repeat number therefore alters the rate of yeast prion conversion in vivo. Furthermore, there appears to be evolutionary conservation of function of the N-terminally located oligopeptide repeats in prion propagation. PMID: 11331577 [PubMed - indexed for MEDLINE] 202: Biochemistry 2001 Feb 20;40(7):2080-6 Mitochondrial phosphate transport protein. Reversions of inhibitory conservative mutations identify four helices and a nonhelix protein segment with transmembrane interactions and Asp39, Glu137, and Ser158 as nonessential for transport. Phelps A, Briggs C, Haefele A, Mincone L, Ligeti E, Wohlrab H. Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA. The mitochondrial phosphate transport protein (PTP) has six (A--F) transmembrane (TM) helices per subunit of functional homodimer with all mutations referring to the subunit of the homodimer. In earlier studies, conservative replacements of several residues located either at the matrix end (Asp39/helix A, Glu137/helix C, Asp236/helix E) or at the membrane center (His32/helix A, Glu136/helix C) of TM helices yielded inactive single mutation PTPs. Some of these residues were suggested to act as phosphate ligands or as part of the proton cotransport path. We now show that the mutation Ser158Thr, not part of a TM helix but located near the center of the matrix loop (Ile141--Ser171) between TM helices C and D, inactivates PTP and is thus also functionally relevant. On the other side of the membrane, the single mutation Glu192Asp at the intermembrane space end of TM helix D yields a PTP with 33% wild-type activity. We constructed double mutants by adding this mutation to the six transport-inactivating mutations. Transport was detected only in those with Asp39Asn, Glu137Gln, or Ser158Thr. We conclude that TM helix D can interact with TM helices A and C and matrix loop Ile141--Ser171 and that Asp39, Glu137, and Ser158 are not essential for phosphate transport. Since our results are consistent with residues present in all 12 functionally identified members of the mitochondrial transport protein (MTP) family, they lead to a general rule that specifies MTP residue types at 7 separate locations. The conformations of all the double mutation PTPs (except that with the matrix loop Ser158Thr) are significantly different from those of the single mutation PTPs, as indicated by their very low liposome incorporation efficiency and their requirement for less detergent (Triton X-100) to stay in solution. These dramatic conformational differences also suggest an interaction between TM helices D and E. The results are discussed in terms of TM helix movements and changes in the PTP monomer/dimer ratio. PMID: 11329276 [PubMed - indexed for MEDLINE] 203: Biochemistry 2001 Feb 20;40(7):1930-6 Covariation of a specificity-determining structural motif in an aminoacyl-tRNA synthetase and a tRNA identity element. Hawko SA, Francklyn CS. Department of Biochemistry, University of Vermont, Health Sciences Complex, Burlington, Vermont 05405, USA. Transfer RNA (tRNA) identity determinants help preserve the specificity of aminoacylation in vivo, and prevent cross-species interactions. Here, we investigate covariation between the discriminator base (N73) element in histidine tRNAs and residues in the histidyl-tRNA synthetase (HisRS) motif 2 loop. A model of the Escherichia coli HisRS--tRNA(His) complex predicts an interaction between the prokaryotic conserved glutamine 118 of the motif 2 loop and cytosine 73. The substitution of Gln 118 in motif 2 with glutamate decreased discrimination between cytosine and uracil some 50-fold, but left overall rates of adenylation and aminoacylation unaffected. By contrast, substitutions at neighboring Glu 115 and Arg 121 affected both adenylation and aminoacylation, consistent with their predicted involvement in both half-reactions. Additional evidence for the involvement of the motif 2 loop was provided by functional analysis of a hybrid Saccharomyces cerevisiae-- E. coli HisRS possessing the 11 amino acid motif 2 loop of the yeast enzyme. Despite an overall decreased activity of nearly 1000-fold relative to the E. coli enzyme, the chimera nevertheless exhibited a modest preference for the yeast tRNA(His) over the E. coli tRNA, and preferred wild-type yeast tRNA(His) to a variant with C at the discriminator position. These experiments suggest that part of, but not all of, the specificity is provided by the motif 2 loop. The close interaction between enzyme loop and RNA sequence elements suggested by these experiments reflects a covariation between enzyme and tRNA that may have acted to preserve aminoacylation fidelity over evolutionary time. PMID: 11329259 [PubMed - indexed for MEDLINE] 204: Nat Med 2001 May;7(5):625-9 Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity. Stubbs AC, Martin KS, Coeshott C, Skaates SV, Kuritzkes DR, Bellgrau D, Franzusoff A, Duke RC, Wilson CC. Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado, USA. There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses. PMID: 11329066 [PubMed - indexed for MEDLINE] 205: Methods 2001 May;24(1):29-34 Protein recruitment systems for the analysis of protein +/- protein interactions. Aronheim A. Department of Molecular Genetics, Rappaport Family Institute for Research in the Medical Sciences, Bat-Galim, Haifa 31096, Israel. aronheim@tx.technion.ac.il The yeast Saccharomyces cerevisiae serves as an excellent genetic tool for the analysis of protein +/- protein interactions. The most common system, used to date, is the two-hybrid system. Although proven very powerful, the two-hybrid system exhibits several inherent problems and limitations. Recently, two alternative systems have been described that take advantage of the fact that localization of signal transduction effectors to the inner leaflet of the plasma membrane is absolutely necessary for yeast viability. These effectors can either be the Ras guanyl nucleotide exchange factor or Ras itself. The yeast strain used in both systems is a temperature-sensitive mutant in the yeast Ras guanyl nucleotide exchange factor, CDC25. Membrane localization of these effectors is achieved via protein +/- protein interaction. Each system can be used to test interaction between known protein pairs, as well as for isolation of novel protein interactions. Described here are the scientific and technical steps to be considered for both protein recruitment systems. Copyright 2001 Academic Press. PMID: 11327799 [PubMed - indexed for MEDLINE] 206: Nat Struct Biol 2001 May;8(5):417-22 UBA domains of DNA damage-inducible proteins interact with ubiquitin. Bertolaet BL, Clarke DJ, Wolff M, Watson MH, Henze M, Divita G, Reed SI. Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA. Rad23 is a highly conserved protein involved in nucleotide excision repair (NER) that associates with the proteasome via its N-terminus. Its C-terminal ubiquitin-associated (UBA) domain is evolutionarily conserved from yeast to humans. However, the cellular function of UBA domains is not completely understood. Recently, RAD23 and DDI1, both DNA damage-inducible genes encoding proteins with UBA domains, were implicated genetically in Pds1-dependent mitotic control in yeast. The UBA domains of RAD23 and DDI1 are required for these interactions. Timely degradation of Pds1 via the ubiquitin/proteasome pathway allows anaphase onset and is crucial for chromosome maintenance. Here, we show that Rad23 and Ddi1 interact directly with ubiquitin and that this interaction is dependent on their UBA domains, providing a possible mechanism for UBA-dependent cell cycle control. Moreover, we show that a hydrophobic surface on the UBA domain, which from structural work had been predicted to be a protein-protein interaction interface, is indeed required for ubiquitin binding. By demonstrating that UBA domains interact with ubiquitin, we have provided the first indication of a cellular function for the UBA domain. PMID: 11323716 [PubMed - indexed for MEDLINE] 207: J Biol Chem 2001 Jul 13;276(28):26666-73 Assembly of the human origin recognition complex. Vashee S, Simancek P, Challberg MD, Kelly TJ. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits. To explore the subunit-subunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication. PMID: 11323433 [PubMed - indexed for MEDLINE] 208: Genes Dev 2001 Apr 15;15(8):1007-20 Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain. Hidalgo P, Ansari AZ, Schmidt P, Hare B, Simkovich N, Farrell S, Shin EJ, Ptashne M, Wagner G. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA. The GAL4 dimerization domain (GAL4-dd) is a powerful transcriptional activator when tethered to DNA in a cell bearing a mutant of the GAL11 protein, named GAL11P. GAL11P (like GAL11) is a component of the RNA-polymerase II holoenzyme. Nuclear magnetic resonance (NMR) studies of GAL4-dd revealed an elongated dimer structure with C(2) symmetry containing three helices that mediate dimerization via coiled-coil contacts. The two loops between the three coiled coils form mobile bulges causing a variation of twist angles between the helix pairs. Chemical shift perturbation analysis mapped the GAL11P-binding site to the C-terminal helix alpha3 and the loop between alpha1 and alpha2. One GAL11P monomer binds to one GAL4-dd dimer rendering the dimer asymmetric and implying an extreme negative cooperativity mechanism. Alanine-scanning mutagenesis of GAL4-dd showed that the NMR-derived GAL11P-binding face is crucial for the novel transcriptional activating function of the GAL4-dd on GAL11P interaction. The binding of GAL4 to GAL11P, although an artificial interaction, represents a unique structural motif for an activating region capable of binding to a single target to effect gene expression. PMID: 11316794 [PubMed - indexed for MEDLINE] 209: Oncogene 2001 Jan 25;20(4):501-13 p53 mutants exhibiting enhanced transcriptional activation and altered promoter selectivity are revealed using a sensitive, yeast-based functional assay. Inga A, Monti P, Fronza G, Darden T, Resnick MA. Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences (NIEHS), PO Box 12233, Research Triangle Park, North Carolina, NC 27709, USA. Changes in promoter specificity and binding affinity that may be associated with p53 mutations or post-translational modifications are useful in understanding p53 structure/function relationships and categorizing tumor mutations. We have exploited variable expression of human p53 in yeast to identify mutants with novel phenotypes that would correspond to altered promoter selectivity and affinity. The p53 cDNA regions coding for the DNA binding and tetramerization domains were subjected to random PCR mutagenesis and were cloned directly by recombination in yeast into a vector with a GAL1 promoter whose level of expression could be easily varied. p53 variants exhibiting higher than wild type levels of transactivation (supertrans) for the RGC responsive element were identified at low level of p53 protein expression. All the p53 mutants obtained with this screen were located in the DNA binding domain. Two out of 17 supertrans mutants have been found in tumors. Six mutations were in the L1 loop region between amino acids 115 and 124. The transactivation potential of a panel of supertrans p53 mutants on different promoters was evaluated using the p53 responsive elements, RGC, PIG3, p21 and bax. Although all mutants retained some activity with all promoters, we found different patterns of induction based on strength and promoter specificity. In particular none of the mutants was supertrans for the p21 responsive element. Interestingly, further analysis in yeast showed that the transactivation function could be retained even in the presence of dominant-negative p53 tumor mutations that could inhibit wild type p53. Five mutants were also characterized in human cells in terms of growth suppression and transactivation of various promoters. These novel supertrans p53 mutants may be useful in studies aimed at dissecting p53 downstream pathways, understanding specific interactions between p53 and the DNA, and could replace wild type p53 in cancer gene therapy protocols. The approach may also prove useful in identifying p53 tumor mutations. PMID: 11313981 [PubMed - indexed for MEDLINE] 210: Trends Cell Biol 2001 Mar;11(3):102-6 Towards an understanding of complex protein networks. Tucker CL, Gera JF, Uetz P. Dept of Genetics, University of Washington Box 357360, Seattle, WA 98195, USA. ctucker@u.washington.edu Large-scale two-hybrid screens have generated a wealth of information describing potential protein--protein interactions. When compiled with data from systematic localizations of proteins, mutant screens and other functional tests, a network of interactions among proteins and between proteins and other components of eukaryotic cells can be deduced. These networks can be viewed as maps of the cell, depicting potential signaling pathways and interactive complexes. Most importantly, they provide potential clues to the function of previously uncharacterized proteins. Focusing on recent experiments, we explore these protein-interaction studies and the maps derived from such efforts. Publication Types: Review Review, Tutorial PMID: 11306254 [PubMed - indexed for MEDLINE] 211: Genome Biol 2001;2(4):REVIEWS1013 Genome-wide analysis of protein-DNA interactions in living cells. Pugh BF, Gilmour DS. Center for Gene Regulation, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA. Understanding the regulation of gene expression requires an analysis of gene-specific transcription factors. This review highlights recent work that uses protein-DNA crosslinking, immunoprecipitation and DNA microarrays to determine the binding sites for specific transcription factors throughout the yeast genome. Publication Types: Review Review, Tutorial PMID: 11305945 [PubMed - indexed for MEDLINE] 212: Methods Enzymol 2001;332:277-300 Two-hybrid dual bait system to discriminate specificity of protein interactions in small GTPases. Serebriiskii IG, Mitina OV, Chernoff J, Golemis EA. Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. PMID: 11305104 [PubMed - indexed for MEDLINE] 213: Methods Enzymol 2001;332:260-70 Ras signaling pathway for analysis of protein-protein interactions. Aronheim A. Department of Molecular Genetics, B. Rappaport Faculty of Medicine, Israel Institute of Technology, Haifa 31096, Israel. PMID: 11305102 [PubMed - indexed for MEDLINE] 214: J Biol Chem 2001 Apr 20;276(16):12636-44 The interactions of yeast SWI/SNF and RSC with the nucleosome before and after chromatin remodeling. Sengupta SM, VanKanegan M, Persinger J, Logie C, Cairns BR, Peterson CL, Bartholomew B. Program in Molecular Biology, Microbiology, and Molecular Biology and Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901-4413, USA. Interactions of the yeast chromatin-remodeling complexes SWI/SNF and RSC with nucleosomes were probed using site-specific DNA photoaffinity labeling. 5 S rDNA was engineered with photoreactive nucleotides incorporated at different sites in DNA to scan for the subunits of SWI/SNF in close proximity to DNA when SWI/SNF is bound to the 5 S nucleosome or to the free 5 S rDNA. The Swi2/Snf2 and Snf6 subunits of SWI/SNF were efficiently cross-linked at several positions in the nucleosome, whereas only Snf6 was efficiently cross-linked when SWI/SNF was bound to free DNA. DNA photoaffinity labeling of RSC showed that the Rsc4 subunit is in close proximity to nucleosomal DNA and not when RSC is bound to free DNA. After remodeling, the Swi2/Snf2 and Rsc4 subunits are no longer detected near the nucleosomal DNA and are evidently displaced from the surface of the nucleosome, indicating significant changes in SWI/SNF and RSC contacts with DNA after remodeling. PMID: 11304548 [PubMed - indexed for MEDLINE] 215: Biochem Biophys Res Commun 2001 Apr 20;282(5):1211-9 The role of heat shock protein 70 in vitamin D receptor function. Lutz W, Kohno K, Kumar R. Department of Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA. We previously demonstrated that the 1alpha,25-dihydroxyvitamin D(3) receptor (VDR) interacts with the constitutive heat shock protein, hsc70 in vitro, and with DnaK (Biochem. Biophys. Res. Commun. 260, 446-452, 1999). The biological significance of VDR-heat shock protein interactions, however, is unknown. To examine the role of such interactions in eukaryotic cells, we heterologously expressed VDR and RXRalpha together with a vitamin D-responsive reporter system in Saccharomyces cerevisiae and examined the consequences of heat shock protein 70 gene (SSA) deletion in these cells. We show that heterologously expressed VDR associates with the yeast cytosolic hsp70 protein, Ssa1p. Deletion of the SSA2, SSA3, and SSA4 genes and reduction of Ssa1p activity, reduces the intracellular concentrations of the VDR and its heterodimeric partner, RXRalpha and reduces the activity of a vitamin D-dependent gene. Hsp70-like chaperone proteins play a role in controlling concentrations of the VDR within the cell. Copyright 2001 Academic Press. PMID: 11302745 [PubMed - indexed for MEDLINE] 216: J Biol Chem 2001 May 11;276(19):16520-7 5-Lipoxygenase interacts with coactosin-like protein. Provost P, Doucet J, Hammarberg T, Gerisch G, Samuelsson B, Radmark O. Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institute, S-171 77 Stockholm, Sweden. We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. In this report, we demonstrate a direct interaction between 5LO and CLP. 5LO associated with CLP, which was expressed as a glutathione S-transferase fusion protein, in a dose-dependent manner. Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates. In reciprocal experiments, 5LO was detected in CLP immunoprecipitates. Non-denaturing polyacrylamide gel electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar stoichiometry in a Ca(2+)-independent manner. Site-directed mutagenesis suggested an important role for lysine 131 of CLP in mediating 5LO binding. In view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments. However, no F-actin-CLP.5LO ternary complex was observed. In contrast, 5LO appeared to compete with F-actin for the binding of CLP. Moreover, 5LO was found to interfere with actin polymerization. Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusive and suggest a modulatory role for 5LO in actin dynamics. PMID: 11297527 [PubMed - indexed for MEDLINE] 217: Appl Biochem Biotechnol 2001 Feb;90(2):155-86 Thermozymes and their applications: a review of recent literature and patents. Bruins ME, Janssen AE, Boom RM. Department of Food Technology and Nutritional Sciences, Wageningen University, The Netherlands. marieke.bruins@algemeen.pk.wau.nl Enzymes from thermophilic microorganisms, thermozymes, have unique characteristics such as temperature, chemical, and pH stability. They can be used in several industrial processes, in which they replace mesophilic enzymes or chemicals. Thermozymes are often used when the enzymatic process is compatible with existing (high-temperature) process conditions. The main advantages of performing processes at higher temperatures are reduced risk of microbial contamination, lower viscosity, improved transfer rates, and improved solubility of substrates. However, cofactors, substrates, or products might be unstable or other side reactions may occur. Recent developments show that thermophiles are a good source of novel catalysts that are of great industrial interest. Thermostable polymer-degrading enzymes such as amylases, pullulanases, xylanases, proteases, and cellulases are expected to play an important role in food, chemical, pharmaceutical, paper, pulp, and waste-treatment industries. Considerable research efforts have been made to better understand the stability of thermozymes. There are no major conformational differences with mesophilic enzymes, and a small number of extra salt bridges, hydrophobic interactions, or hydrogen bounds seem to confer the extra degree of stabilization. Currently, overexpression of thermozymes in standard Escherichia coli allows the production of much larger quantities of enzymes, which are easy to purify by heat treatment. With wider availability and lower cost, thermophilic enzymes will see more application in industry. Publication Types: Review Review, Tutorial PMID: 11297390 [PubMed - indexed for MEDLINE] 218: Mol Biol Cell 2001 Apr;12(4):1177-88 Cadherin sequences that inhibit beta-catenin signaling: a study in yeast and mammalian cells. Simcha I, Kirkpatrick C, Sadot E, Shtutman M, Polevoy G, Geiger B, Peifer M, Ben-Ze'ev A. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel, 76100. Drosophila Armadillo and its mammalian homologue beta-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles. They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis. To learn more about Armadillo/beta-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/beta-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence. We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or beta-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells. We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/beta-catenin. Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited beta-catenin-mediated signaling. This suggests that the interaction between beta-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression. PMID: 11294915 [PubMed - indexed for MEDLINE] 219: Nucleic Acids Res 2001 Apr 15;29(8):1715-23 The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is required for full activity. Cicero MP, Hubl ST, Harrison CJ, Littlefield O, Hardy JA, Nelson HC. Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6089, USA. The yeast heat shock transcription factor (HSF) belongs to the winged helix family of proteins. HSF binds DNA as a trimer, and additional trimers can bind DNA co-operatively. Unlike other winged helix-turn-helix proteins, HSF's wing does not appear to contact DNA, as based on a previously solved crystal structure. Instead, the structure implies that the wing is involved in protein-protein interactions, possibly within a trimer or between adjacent trimers. To understand the function of the wing in the HSF DNA-binding domain, a Saccharomyces cerevisiae strain was created that expresses a wingless HSF protein. This strain grows normally at 30 degrees C, but shows a decrease in reporter gene expression during constitutive and heat-shocked conditions. Removal of the wing does not affect the stability or trimeric nature of a protein fragment containing the DNA-binding and trimerization domains. Removal of the wing does result in a decrease in DNA-binding affinity. This defect was mainly observed in the ability to form the first trimer-bound complex, as the formation of larger complexes is unaffected by the deletion. Our results suggest that the wing is not involved in the highly co-operative nature of HSF binding, but may be important in stabilizing the first trimer bound to DNA. PMID: 11292844 [PubMed - indexed for MEDLINE] 220: J Mol Biol 2001 Apr 13;307(5):1207-21 Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament. Solinger JA, Lutz G, Sugiyama T, Kowalczykowski SC, Heyer WD. Institute of General Microbiology, University of Bern, Bern, CH-3012, Switzerland. RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity. Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA. Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation. We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments. Copyright 2001 Academic Press. PMID: 11292336 [PubMed - indexed for MEDLINE] 221: Plant Mol Biol 2001 Feb;45(3):365-76 Further analysis of the interactions between the Brassica S receptor kinase and three interacting proteins (ARC1, THL1 and THL2) in the yeast two-hybrid system. Mazzurco M, Sulaman W, Elina H, Cock JM, Goring DR. Biology Department, York University, Toronto, Ontario, Canada. The yeast two-hybrid system was used to further characterize the interactions between the Brassica S receptor kinase (SRK) and three putative substrates, ARC1 and the two thioredoxin h proteins, THL1 and THL2. Interactions were generally detectable with kinase domains of both Class I and Class II SRKs. Chimeric constructs were made between the SRK910 kinase domain and the non-interacting Arabidopsis RLK5 kinase domain. Only one chimeric construct, SRR2, interacted with THL1 and THL2, while none of the chimeras were able to interact with ARC1. SRR2 is largely made up of RLK5 kinase domain with the N-terminal end being derived from the SRK910 kinase domain and was the only chimeric construct that retained kinase activity. Deletion or substitution of a conserved cysteine at the N-terminal end of the SRK910 kinase domain resulted in loss of interaction with THL1 and THL2, while the addition of this cysteine to a related receptor kinase, SFR1, conferred the ability to interact with the thioredoxin h proteins. In addition, substitution of the cysteines in the THL1 active site abolished the interaction. Lastly, the two Arabidopsis thioredoxin h clones most closely related to THL1 and THL2 were found to interact with the SRK kinase domains. Thus, the nature of the interaction of the thioredoxin h clones with SRK involves the reducing activity of these proteins and is restricted to the class of thioredoxin h proteins which have the variant CPPC active site. PMID: 11292081 [PubMed - indexed for MEDLINE] 222: Genetics 2001 Apr;157(4):1451-67 Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae. van Nues RW, Beggs JD. Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom. Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex. PMID: 11290703 [PubMed - indexed for MEDLINE] 223: Development 2001 May;128(9):1697-707 Orc mutants arrest in metaphase with abnormally condensed chromosomes. Pflumm MF, Botchan MR. Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. The origin recognition complex (ORC) is a six subunit complex required for eukaryotic DNA replication initiation and for silencing of the heterochromatic mating type loci in Saccharomyces cerevisiae. Our discovery of the Drosophila ORC complex concentrated in the centric heterochromatin of mitotic cells in the early embryo and its interactions with heterochromatin protein 1 (HP-1) lead us to speculate that ORC may play some general role in chromosomal folding. To explore the role of ORC in chromosomal condensation, we have identified a mutant of subunit 5 of the Drosophila melanogaster origin recognition complex (Orc5) and have characterized the phenotypes of both the Orc5 and the previously identified Orc2 mutant, k43. Both Orc mutants died at late larval stages and surprisingly, despite a reduced number of S-phase cells, an increased fraction of cells were also detected in mitosis. For this latter population of cells, Orc mutants arrest in a defective metaphase with shorter and thicker chromosomes that fail to align at the metaphase plate within a poorly assembled mitotic spindle. In addition, sister chromatid cohesion was frequently lost. PCNA and MCM4 mutants had similar phenotypes to Orc mutants. We propose that DNA replication defects trigger the mitotic arrest, due to the fact that frequent fragmentation was observed. Thus, cells have a mitotic checkpoint that senses chromosome integrity. These studies also suggest that the density of functional replication origins and completion of S phase are requirements for proper chromosomal condensation. PMID: 11290306 [PubMed - indexed for MEDLINE] 224: Proc Natl Acad Sci U S A 2001 Apr 10;98(8):4391-6 HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1. Wu J, Carmen AA, Kobayashi R, Suka N, Grunstein M. Department of Biological Chemistry, University of California School of Medicine and the Molecular Biology Institute, Boyer Hall, University of California, Los Angeles, CA 90095, USA. Histone deacetylase HDA1, the prototype for the class II mammalian deacetylases, is likely the catalytic subunit of the HDA1-containing complex that is involved in TUP1-specific repression and global deacetylation in yeast. Although the class I RPD3-like enzymatic complexes have been well characterized, little is known about the identity and interactions of the factors that associate to form the HDA1 complex. In this paper, we identify related HDA2 and HDA3 proteins that are found in the HDA1 complex and show that HDA1 interacts with itself and with the HDA2-HDA3 subcomplex to form a likely tetramer. These interactions are necessary for catalytic activity because mutations in any of the three components disrupt activity both in vitro and in vivo. In this respect the HDA1 complex differs from yeast RPD3, which has components such as SIN3 that are not essential for activity in vitro, and yeast HOS3, which has intrinsic in vitro activity as a homodimer in the absence of other subunits. PMID: 11287668 [PubMed - indexed for MEDLINE] 225: Mol Cell Biol 2001 May;21(9):3144-58 Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion. Hanna JS, Kroll ES, Lundblad V, Spencer FA. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both exhibit genetic and physical ties to replication fork constituents. We find that absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint. The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion. We find that Ctf18p, an RFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of the budding yeast polymerase kappa, encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion. Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-C(CTF18) may be involved in a polymerase switch event that facilities sister chromatid cohesion. The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process. PMID: 11287619 [PubMed - indexed for MEDLINE] 226: Am J Physiol Cell Physiol 2001 May;280(5):C1284-92 A store-operated nonselective cation channel in lymphocytes is activated directly by Ca(2+) influx factor and diacylglycerol. Su Z, Csutora P, Hunton D, Shoemaker RL, Marchase RB, Blalock JE. Departments of Physiology and Biophysics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms. PMID: 11287342 [PubMed - indexed for MEDLINE] 227: J Cell Biol 2001 Apr 2;153(1):159-68 The surveillance mechanism of the spindle position checkpoint in yeast. Adames NR, Oberle JR, Cooper JA. Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. nadames@cellbio.wustl.edu The spindle position checkpoint in Saccharomyces cerevisiae delays mitotic exit until the spindle has moved into the mother-bud neck, ensuring that each daughter cell inherits a nucleus. The small G protein Tem1p is critical in promoting mitotic exit and is concentrated at the spindle pole destined for the bud. The presumed nucleotide exchange factor for Tem1p, Lte1p, is concentrated in the bud. These findings suggested the hypothesis that movement of the spindle pole through the neck allows Tem1p to interact with Lte1p, promoting GTP loading of Tem1p and mitotic exit. However, we report that deletion of LTE1 had little effect on the timing of mitotic exit. We also examined several mutants in which some cells inappropriately exit mitosis even though the spindle is within the mother. In some of these cells, the spindle pole body did not interact with the bud or the neck before mitotic exit. Thus, some alternative mechanism must exist to coordinate mitotic exit with spindle position. In both wild-type and mutant cells, mitotic exit was preceded by loss of cytoplasmic microtubules from the neck. Thus, the spindle position checkpoint may monitor such interactions. PMID: 11285282 [PubMed - indexed for MEDLINE] 228: Nat Cell Biol 2001 Apr;3(4):384-91 Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly. Seol JH, Shevchenko A, Shevchenko A, Deshaies RJ. Division of Biology and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA. SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a large family of substrate receptors known as F-box proteins (FBPs). Here we report the identification, using sequential rounds of epitope tagging, affinity purification and mass spectrometry, of 16 Skp1 and Cdc53-associated proteins in budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202 (Rav2). Rav1, Rav2 and Skp1 form a complex that we have named 'regulator of the (H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase). V-ATPases are conserved throughout eukaryotes, and have been implicated in tumour metastasis and multidrug resistance, and here we show that RAVE promotes glucose-triggered assembly of the V-ATPase holoenzyme. Previous systematic genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and Cdc53, only 3 of which overlap with those reported here. Thus, our results provide a distinct view of the interactions that link proteins into a comprehensive cellular network. PMID: 11283612 [PubMed - indexed for MEDLINE] 229: Proc Natl Acad Sci U S A 2001 Apr 10;98(8):4569-74 Comment in: Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4277-8. A comprehensive two-hybrid analysis to explore the yeast protein interactome. Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, Sakaki Y. Division of Genome Biology, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan. titolab@kenroku.kanazawa-u.ac.jp Protein-protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the approximately 6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623-627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system. PMID: 11283351 [PubMed - indexed for MEDLINE] 230: J Biol Chem 2001 May 18;276(20):17448-54 A-kinase-anchoring protein AKAP95 is targeted to the nuclear matrix and associates with p68 RNA helicase. Akileswaran L, Taraska JW, Sayer JA, Gettemy JM, Coghlan VM. Neurological Sciences Institute, Oregon Health Sciences University, Beaverton, Oregon 97006, USA. The cell nucleus is structurally and functionally organized by the nuclear matrix. We have examined whether the nuclear cAMP-dependent protein kinase-anchoring protein AKAP95 contains specific signals for targeting to the subnuclear compartment and for interaction with other proteins. AKAP95 was expressed in mammalian cells and found to localize exclusively to the nuclear matrix. Mutational analysis was used to identify determinants for nuclear localization and nuclear matrix targeting of AKAP95. These sites were found to be distinct from previously identified DNA and protein kinase A binding domains. The nuclear matrix-targeting site is unique but conserved among members of the AKAP95 family. Direct binding of AKAP95 to isolated nuclear matrix was demonstrated in situ and found to be dependent on the nuclear matrix-targeting site. Moreover, Far Western blot analysis identified at least three AKAP95-binding proteins in nuclear matrix isolated from rat brain. Yeast two-hybrid cloning identified one binding partner as p68 RNA helicase. The helicase and AKAP95 co-localized in the nuclear matrix of mammalian cells, associated in vitro, and were precipitated as a complex from solubilized cell extracts. The results define novel protein-protein interactions among nuclear matrix proteins and suggest a potential role of AKAP95 as a scaffold for coordinating assembly of hormonally responsive transcription complexes. PMID: 11279182 [PubMed - indexed for MEDLINE] 231: J Biol Chem 2001 May 18;276(20):17261-6 Structure-function analysis of the active site tunnel of yeast RNA triphosphatase. Bisaillon M, Shuman S. Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA. Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus, catalyzes metal-dependent gamma phosphate hydrolysis within the hydrophilic interior of a topologically closed 8-strand beta barrel (the "triphosphate tunnel"). We used structure-guided alanine scanning to identify 6 side chains within the triphosphate tunnel that are essential for phosphohydrolase activity in vitro and in vivo: Arg393, Glu433, Arg458, Arg469, Asp471 and Thr473. Alanine substitutions at two positions, Asp377 and Lys409, resulted in partial catalytic defects and a thermosensitive growth phenotype. Structure-function relationships were clarified by introducing conservative substitutions. Five residues were found to be nonessential: Lys309, Ser395, Asp397, Lys427 Asn431, and Lys474. The present findings, together with earlier mutational analyses, reveal an unusually complex active site in which 15 individual side chains in the tunnel cavity are important for catalysis, and each of the 8 strands of the beta barrel contributes at least one functional constituent. The active site residues fall into three classes: (i) those that participate directly in catalysis via coordination of the gamma phosphate or the metal; (ii) those that make critical water-mediated contacts with the gamma phosphate or the metal; and (iii) those that function indirectly via interactions with other essential side chains or by stabilization of the tunnel structure. PMID: 11279161 [PubMed - indexed for MEDLINE] 232: J Biol Chem 2001 May 4;276(18):14996-5002 Importance of homodimerization for the in vivo function of yeast RNA triphosphatase. Lehman K, Ho CK, Shuman S. Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA. Saccharomyces cerevisiae RNA triphosphatase Cet1 is an essential component of the yeast mRNA capping apparatus. The active site of Cet1 resides within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that is supported by a globular hydrophobic core. The homodimeric quaternary structure of Cet1 is formed by a network of contacts between the partner protomers. By studying the effects of alanine-cluster mutations, we highlight the contributions of two separate facets of the crystallographic dimer interface to Cet1 function in vivo. One essential facet of the interface entails hydrophobic cross-dimer interactions of Cys(330) and Val(331) and a cross-dimer hydrogen bond of Asp(280) with the backbone amide of Gln(329). The second functionally relevant dimer interface involves hydrophobic side-chain interactions of Phe(272) and Leu(273). Ala-cluster mutations involving these residues elicited lethal or severe temperature-sensitive phenotypes that were suppressed completely by fusion of the mutated triphosphatases to the guanylyltransferase domain of mammalian capping enzyme. The recombinant D279A-D280A and F272A-L273A proteins retained phosphohydrolase activity but sedimented as monomers. These results indicate that a disruption of the dimer interface is uniquely deleterious when the yeast RNA triphosphatase must function in concert with the endogenous yeast guanylyltransferase. We also identify key residue pairs in the hydrophobic core of the Cet1 protomer that support the active site tunnel and stabilize the triphosphatase in vivo. PMID: 11279098 [PubMed - indexed for MEDLINE] 233: J Biol Chem 2001 May 11;276(19):16207-15 DNA binding by the ETS-domain transcription factor PEA3 is regulated by intramolecular and intermolecular protein.protein interactions. Greenall A, Willingham N, Cheung E, Boam DS, Sharrocks AD. School of Biochemistry and Genetics, The Medical School, University of Newcastle Upon Tyne, Newcastle Upon Tyne NE2 4HH, United Kingdom. The control of DNA binding by eukaryotic transcription factors represents an important regulatory mechanism. Many transcription factors are controlled by cis-acting autoinhibitory modules that are thought to act by blocking promiscuous DNA binding in the absence of appropriate regulatory cues. Here, we have investigated the determinants and regulation of the autoinhibitory mechanism employed by the ETS-domain transcription factor, PEA3. DNA binding is inhibited by a module composed of a combination of two short motifs located on either side of the ETS DNA-binding domain. A second type of protein, Ids, can act in trans to mimic the effect of these cis-acting inhibitory motifs and reduce DNA binding by PEA3. By using a one-hybrid screen, we identified the basic helix-loop-helix-leucine zipper transcription factor USF-1 as an interaction partner for PEA3. PEA3 and USF-1 form DNA complexes in a cooperative manner. Moreover, the formation of ternary PEA3.USF-1.DNA complexes requires parts of the same motifs in PEA3 that form the autoinhibitory module. Thus the binding of USF-1 to PEA3 acts as a switch that modifies the autoinhibitory motifs in PEA3 to first relieve their inhibitory action, and second, promote ternary nucleoprotein complex assembly. PMID: 11278941 [PubMed - indexed for MEDLINE] 234: J Biol Chem 2001 Apr 13;276(15):12135-9 Biochemical characterization of Gyp6p, a Ypt/Rab-specific GTPase-activating protein from yeast. Will E, Gallwitz D. Max Planck Institute for Biophysical Chemistry, Department of Molecular Genetics, D-37070 Gottingen, Germany. Gyp6p from yeast belongs to the GYP family of Ypt/Rab-specific GTPase-activating proteins, and Ypt6p is its preferred substrate (Strom, M., Vollmer, P., Tan, T. J., and Gallwitz, D. (1993) Nature 361, 736-739). We have investigated the kinetic parameters of Gyp6p/Ypt6p interactions and find that Gyp6p accelerates the intrinsic GTPase activity of Ypt6p (0.0002 min(-1)) by a factor of 5 x 10(6) and that they have a very low affinity for its preferred substrate (K(m) = 592 micrometer). Substitution with alanine of several arginines, which Gyp6p shares with other GYP family members, resulted in significant inhibition of GAP activity. Replacement of arginine-155 with either alanine or lysine abolished its GAP activity, indicating a direct involvement of this strictly conserved arginine in catalysis. Physical interaction of the catalytically inactive Gyp6(R155A) mutant GAP with Ypt6 wild-type and Ypt6 mutant proteins could be demonstrated with the two-hybrid system. Short N-terminal and C-terminal truncations of Gyp6p resulted in a complete loss of GAP activity and Ypt6p binding, showing that in contrast to two other Gyp proteins studied previously, most of the 458 amino acid-long Gyp6p sequence is required to form a three-dimensional structure that allows substrate binding and catalysis. PMID: 11278907 [PubMed - indexed for MEDLINE] 235: J Biol Chem 2001 May 11;276(19):16265-70 Helical stalk segments S4 and S5 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae are optimized to impact catalytic site environment. Soteropoulos P, Valiakhmetov A, Kashiwazaki R, Perlin DS. Public Health Research Institute, New York, New York 10016, USA. The stalk segments of P-type ion-translocating enzymes are presumed to play important roles in energy coupling. In this work, stalk segments S4 and S5 of the yeast H(+)-ATPase were examined for helical character, optimal length, and segment orientation by a combination of proline substitution, insertion/deletion mutagenesis, and second-site suppressor analyses. The substitution of various residues for helix-disrupting proline in both S4 (L353P,L353G; A354P; and G371P) and S5 (D676P and I684P) resulted in highly defective or inactive enzymes supporting the importance of helical character and/or the maintenance of essential interactions. The contiguous helical nature of transmembrane segment M5 and stalk element S5 was explored and found to be favorable, although not essential. The deletion or addition of one or more amino acids at positions Ala(354) in S4 and Asp(676) in S5, which were intended to either rotate helical faces or extend/reduce the length of helical segments, resulted in enzyme destabilization that abolished most enzyme assembly. Second-site suppressor mutations were obtained to primary site mutations G371A (S4) and D676G (S5) and were analyzed with a molecular structure model of the H(+)-ATPase. Primary site mutations were predicted to alter the site of phosphorylation either directly or indirectly. The suppressor mutations either directly changed packing around the primary site or altered the environment of the site of phosphorylation. Overall, these data support the view that stalk segments S4 and S5 of the H(+)-ATPase are helical elements that are optimized for length and interactions with other stalk elements and can influence the phosphorylation domain. PMID: 11278840 [PubMed - indexed for MEDLINE] 236: J Biol Chem 2001 May 18;276(20):17524-32 J-domain protein, Jac1p, of yeast mitochondria required for iron homeostasis and activity of Fe-S cluster proteins. Kim R, Saxena S, Gordon DM, Pain D, Dancis A. Department of Medicine, Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. J-proteins are molecular chaperones with a characteristic domain predicted to mediate interaction with Hsp70 proteins. We have previously isolated yeast mutants of the mitochondrial Hsp70, Ssq1p, in a genetic screen for mutants with altered iron homeostasis. Here we describe the isolation of mutants of the J-domain protein, Jac1p, using the same screen. Mutant jac1 alleles predicted to encode severely truncated proteins (lacking 70 or 152 amino acids) were associated with phenotypes strikingly similar to the phenotypes of ssq1 mutants. These phenotypes include activation of the high affinity cellular iron uptake system and iron accumulation in mitochondria. In contrast to iron accumulation, Fe-S proteins of mitochondria were specifically deficient. In jac1 mutants, like in ssq1 mutants, processing of the Yfh1p precursor protein from intermediate to mature forms was delayed. In the genetic backgrounds used in this study, jac1 null mutants were found to be viable, permitting analysis of genetic interactions. The Deltajac1 Deltassq1 double mutant was more severely compromised for growth than either single mutant, suggesting a synthetic or additive effect of these mutations. Overexpression of Jac1p partially suppressed ssq1 slow growth and vice versa. Similar mitochondrial localization and similar mutant phenotypes suggest that Ssq1p and Jac1p are functional partners in iron homeostasis. PMID: 11278728 [PubMed - indexed for MEDLINE] 237: J Biol Chem 2001 May 11;276(19):15768-75 Delineation of functional regions within the subunits of the Saccharomyces cerevisiae cell adhesion molecule a-agglutinin. Shen ZM, Wang L, Pike J, Jue CK, Zhao H, de Nobel H, Kurjan J, Lipke PN. Department of Biological Sciences and the Institute for Biomolecular Structure and Function, Hunter College of the City University of New York, New York 10021, USA. a-Agglutinin from Saccharomyces cerevisiae is a cell adhesion glycoprotein expressed on the surface of cells of a mating type and consists of an anchorage subunit Aga1p and a receptor binding subunit Aga2p. Cell wall attachment of Aga2p is mediated through two disulfide bonds to Aga1p (Cappellaro, C., Baldermann, C., Rachel, R., and Tanner, W. (1994) EMBO J. 13, 4737-4744). We report here that purified Aga2p was unstable and had low molar specific activity relative to its receptor alpha-agglutinin. Aga2p co-expressed with a 149-residue fragment of Aga1p formed a disulfide-linked complex with specific activity 43-fold higher than Aga2p expressed alone. Circular dichroism of the complex revealed a mixed alpha/beta structure, whereas Aga2p alone had no periodic secondary structure. A 30-residue Cys-rich Aga1p fragment was partially active in stabilization of Aga2p activity. Mutation of either or both Aga2p cysteine residues eliminated stabilization of Aga2p. Thus the roles of Aga1p include both cell wall anchorage and cysteine-dependent conformational restriction of the binding subunit Aga2p. Mutagenesis of AGA2 identified only C-terminal residues of Aga2p as being essential for binding activity. Aga2p residues 45-72 are similar to sequences in soybean Nod genes, and include residues implicated in interactions with both Aga1p (including Cys(68)) and alpha-agglutinin. PMID: 11278672 [PubMed - indexed for MEDLINE] 238: J Biol Chem 2001 Apr 13;276(15):11980-7 Genetic analysis of the Escherichia coli FtsZ.ZipA interaction in the yeast two-hybrid system. Characterization of FtsZ residues essential for the interactions with ZipA and with FtsA. Haney SA, Glasfeld E, Hale C, Keeney D, He Z, de Boer P. Department of Infectious Disease, Wyeth-Ayerst Research, Pearl River, New York 10965 and the Department of Molecular Biology and Microbiology, Case Western Reserve University Medical School, Cleveland, Ohio 44106-4960. The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division in Escherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZ(D373G)) identified eight different changes at two residues within this sequence. In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZ(D373G) failed to interact. Two mutant proteins examined restored this interaction significantly. In vivo, the alleles tested are significantly more toxic than the wild type ftsZ and cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZ(D373G), no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently. PMID: 11278571 [PubMed - indexed for MEDLINE] 239: J Biol Chem 2001 Apr 20;276(16):13256-63 Trax (translin-associated factor X), a primarily cytoplasmic protein, inhibits the binding of TB-RBP (translin) to RNA. Chennathukuzhi VM, Kurihara Y, Bray JD, Hecht NB. Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women's Health, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6142, USA. Trax (Translin-associated factor X) has been shown to interact with TB-RBP/Translin by its coimmunoprecipitation and in yeast two-hybrid assays. Here we demonstrate that Trax is widely expressed, does not bind to DNA or RNA, but forms heterodimers with TB-RBP under reducing conditions. The heterodimer of TB-RBP and Trax inhibits TB-RBP binding to RNA, but enhances TB-RBP binding to specific single stranded DNA sequences. The in vitro interactions between TB-RBP and Trax are confirmed by similar interactions in the yeast two-hybrid system. Cell fractionation and confocal microscope studies reveal that Trax is predominantly cytoplasmic. In contrast, TB-RBP is present in both the nuclei and cytoplasm of transfected cells and uses a highly conserved nuclear export signal to exit nuclei. In addition to a leucine zipper, two basic domains in TB-RBP are essential for RNA binding, but only one of these domains is needed for DNA binding. Trax restores DNA binding to TB-RBP containing an altered form of this domain. These data suggest that Trax-TB.RBP interactions modulate the DNA- and RNA-binding activity of TB-RBP. PMID: 11278549 [PubMed - indexed for MEDLINE] 240: J Biol Chem 2001 Jun 29;276(26):24212-22 The dimerization interface of the metastasis-associated protein S100A4 (Mts1): in vivo and in vitro studies. Tarabykina S, Scott DJ, Herzyk P, Hill TJ, Tame JR, Kriajevska M, Lafitte D, Derrick PJ, Dodson GG, Maitland NJ, Lukanidin EM, Bronstein IB. Department of Molecular Cancer Biology, Danish Cancer Society, Copenhagen DK-2100, Denmark. The S100 calcium-binding proteins are implicated in signal transduction, motility, and cytoskeletal dynamics. The three-dimensional structure of several S100 proteins revealed that the proteins form non-covalent dimers. However, the mechanism of the S100 dimerization is still obscure. In this study we characterized the dimerization of S100A4 (also named Mts1) in vitro and in vivo. Analytical ultracentrifugation revealed that apoS100A4 was present in solution as a mixture of monomers and dimers in a rapidly reversible equilibrium (K(d) = 4 +/- 2 microm). The binding of calcium promoted dimerization. Replacement of Tyr-75 by Phe resulted in the stabilization of the dimer. Helix IV is known to form the major part of the dimerization interface in homologous S100 proteins. By using the yeast two-hybrid system we showed that only a few residues of helix IV, namely Phe-72, Tyr-75, Phe-78, and Leu-79, are essential for dimerization in vivo. A homology model demonstrated that these residues form a hydrophobic cluster on helix IV. Their role is to stabilize the structure of individual subunits rather than provide specific interactions across the dimerization surface. Our mutation data showed that the specificity at the dimerization surface is not particularly stringent, which is consistent with recent data indicating that S100 proteins can form heterodimers. PMID: 11278510 [PubMed - indexed for MEDLINE] 241: J Biol Chem 2001 Apr 20;276(16):13127-35 Gaf-1, a gamma -SNAP-binding protein associated with the mitochondria. Chen D, Xu W, He P, Medrano EE, Whiteheart SW. Department of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536, USA. The role of alpha/beta-SNAP (Soluble NSF Attachment Protein) in vesicular trafficking is well established; however, the function of the ubiquitously expressed gamma-SNAP remains unclear. To further characterize the cellular role of this enigmatic protein, a two-hybrid screen was used to identify new, gamma-SNAP-binding proteins and to uncover potentially novel functions for gamma-SNAP. One such SNAP-binding protein, termed Gaf-1 (gamma-SNAP associate factor-1) specifically binds gamma- but not alpha-SNAP. The full-length Gaf-1 (75 kDa) is ubiquitously expressed and is found stoichiometrically associated with gamma-SNAP in cellular extracts. This binding is distinct from other SNAP interactions since no alpha-SNAP or NSF coprecipitated with Gaf-1. Subcellular fractionation and immunofluorescence analysis show that Gaf-1 is peripherally associated with the outer mitochondrial membrane. Only a fraction of gamma-SNAP was mitochondrial with the balance being either cytosolic or associated with other membrane fractions. GFP-gamma-SNAP and the C-terminal domain of Gaf-1 both show a reticular distribution in HEK-293 cells. This reticular structure colocalizes with Gaf-1 and mitochondria as well as with microtubules but not with other cytoskeletal elements. These data identify a class of gamma-SNAP interactions that is distinct from other members of the SNAP family and point to a potential role for gamma-SNAP in mitochondrial dynamics. PMID: 11278501 [PubMed - indexed for MEDLINE] 242: Cell Physiol Biochem 2001;11(1):55-60 Determination of protein-protein interactions of ICIn by the yeast two-hybrid system. Schmarda A, Fresser F, Gschwentner M, Furst J, Ritter M, Lang F, Baier G, Paulmichl M. Department of Physiology, Institute for Medical Biology and Human Genetics, University of Innsbruck, Austria. ICln is a ubiquitously expressed eukaryotic protein. Expression of the protein in Xenopus laevis oocytes, the knocking-down of the protein in fibroblasts, or the reconstitution of the protein in lipid bilayer led to the assumption that this protein is an ionic channel or a significant part thereof. However, other possible roles for ICln in potential regulatory mechanisms have been postulated, as diverse as regulator of cell morphology by interacting with the Skb1 protein and/or interaction with core spliceosomal proteins. Here we show that ICln is able to interact with SnRNP core proteins SmD1, SmD2, SmD3, SmX5 and SmB/B'. PMID: 11275683 [PubMed - indexed for MEDLINE] 243: J Biol Chem 2001 Jun 15;276(24):21594-600 Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ (PSD95/Dlg/ZO-1) domain-containing proteins. DeMarco SJ, Strehler EE. Program in Molecular Neuroscience, Department of Biochemistry, Mayo Graduate School, Mayo Clinic, Rochester, Minnesota 55905, USA. Spatial and temporal regulation of intracellular Ca(2+) signaling depends on localized Ca(2+) microdomains containing the requisite molecular components for Ca(2+) influx, efflux, and signal transmission. Plasma membrane Ca(2+)-ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains. The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen. The related MAGUKs SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b. Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95. These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca(2+) regulation. PMID: 11274188 [PubMed - indexed for MEDLINE] 244: J Mol Biol 2001 Mar 30;307(3):929-38 Mapping protein family interactions: intramolecular and intermolecular protein family interaction repertoires in the PDB and yeast. Park J, Lappe M, Teichmann SA. European Bioinformatics Institute, Hinxton, Cambridgeshire, CB10 1SD, UK. In the postgenomic era, one of the most interesting and important challenges is to understand protein interactions on a large scale. The physical interactions between protein domains are fundamental to the workings of a cell: in multi-domain polypeptide chains, in multi-subunit proteins and in transient complexes between proteins that also exist independently. To study the large-scale patterns and evolution of interactions between protein domains, we view interactions between protein domains in terms of the interactions between structural families of evolutionarily related domains. This allows us to classify 8151 interactions between individual domains in the Protein Data Bank and the yeast Saccharomyces cerevisiae in terms of 664 types of interactions, between protein families. At least 51 interactions do not occur in the Protein Data Bank and can only be derived from the yeast data. The map of interactions between protein families has the form of a scale-free network, meaning that most protein families only interact with one or two other families, while a few families are extremely versatile in their interactions and are connected to many families. We observe that almost half of all known families engage in interactions with domains from their own family. We also see that the repertoires of interactions of domains within and between polypeptide chains overlap mostly for two specific types of protein families: enzymes and same-family interactions. This suggests that different types of protein interaction repertoires exist for structural, functional and regulatory reasons. Copyright 12001 Academic Press. PMID: 11273711 [PubMed - indexed for MEDLINE] 245: Protein Sci 2001 Jan;10(1):12-6 Second virial coefficients as a measure of protein--osmolyte interactions. Weatherly GT, Pielak GJ. Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. The cytoplasm contains high concentrations of cosolutes. These cosolutes include macromolecules and small organic molecules called osmolytes. However, most biophysical studies of proteins are conducted in dilute solutions. Two broad classes of models have been used to describe the interaction between osmolytes and proteins. One class focuses on excluded volume effects, while the other focuses on binding between the protein and the osmolyte. To better understand protein--smolyte interactions, we have conducted sedimentation equilibrium analytical ultracentrifugation experiments using ferricytochrome c as a model protein. From these experiments, we determined the second virial coefficients for a series of osmolytes. We have interpreted the second virial coefficient as a measure of both excluded volume and protein--osmolyte binding. We conclude that simple models are not sufficient to understand the interactions between osmolytes and proteins. PMID: 11266589 [PubMed - indexed for MEDLINE] 246: Nucleic Acids Res 2001 Apr 1;29(7):1524-33 Transcriptional adaptor and histone acetyltransferase proteins in Arabidopsis and their interactions with CBF1, a transcriptional activator involved in cold-regulated gene expression. Stockinger EJ, Mao Y, Regier MK, Triezenberg SJ, Thomashow MF. Department of Crop and Soil Sciences and Department of Biochemistry, Michigan State University, East Lansing, MI 48824, USA. The ARABIDOPSIS CBF transcriptional activators bind to the CRT/DRE regulatory element present in the promoters of many cold-regulated genes and stimulate their transcription. Expression of the CBF1 proteins in yeast activates reporter genes carrying a minimal promoter with the CRT/DRE as an upstream regulatory element. Here we report that this ability of CBF1 is dependent upon the activities of three key components of the yeast Ada and SAGA complexes, namely the histone acetyltransferase (HAT) Gcn5 and the transcriptional adaptor proteins Ada2 and Ada3. This result suggested that CBF1 might function through the action of similar complexes in ARABIDOPSIS In support of this hypothesis we found that ARABIDOPSIS has a homolog of the GCN5 gene and two homologs of ADA2, the first report of multiple ADA2 genes in an organism. The ARABIDOPSIS GCN5 protein has intrinsic HAT activity and can physically interact in vitro with both the ARABIDOPSIS ADA2a and ADA2b proteins. In addition, the CBF1 transcriptional activator can interact with the ARABIDOPSIS GCN5 and ADA2 proteins. We conclude that ARABIDOPSIS encodes HAT-containing adaptor complexes that are related to the Ada and SAGA complexes of yeast and propose that the CBF1 transcriptional activator functions through the action of one or more of these complexes. PMID: 11266554 [PubMed - indexed for MEDLINE] 247: Nucleic Acids Res 2001 Apr 1;29(7):1410-9 All Tcf HMG box transcription factors interact with Groucho-related co-repressors. Brantjes H, Roose J, van De Wetering M, Clevers H. Department of Immunology, University Medical Center Utrecht, PO Box 85500, 3508 GA, Utrecht, The Netherlands. Tcf/Lef family transcription factors are the downstream effectors of the Wingless/Wnt signal transduction pathway. Upon Wingless/Wnt signalling, beta-catenin translocates to the nucleus, interacts with Tcf (1-3) and thus activates transcription of target genes (4,5). Tcf factors also interact with members of the Groucho (Grg/TLE) family of transcriptional co-repressors (6). We have now tested all known mammalian Groucho family members for their ability to interact specifically with individual Tcf/Lef family members. Transcriptional activation by any Tcf could be repressed by Grg-1, Grg-2/TLE-2, Grg-3 and Grg-4 in a reporter assay. Specific interactions between Tcf and Grg proteins may be achieved in vivo by tissue- or cell type-limited expression. To address this, we determined the expression of all Tcf and Grg/TLE family members in a panel of cell lines. Within any cell line, several Tcfs and TLEs are co-expressed. Thus, redundancy in Tcf/Grg interactions appears to be the rule. The 'long' Groucho family members containing five domains are repressors of Tcf-mediated transactivation, whereas Grg-5, which only contains the first two domains, acts as a de-repressor. As previously shown for DROSOPHILA: Groucho, we show that long Grg proteins interact with histone deacetylase-1. Although Grg-5 contains the GP homology domain that mediates HDAC binding in long Grg proteins, Grg-5 fails to bind this co-repressor, explaining how it can de-repress transcription. PMID: 11266540 [PubMed - indexed for MEDLINE] 248: Pac Symp Biocomput 2001;:115-26 SAMIE: statistical algorithm for modeling interaction energies. Benos PV, Lapedes AS, Fields DS, Stormo GD. Dept. of Genetics, Campus Box 8232, Medical School, Washington University, 4566 Scott Ave., St. Louis, MO 63110, USA. benos@genetics.wustl.edu We are investigating the rules that govern protein-DNA interactions, using a statistical mechanics based formalism that is related to the Boltzmann Machine of the neural net literature. Our approach is data-driven, in which probabilistic algorithms are used to model protein-DNA interactions, given SELEX and/or phage data as input. In the current report, we trained the network using SELEX data, under the "one-to-one" model of interactions (i.e. one amino acid contacts one base). The trained network was able to successfully identify the wild-type binding sites of EGR and MIG protein families. The predictions using our method are the same or better than that of methods existing in the literature. However our methodology offers the potential to capitalise in quantitative detail, as well as to be used to explore more general model of interactions, given availability of data. PMID: 11262933 [PubMed - indexed for MEDLINE] 249: Mol Cell Biol 2001 Apr;21(7):2337-48 Protein import channel of the outer mitochondrial membrane: a highly stable Tom40-Tom22 core structure differentially interacts with preproteins, small tom proteins, and import receptors. Meisinger C, Ryan MT, Hill K, Model K, Lim JH, Sickmann A, Muller H, Meyer HE, Wagner R, Pfanner N. Institut fur Biochemie und Molekularbiologie, Universitat Freiburg, Germany. The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a approximately 400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5. We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH. Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex. A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex. Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex. Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex. Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins. The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores. PMID: 11259583 [PubMed - indexed for MEDLINE] 250: Mol Cell Biol 2001 Apr;21(7):2281-91 Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide excision repair, without a preassembled repairosome. Araujo SJ, Nigg EA, Wood RD. Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom. In mammalian cells, the core factors involved in the damage recognition and incision steps of DNA nucleotide excision repair are XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF. Many interactions between these components have been detected, using different physical methods, in human cells and for the homologous factors in Saccharomyces cerevisiae. Several human nucleotide excision repair (NER) complexes, including a high-molecular-mass repairosome complex, have been proposed. However, there have been no measurements of activity of any mammalian NER protein complex isolated under native conditions. In order to assess relative strengths of interactions between NER factors, we captured TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in active form attached to magnetic beads. Coimmunoprecipitation of other NER proteins was then monitored functionally in a reconstituted repair system with purified proteins. We found that all detectable TFIIH in gently prepared human cell extracts was present in the intact nine-subunit form. There was no evidence for a repair complex that contained all of the NER components. At low ionic strength TFIIH could associate with functional amounts of each NER factor except RPA. At physiological ionic strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not other repair factors. The strongest interaction was between TFIIH and XPC-HR23B, indicating a coupled role of these proteins in early steps of repair. A panel of antibodies was used to estimate that there are on the order of 10(5) molecules of each core NER factor per HeLa cell. PMID: 11259578 [PubMed - indexed for MEDLINE] 251: Infect Immun 2001 Apr;69(4):2037-44 Interactions of surfactant proteins A and D with Saccharomyces cerevisiae and Aspergillus fumigatus. Allen MJ, Voelker DR, Mason RJ. Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206, USA. Surfactant proteins A (SP-A) and D (SP-D) are members of the collectin family of calcium-dependent lectins and are important pulmonary host defense molecules. Human SP-A and SP-D and rat SP-D bind to Aspergillus fumigatus conidia, but the ligand remains unidentified. To identify a fungal ligand for SP-A and/or SP-D, we examined the interactions of the proteins with Saccharomyces cerevisiae. SP-D but not SP-A bound yeast cells, and EDTA inhibited the binding. SP-D also aggregated yeast cells and isolated yeast cell walls. Treating yeast cells to remove cell wall mannoprotein did not reduce SP-D binding, and SP-D failed to aggregate chitin. However, SP-D aggregated yeast glucan before and after treatment with a beta(1-->3)-glucanase, suggesting a specific interaction between the collectin and beta(1-->6)-glucan. In support of this idea, SP-D-induced yeast aggregation was strongly inhibited by pustulan [a beta(1-->6)-linked glucose homopolymer] but was not inhibited by laminarin [a beta(1-->3)-linked glucose homopolymer]. Additionally, pustulan but not laminarin strongly inhibited SP-D binding to A. fumigatus. The pustulan concentration for 50% inhibition of SP-D binding to A. fumigatus is 1.0 +/- 0.3 microM glucose equivalents. Finally, SP-D showed reduced binding to the beta(1-->6)-glucan-deficient kre6 yeast mutant. Taken together, these observations demonstrate that beta(1-->6)-glucan is an important fungal ligand for SP-D and that glycosidic bond patterns alone can determine if an extended carbohydrate polymer is recognized by SP-D. PMID: 11254556 [PubMed - indexed for MEDLINE] 252: Mol Gen Genet 2001 Feb;264(6):842-51 A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of global genome nucleotide excision repair. Wong JM, Ingles CJ. Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada. Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation. In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively. A RAD7/16-dependent reduction in survival after UV irradiation was also seen in the presence of mutations in RNA Pol II that confer a defect in its response to SII, as well as with other mutations which reside in regions of the largest subunit of Pol II not involved in SII interactions. Indeed, an increase in UV sensitivity was achieved by simply decreasing the steadystate level of RNA Pol II. Truncation of the C-terminal domain and other RNA Pol II mutations conferred sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and RNR2 mRNAs after UV irradiation was attenuated in these mutant cells. That UV sensitivity can be a consequence of mutations in the RNA Pol II machinery in yeast cells suggests that alterations in transcriptional programs could underlie some of the pathophysiological defects seen in the human disease Cockayne syndrome. PMID: 11254132 [PubMed - indexed for MEDLINE] 253: Biotechniques 2001 Mar;30(3):634-6, 638, 640 passim Redefinition of the yeast two-hybrid system in dialogue with changing priorities in biological research. Serebriiskii IG, Khazak V, Golemis EA. Fox Chase Cancer Center, Philadelphia, PA, USA. Examination of the pattern of reagent creation and application in the two-hybrid system since 1989 reveals the expansion of a simple core technology to address increasingly sophisticated problems in protein interaction. As the technology has matured, its clear suitability for large-scale proteomic projects has made a major focus of its application the generation of global organismal protein interaction networks. In an inversion of emphasis, the increasing availability of such information now provides a master plan with the potential to specify the most promising directions for biological investigations (i.e., by directing the physiological validation of predicted critical protein-protein interactions). Recent derivatives of the two-hybrid system enable the targeting of such key interactions by facilitating the identification of essential amino acids conferring protein interaction specificity and of small molecules that selectively disrupt defined interaction pairs. Finally, the creation of mammalian expression systems based on two-hybrid principles became a new tool to create and probe novel biological systems. Taken in sum, this trajectory emphasizes the point that the creation of tools and the evolution of the idea of what is an interesting biological problem are in intimate dialogue. Publication Types: Review Review, Tutorial PMID: 11252799 [PubMed - indexed for MEDLINE] 254: Mol Biol Cell 2001 Mar;12(3):521-38 Selective formation of Sed5p-containing SNARE complexes is mediated by combinatorial binding interactions. Tsui MM, Tai WC, Banfield DK. Department of Biology, The Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong, China. Sed5p is the only syntaxin family member required for protein transport through the yeast Golgi and it is known to bind up to nine other soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in vivo. We describe in vitro binding experiments in which we identify ternary and quaternary Sed5p-containing SNARE complexes. The formation of SNARE complexes among these endoplasmic reticulum- and Golgi-localized proteins requires Sed5p and is syntaxin-selective. In addition, Sed5p-containing SNARE complexes form selectively and this selectivity is mediated by Sed5p-containing intermediates that discriminate among subsequent binding partners. Although many of these SNAREs have overlapping distributions in vivo, the SNAREs that form complexes with Sed5p in vitro reflect their functionally distinct locales. Although SNARE-SNARE interactions are promiscuous and a single SNARE protein is often found in more than one complex, both the biochemical as well as genetic analyses reported here suggest that this is not a result of nonselective direct substitution of one SNARE for another. Rather our data are consistent with the existence of multiple (perhaps parallel) trafficking pathways where Sed5p-containing SNARE complexes play overlapping and/or distinct functional roles. PMID: 11251068 [PubMed - indexed for MEDLINE] 255: J Bacteriol 2001 Apr;183(7):2306-15 Functional domains of yeast plasmid-encoded Rep proteins. Sengupta A, Blomqvist K, Pickett AJ, Zhang Y, Chew JS, Dobson MJ. Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. Both of the Saccharomyces cerevisiae 2 microm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2 microm-based stability vector with rep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2 microm circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex. PMID: 11244071 [PubMed - indexed for MEDLINE] 256: Cell 2001 Feb 9;104(3):397-408 Comment in: Cell. 2001 Feb 9;104(3):329-32. Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae. Myung K, Datta A, Kolodner RD. Ludwig Institute for Cancer Research, Cancer Center and Department of Medicine, University of California-San Diego School of Medicine, La Jolla, CA 92093, USA. Cancer cells show increased genome rearrangements, although it is unclear what defects cause these rearrangements. Mutations in Saccharomyces cerevisiae RFC5, DPB11, MEC1, DDC2 MEC3, RAD53, CHK1, PDS1, and DUN1 increased the rate of genome rearrangements up to 200-fold whereas mutations in RAD9, RAD17, RAD24, BUB3, and MAD3 had little effect. The rearrangements were primarily deletion of a portion of a chromosome arm along with TEL1-dependent addition of a new telomere. tel1 mutations increased the proportion of translocations observed, and in some cases showed synergistic interactions when combined with mutations that increased the genome rearrangement rate. These data suggest that one role of S phase checkpoint functions in normal cells is to suppress spontaneous genome rearrangements resulting from DNA replication errors. PMID: 11239397 [PubMed - indexed for MEDLINE] 257: Mol Cell Biol 2001 Mar;21(6):2098-106 Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates. Gelbart ME, Rechsteiner T, Richmond TJ, Tsukiyama T. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024, USA. To facilitate the biochemical characterization of chromatin-associated proteins in the budding yeast Saccharomyces cerevisiae, we have developed a system to assemble nucleosomal arrays on immobilized templates using recombinant yeast core histones. This system enabled us to analyze the interaction of Isw2 ATP-dependent chromatin remodeling complex with nucleosomal arrays. We found that Isw2 complex interacts efficiently with both naked DNA and nucleosomal arrays in an ATP-independent manner, suggesting that ATP is required at steps subsequent to this physical interaction. We identified the second subunit of Isw2 complex, encoded by open reading frame YGL 133w (herein named ITC1), and found that both subunits of the complex, Isw2p and Itc1p, are essential for efficient interaction with DNA and nucleosomal arrays. Both subunits are also required for nucleosome-stimulated ATPase activity and chromatin remodeling activity of the complex. Finally, we found that ITC1 is essential for function of Isw2 complex in vivo, since isw2 and itc1 deletion mutants exhibit virtually identical phenotypes. These results demonstrate the utility of our in vitro system in studying interactions between chromatin-associated proteins and nucleosomal arrays. PMID: 11238944 [PubMed - indexed for MEDLINE] 258: Mol Cell Biol 2001 Mar;21(6):2026-37 Fip1 regulates the activity of Poly(A) polymerase through multiple interactions. Helmling S, Zhelkovsky A, Moore CL. Department of Biochemistry, Tufts University, School of Medicine, Boston, Massachusetts 02111, USA. Fip1 is an essential component of the Saccharomyces cerevisiae polyadenylation machinery and the only protein known to interact directly with poly(A) polymerase (Pap1). Its association with Pap1 inhibits the extension of an oligo(A) primer by limiting access of the RNA substrate to the C-terminal RNA binding domain (C-RBD) of Pap1. We present here the identification of separate functional domains of Fip1. Amino acids 80 to 105 are required for binding to Pap1 and for the inhibition of Pap1 activity. This region is also essential for viability, suggesting that Fip1-mediated repression of Pap1 has a crucial physiological function. Amino acids 206 to 220 of Fip1 are needed for the interaction with the Yth1 subunit of the complex and for specific polyadenylation of the cleaved mRNA precursor. A third domain within amino acids 105 to 206 helps to limit RNA binding at the C-RBD of Pap1. Our data demonstrate that the C terminus of Fip1 is required to relieve the Fip1-mediated repression of Pap1 in specific polyadenylation. In the absence of this domain, Pap1 remains in an inhibited state. These findings show that Fip1 has a crucial regulatory function in the polyadenylation reaction by controlling the activity of poly(A) tail synthesis through multiple interactions within the polyadenylation complex. PMID: 11238938 [PubMed - indexed for MEDLINE] 259: J Virol 2001 Apr;75(7):3207-19 Brome mosaic virus Protein 1a recruits viral RNA2 to RNA replication through a 5' proximal RNA2 signal. Chen J, Noueiry A, Ahlquist P. Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA. Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions. 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain. In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane. Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication. To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo. In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a. In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5'-proximal third of RNA2. The RNA2 5' untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA. A 5'-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TPsiC stem-loop of host tRNAs. The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates. These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication. PMID: 11238847 [PubMed - indexed for MEDLINE] 260: Genetics 2001 Mar;157(3):1179-89 The Saccharomyces cerevisiae MUM2 gene interacts with the DNA replication machinery and is required for meiotic levels of double strand breaks. Davis L, Barbera M, McDonnell A, McIntyre K, Sternglanz R, Jin Q, Loidl J, Engebrecht J. Department of Pharmacological Sciences, Graduate Program in Genetics, State University of New York, Stony Brook, New York 11794-8651, USA. The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not mitotic, DNA replication and thus sporulation. Genetic interactions between MUM2 and a component of the origin recognition complex and polymerase alpha-primase suggest that MUM2 influences the function of the DNA replication machinery. Early meiotic gene expression is induced to a much greater extent in mum2 cells than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea. This result indicates that the mum2 meiotic arrest is downstream of the arrest induced by hydroxyurea and suggests that DNA synthesis is initiated in the mutant. Genetic analyses indicate that the recombination that occurs in mum2 mutants is dependent on the normal recombination machinery and on synaptonemal complex components and therefore is not a consequence of lesions created by incompletely replicated DNA. Both meiotic ectopic and allelic recombination are similarly reduced in the mum2 mutant, and the levels are consistent with the levels of meiosis-specific DSBs that are generated. Cytological analyses of mum2 mutants show that chromosome pairing and synapsis occur, although at reduced levels compared to wild type. Given the near-wild-type levels of meiotic gene expression, pairing, and synapsis, we suggest that the reduction in DNA replication is directly responsible for the reduced level of DSBs and meiotic recombination. PMID: 11238403 [PubMed - indexed for MEDLINE] 261: Genetics 2001 Mar;157(3):1107-16 Genes encoding ribosomal proteins Rps0A/B of Saccharomyces cerevisiae interact with TOM1 mutants defective in ribosome synthesis. Tabb AL, Utsugi T, Wooten-Kee CR, Sasaki T, Edling SA, Gump W, Kikuchi Y, Ellis SR. Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky 40292, USA. The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA. We show here that the RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants. Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits. Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family. PMID: 11238398 [PubMed - indexed for MEDLINE] 262: Biochem J 2001 Mar 15;354(Pt 3):655-61 Identification of Cdc6 protein domains involved in interaction with Mcm2 protein and Cdc4 protein in budding yeast cells. Jang SW, Elsasser S, Campbell JL, Kim J. Graduate School of Biotechnology, Department of Genetic Engineering, Kyung Hee University, Yongin, Kyonggi-Do, 449-701, Korea. The Cdc6 protein (Cdc6p) has essential roles in regulating initiation of DNA replication. Cdc6p is recruited to origins of replication by the origin recognition complex (ORC) late in mitosis; Cdc6p in turn recruits minichromosome maintenance (Mcm) proteins to form the pre-replicative complex. Cdc6p is thought to interact with one or more Mcm proteins but this point has not yet been demonstrated. In the present study we observed that Cdc6p interacted significantly only with Mcm2p out of six Mcm proteins in yeast two-hybrid cells. Our results indicate that the interaction of Cdc6p with Mcm2p is specific, although we cannot exclude the possibility that the interaction might not be direct. In attempts to identify domains of Cdc6p important for interaction with Mcm2p, we tested interactions of various deleted versions of Cdc6p with Mcm2p and also with Cdc4p, which was previously known to interact with Cdc6p. The portion of Cdc6p from amino acid residues 51 to 394 was able to interact with Mcm2p. During the course of the studies we also discovered a previously undetected Cdc4p interaction domain between residues 51 and 394. Interestingly, when all six putative Cdc28 phosphorylation sites in Cdc6p were changed to alanine, a 6-7-fold increase in binding to Mcm2p was observed. This result suggests that unphosphorylated Cdc6p has higher affinity than phosphorylated Cdc6p for Mcm2p; this might partly explain the previous observation that Cdc6p failed to load Mcm proteins on replication origins during S phase when the cyclin-dependent protein kinase was active, thus helping to prevent the reinitiation of activated replicons. PMID: 11237870 [PubMed - indexed for MEDLINE] 263: J Mol Biol 2001 Mar 9;306(5):903-13 Specific interactions of the telomeric protein Rap1p with nucleosomal binding sites. Rossetti L, Cacchione S, De Menna A, Chapman L, Rhodes D, Savino M. Dipartimento di Genetica e Biologia Molecolare, Fondazione Istituto Pasteur -Fondazione Cenci Bolognetti, Universita di Roma La Sapienza, Piazzale A Moro 5,00185, Roma, Italy. The telomeres of Saccharomyces cerevisiae are structurally and functionally well characterized. Their telomeric DNA is packaged by the protein Rap1p (repressor activator protein 1). Rap1p is a multifunctional, sequence-specific, DNA-binding protein which, besides participating in the regulation of telomeres structure and length, is also involved in transcriptional regulation of genes essential for cell growth and in silencing. Whereas the long tracts of telomeric DNA repeats of higher eukaryotes are mostly organized in closely spaced canonical nucleosomal arrays, it has been proposed that the 300 base-pairs of S. cerevisiae telomeric DNA are organized in a large non-nucleosomal structure that has been called the telosome. Recently, nucleosomes have been found also in Tetrahymena thermophila telomeres, suggesting that, in general, telomere structural differences between lower and higher eukaryotes could be quantitative, rather than qualitative. Using an in vitro model system, we have addressed the question of whether Rap1p can form a stable ternary complex with nucleosomes containing telomeric binding sites, or competes with nucleosome core formation. The approach we have taken is to place a single Rap1p-binding site at different positions within a nucleosome core and then test the binding of Rap1p and its DNA-binding domain (Rap1p-DBD). We show here that both proteins are able to specifically recognize their nucleosomal binding site, but that binding is dependent on the location of the site within the nucleosome core structure. These results show that a ternary complex between a nucleosome and Rap1p is stable and could be a possible intermediate between telomeric nucleosomes and telosomes in the dynamics of S. cerevisiae telomere organization. PMID: 11237607 [PubMed - indexed for MEDLINE] 264: Biochem Cell Biol 2001;79(1):83-91 A tale of two charges: distinct roles for an acidic and a basic amino acid in the structure and function of cytochrome c. Parrish JC, Guillemette JG, Wallace CJ. Department of Biochemistry, Dalhousie University, Halifax, NS, Canada. Cytochrome c is a small electron transport protein found in the intermembrane space of mitochondria. As it interacts with a number of different physiological partners in a specific fashion, its structure varies little over eukaryotic evolutionary history. Two highly conserved residues found within its sequence are those at positions 13 and 90 (numbering is based on the standard horse cytochrome c); with single exceptions, residue 13 is either Lys or Arg, and residue 90 is either Glu or Asp. There have been conflicting views on the roles to be ascribed to these residues, particularly residue 13, so the functional properties of a number of site-directed mutants of Saccaromyces cerevisiae iso-1 cytochrome c have been examined. Results indicate that the two residues do not interact specifically with each other; however, residue 13 (Arg) is likely to be involved in interactions between cytochrome c and other electrostatically oriented physiological partners (intermolecular), whereas residue 90 (Asp) is involved in maintaining the intrinsic structure and stability of cytochrome c (intramolecular). This is supported by molecular dynamics simulations carried out for these mutants where removal of the negative charge at position 90 leads to significant shifts in the conformations of neighboring residues, particularly lysine 86. Both charged residues appear to exert their effects through electrostatics; however, biological activity is significantly more sensitive to substitutions of residue 13 than of residue 90. PMID: 11235919 [PubMed - indexed for MEDLINE] 265: EMBO J 2001 Jan 15;20(1-2):118-27 Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1. Holm M, Hardtke CS, Gaudet R, Deng XW. Department of Molecular, Cellular and Developmental Biology, Yale University, OML 354, Yale University, PO Box 20-8104, 165 Prospect Street, New Haven, CT 06520-8104, USA. Arabidopsis COP1 is a photomorphogenesis repressor capable of directly interacting with the photomorphogenesis-promoting factor HY5. This interaction between HY5 and COP1 results in targeted deg radation of HY5 by the 26S proteasome. Here we characterized the WD40 repeat domain-mediated interactions of COP1 with HY5 and two new proteins. Mutational analysis of those interactive partners revealed a conserved motif responsible for the interaction with the WD40 domain. This novel motif, with the core sequence V-P-E/D-φ-G (φ = hydrophobic residue) in conjunction with an upstream stretch of 4-5 negatively charged residues, interacts with a defined surface area of the ss-propeller assembly of the COP1 WD40 repeat domain through both hydrophobic and ionic interactions. Several residues in the COP1 WD40 domain that are critical for the interaction with this motif have been revealed. The fact that point mutations either in the COP1 WD40 domain or in the HY5 motif that abolish the interaction between COP1 and HY5 in yeast result in a dramatic reduction of HY5 degradation in transgenic plants validates the biological significance of this defined interaction. PMID: 11226162 [PubMed - indexed for MEDLINE] 266: Acta Crystallogr D Biol Crystallogr 2001 Mar;57(Pt 3):459-61 Crystallization and preliminary X-ray diffraction studies of FHA domains of Dun1 and Rad53 protein kinases. Blanchard H, Fontes MR, Hammet A, Pike BL, Teh T, Gleichmann T, Gooley PR, Kobe B, Heierhorst J. Department of Biochemistry, University of Queensland, St Lucia, Brisbane, Qld 4072, Australia. helenb@biosci.uq.edu.au Forkhead-associated (FHA) domains are modular protein-protein interaction domains of approximately 130 amino acids present in numerous signalling proteins. FHA-domain-dependent protein interactions are regulated by phosphorylation of target proteins and FHA domains may be multifunctional phosphopeptide-recognition modules. FHA domains of the budding yeast cell-cycle checkpoint protein kinases Dun1p and Rad53p have been crystallized. Crystals of the Dun1-FHA domain exhibit the symmetry of the space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 127.3, c = 386.3 A; diffraction data have been collected to 3.1 A resolution on a synchrotron source. Crystals of the N-terminal FHA domain (FHA1) of Rad53p diffract to 4.0 A resolution on a laboratory X-ray source and have Laue-group symmetry 4/mmm, with unit-cell parameters a = b = 61.7, c = 104.3 A. PMID: 11223532 [PubMed - indexed for MEDLINE] 267: RNA 2001 Jan;7(1):133-42 Yeast U1 snRNP-pre-mRNA complex formation without U1snRNA-pre-mRNA base pairing. Du H, Rosbash M. Department of Biology, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454, USA. Base pairing between the 5' end of U1 snRNA and the conserved 5' splice site of pre-mRNA is important for commitment complex formation in vitro. However, the biochemical mechanisms by which pre-mRNA is initially recognized by the splicing machinery is not well understood. To evaluate the role of this base pairing interaction, we truncated U1 snRNA to eliminate the RNA-RNA interaction and surprisingly found that U1 snRNP can still form a nearly normal RNA-protein complex and maintain sequence specificity. We propose that some feature of U1 snRNP, perhaps one or more protein factors, is more important than the base pairing for initial 5' splice site recognition. In addition, at least five sets of interactions contribute to complex formation or stability. Only one of these is base pairing between the 5' splice site and the 5' end of U1 snRNA, without which the U1 snRNP-pre-mRNA complex is less stable and has a somewhat altered conformation. PMID: 11214175 [PubMed - indexed for MEDLINE] 268: Nature 2001 Jan 25;409(6819):533-8 Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Iyer VR, Horak CE, Scafe CS, Botstein D, Snyder M, Brown PO. Department of Biochemistry, Stanford University Medical Center, California 94305, USA. Proteins interact with genomic DNA to bring the genome to life; and these interactions also define many functional features of the genome. SBF and MBF are sequence-specific transcription factors that activate gene expression during the G1/S transition of the cell cycle in yeast. SBF is a heterodimer of Swi4 and Swi6, and MBF is a heterodimer of Mbpl and Swi6 (refs 1, 3). The related Swi4 and Mbp1 proteins are the DNA-binding components of the respective factors, and Swi6 mayhave a regulatory function. A small number of SBF and MBF target genes have been identified. Here we define the genomic binding sites of the SBF and MBF transcription factors in vivo, by using DNA microarrays. In addition to the previously characterized targets, we have identified about 200 new putative targets. Our results support the hypothesis that SBF activated genes are predominantly involved in budding, and in membrane and cell-wall biosynthesis, whereas DNA replication and repair are the dominant functions among MBF activated genes. The functional specialization of these factors may provide a mechanism for independent regulation of distinct molecular processes that normally occur in synchrony during the mitotic cell cycle. PMID: 11206552 [PubMed - indexed for MEDLINE] 269: Med Mycol 2000;38 Suppl 1:125-37 Candida albicans: adherence, signaling and virulence. Calderone R, Suzuki S, Cannon R, Cho T, Boyd D, Calera J, Chibana H, Herman D, Holmes A, Jeng HW, Kaminishi H, Matsumoto T, Mikami T, O'Sullivan JM, Sudoh M, Suzuki M, Nakashima Y, Tanaka T, Tompkins GR, Watanabe T. Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20007, USA. calderor@gunet.georgetown.edu The focus of this symposium was to present new information on the morphogenesis of Candida albicans, particularly how it relates to signal transduction pathways and other genes involved in the regulation of morphogenesis. In addition, we discuss the role of adherence and colonization of the oral cavity by the organism and discuss the role of mannan as an adhesin that recognizes the human red blood cell. C. albicans utilizes at least two signal pathways to regulate its conversion from a yeast form to filamentous growth (hyphae). One of these two pathways is similar to the Saccharomyces cerevisiae pseudohyphal/mating pathway, which utilizes the regulatory protein, Cphlp. The other pathway is not totally defined but requires a second regulatory protein, referred to as Efg1p. Other signal pathways may exist, which include a two-component histidine kinase and response regulator proteins. The latter pathway(s) may include proteins such as Chk1p, Ssk1p, Shi1p and Cos1p/Nik1p. Mutations in strains, which specifically target these proteins, result in morphogenesis defects and avirulence or attenuation of strains. A growth regulatory gene has also been recently defined whose expression is associated with growth cessation and which appears to be a necessary prerequisite in conversion of the organism to a filamentous growth form. Starvation of yeast cells induces exponentially grown cells (and usually non-germinative) to germinate. This phenomenon is also observed in cells that are transiently treated with metabolic inhibitors. During each of these treatments (starvation, metabolic inhibition), expression of a growth regulatory gene (CGRI) increases. Adherence of C. albicans to host cells and tissues is complex; several proteins, which appear to have host recognition functions, have been defined. In the oral cavity, C. albicans selectively adheres to salivary proteins, which are absorbed to many oral surfaces. This mechanism enables the cells to colonize surfaces of the oral cavity. An understanding of these interactions may lead to strategies to prevent oral disease. Mannan from C. albicans may provide a host recognition function for C. albicans. Recent experiments indicate that mannan binds to human red blood cells and causes hemolysis. Binding of mannan to the band 3 protein of human red blood cells has been established. This activity may be associated with the ability of the organism to utilize hemoglobin (and iron). Publication Types: Review Review, Tutorial PMID: 11204138 [PubMed - indexed for MEDLINE] 270: Plant Mol Biol 2000 Nov;44(4):513-27 Two rice MADS domain proteins interact with OsMADS1. Lim J, Moon YH, An G, Jang SK. Department of Life Science, Pohang University of Science and Technology, Kyunghuk, Korea. OsMADS1 is a MADS box gene controlling flower development in rice. In order to learn more about the function of OsMADS1, we searched for cellular proteins interacting with OsMADS1 employing the yeast two-hybrid system. Two novel proteins with MADS domains, which were named OsMADS14 and OsMADS15, were isolated from a rice cDNA library. OsMADS14 and -15 are highly homologous to the maize MADS box gene ZAP1 which is an orthologue of the floral homeotic gene APETALA1 (AP1). Interactions among the three MADS domain proteins were confirmed by in vitro experiments using GST-fused OsMADS1 expressed in Escherichia coli and in vitro translated proteins of OsMADS14 and -15. We determined which domains in OsMADS1, -14, and -15 were required for protein-protein interaction employing the two-hybrid system and pull-down experiments. While the K domain was essential for protein-protein interaction, a region preceded by the K domain augmented this interaction. Interestingly, the C-terminal region of OsMADS1 functioned as a transcriptional activation domain in yeast and mammalian cells, while, on the other hand, the C domains of OsMADS14 and -15 exhibited only very weak transcriptional activator functionality, if any at all. PMID: 11197326 [PubMed - indexed for MEDLINE] 271: Biotechniques 2001 Jan;30(1):94-8, 100 cDNA library screening using the SOS recruitment system. Huang W, Wang SL, Lozano G, de Crombrugghe B. University of Texas M.D. Anderson Cancer Center, Houston, TX, USA. The SOS recruitment system (SRS), a recently developed method for detecting protein-protein interactions, provides an attractive alternative to identify biologically important protein interactions. In SRS, the protein-protein interactions take place in the cytoplasm instead of the nucleus, as is the case in the conventional two-hybrid system. Although the SRS has overcome some of the disadvantages of the conventional two-hybrid system, it still has several problems and limitations. Here, we describe a new protocol for SRS library screening. A new combination of growth media to avoid the tedious step of replica plating greatly increases the number of independent colonies in a single library screening. Furthermore, we designed a pair of ras-specific primers and a one-step simple PCR to rule out the most abundant false positive, the mammalian ras cDNA, in SRS library screening. Publication Types: Technical Report PMID: 11196326 [PubMed - indexed for MEDLINE] 272: Curr Genet 2000 Dec;38(5):248-55 Isolation and molecular characterization of the carboxy-terminal pdr3 mutants in Saccharomyces cerevisiae. Simonics T, Kozovska Z, Michalkova-Papajova D, Delahodde A, Jacq C, Subik J. Department of Microbiology and Virology, Comenius University, Bratislava, Slovak Republic. Multidrug resistance in Saccharomyces cerevisiae mainly results from the overexpression of genes coding for the membrane efflux pumps, the major facilitators and the ABC binding cassette transporters, under the control of key transcription regulators encoded by the PDR1 and PDR3 genes. Pdr3p transcriptional activator contains a weak activation domain near the N-terminal zinc finger, a central regulatory domain, and a strong activation domain near the carboxyl terminus. Here we report the results of the mutational analysis of the C-terminal region of Pdr3p. After in vitro mutagenesis of the PDR3 gene six single amino acid substitutions were identified and resulted in resistance to cycloheximide, sulfomethuron methyl, 4-nitroquinoline oxide, fluconazole, mucidin, chloramphenicol and oligomycin. All the C-terminal pdr3 mutant alleles also conferred multidrug resistance in the presence of the wild-type PDR3 gene. The pdr3 mutations resulted in overexpression of both the PDR3 and PDR5 genes as revealed by transactivation experiments involving the PDR3-lacZ and PDR5-lacZ fusion genes and Western blot analyses using antibodies against Pdr5p. Most of the C-terminal pdr3 mutations were found in two sequence stretches exhibiting a high degree of amino acid identity with Pdr1p indicating that they might play a significant role in protein-protein interactions during the initiation of transcription of genes involved in multidrug resistance. PMID: 11191208 [PubMed - indexed for MEDLINE] 273: Curr Genet 2000 Dec;38(5):233-40 Alterations in the Saccharomyces MAL-activator cause constitutivity but can be suppressed by intragenic mutations. Danzi SE, Zhang B, Michels CA. Department of Biology, Queens College and the Graduate School of CUNY, Flushing, NY 11367, USA. The Saccharomyces MAL-activator regulates the maltose-inducible expression of the MAL structural genes encoding maltose permease and maltase. Constitutive MAL-activator mutant alleles of two types were identified. The first were truncation mutations deleting C-terminal residues 283-470 and the second contained a large number of alterations compared to inducible alleles scattered throughout the C-terminal 200 residues. We used site-directed in vitro mutagenesis of the inducible MAL63 and MAL63/23 genes to identify the residues responsible for the negative regulatory function of the C-terminal domain. Intragenic suppressors that restored the inducible phenotype to the constitutive mutants were identified at closely linked and more distant sites within the MAL-activator protein. MAL63/mal64 fusions of the truncated mutants suggest that residues in the N-terminal 100 residues containing the DNA-binding domain also modulate basal expression. Moreover, a transcription activator protein consisting of LexA(1-87)-Gal4(768-881)-Mal63(200-470) allowed constitutive reporter gene expression, suggesting that the C-terminal regulatory domain is not sufficient for maltose-inducible control of this heterologous activation domain. These results suggest that complex and very specific intramolecular protein-protein interactions regulate the MAL-activator. PMID: 11191206 [PubMed - indexed for MEDLINE] 274: IUBMB Life 2000 Aug;50(2):105-13 A CD2-based model of yeast alpha-agglutinin elucidates solution properties and binding characteristics. Grigorescu A, Chen MH, Zhao H, Kahn PC, Lipke PN. Department of Biological Sciences and The Institute for Biomolecular Structure and Function, Hunter College of the City University of New York, NY 10021, USA. We have previously shown that the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin has sequence characteristics of immunoglobulin-like proteins and have successfully modeled residues 200-325, based on the structure of immunoglobulin variable-type domains. Alignments matching residues 20-200 of alpha-agglutinin with domains I and II of members of the CD2/CD4 subfamily of the immunoglobulin superfamily showed > 80% conservation of key residues despite low sequence similarity overall. Three-dimensional models of two alpha-agglutinin domains constructed on the basis of these alignments were shown to conform to peptide mapping data and biophysical properties of alpha-agglutinin. In addition, the residue volume and surface accessibility characteristics of these models resembled those of the well-packed structures of related proteins. Residue-by-residue analysis showed that packing and accessibility anomalies were largely confined to glycosylated and protease-susceptible loop regions of the domains. Surface accessibility of hydrophobic residues was typical of proteins with extensive domain interactions, a finding compatible with the hydrodynamic properties of alpha -agglutinin and the hydrophobic nature of binding to its peptide ligand alpha-agglutinin. The procedures used to align the alpha-agglutinin sequence and test the quality of the model may be applicable to other proteins, especially those that resist crystallization because of extensive glycosylation. PMID: 11185954 [PubMed - indexed for MEDLINE] 275: Traffic 2000 Oct;1(10):763-8 The use of yeast two-hybrid screens in studies of protein:protein interactions involved in trafficking. Stephens DJ, Banting G. Department of Cell Biology and Cell Biophysics, EMBL-Heidelberg, Germany. The yeast two-hybrid system has provided a convenient means to both screen for proteins that interact with a protein of interest and to characterise the known interaction between two proteins. Several groups with an interest in the molecular mechanisms that underlie discrete steps along trafficking pathways have exploited the yeast two-hybrid system. Here, we provide a brief background to the technology, attempt to point out some of the pitfalls and benefits of the different systems that can be employed, and mention some of the areas (within the trafficking field) where yeast two-hybrid interaction assays have been particularly informative. Publication Types: Review Review, Tutorial PMID: 11208066 [PubMed - indexed for MEDLINE] 276: Cell Microbiol 1999 Jul;1(1):7-17 Identification of the intimin-binding domain of Tir of enteropathogenic Escherichia coli. de Grado M, Abe A, Gauthier A, Steele-Mortimer O, DeVinney R, Finlay BB. Biotechnology Laboratory, University of British Columbia, Vancouver, Canada. Enteropathogenic Escherichia coli (EPEC) attaches intimately to mammalian cells via a bacterial outer membrane adhesion molecule, intimin, and its receptor in the host cell membrane, Tir. Tir is a bacterial protein translocated into the host cell membrane and tyrosine phosphorylated after insertion. Tir-intimin binding induces organized actin polymerization beneath the adherent bacteria, resulting in the formation of pedestal-like structures. A series of Tir deletion derivatives were constructed to analyse which Tir domains are involved in intimin binding. We have localized the intimin-binding domain (IBD) of Tir using a yeast two-hybrid system and a gel-overlay approach to a region of 109 amino acids that is predicted to be exposed on the surface of the plasma membrane. A truncated Tir protein lacking this domain was translocated to the host cell membrane and tyrosine phosphorylated, but failed to bind intimin or to induce either actin polymerization or Tir accumulation beneath the bacteria. These results indicate that only a small region of Tir is needed to bind intimin and support the predicted topology for Tir, with both N- and C-terminal regions in the mammalian cell cytosol. They also confirm that Tir-intimin interactions are needed for cytoskeletal organization. We have also identified N-terminal regions involved in Tir stability and Tir secretion to the media. PMID: 11207537 [PubMed - indexed for MEDLINE] 277: Mol Biol Cell 2001 Feb;12(2):475-85 Vps10p transport from the trans-Golgi network to the endosome is mediated by clathrin-coated vesicles. Deloche O, Yeung BG, Payne GS, Schekman R. Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California, 229 Stanley Hall, Berkeley, California 94720-3206, USA. A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN. PMID: 11179429 [PubMed - indexed for MEDLINE] 278: EMBO J 2001 Feb 15;20(4):891-904 Related eIF3 subunits TIF32 and HCR1 interact with an RNA recognition motif in PRT1 required for eIF3 integrity and ribosome binding. Valasek L, Phan L, Schoenfeld LW, Valaskova V, Hinnebusch AG. Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA. eIF3 binds to 40S ribosomal subunits and stimulates recruitment of Met-tRNAiMet and mRNA to the pre-initiation complex. Saccharomyces cerevisiae contains an ortholog of human eIF3 subunit p35, HCR1, whose interactions with yeast eIF3 are not well defined. We found that HCR1 has a dual function in translation initiation: it binds to, and stabilizes, the eIF3-eIF5- eIF1-eIF2 multifactor complex and is required for the normal level of 40S ribosomes. The RNA recognition motif (RRM) of eIF3 subunit PRT1 interacted simultaneously with HCR1 and with an internal domain of eIF3 subunit TIF32 that has sequence and functional similarity to HCR1. PRT1, HCR1 and TIF32 were also functionally linked by genetic suppressor analysis. We propose that HCR1 stabilizes or modulates interaction between TIF32 and the PRT1 RRM. Removal of the PRT1 RRM resulted in dissociation of TIF32, NIP1, HCR1 and eIF5 from eIF3 in vivo, and destroyed 40S ribosome binding by the residual PRT1-TIF34-TIF35 subcomplex. Hence, the PRT1 RRM is crucial for the integrity and ribosome-binding activity of eIF3. PMID: 11179233 [PubMed - indexed for MEDLINE] 279: EMBO J 2001 Feb 15;20(4):841-51 Gal80-Gal80 interaction on adjacent Gal4p binding sites is required for complete GAL gene repression. Melcher K, Xu HE. Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-8573, USA. K.Melcher@em.uni-frankfurt.de Regulation of the GAL genes of Saccharomyces cerevisiae is determined by the interplay of the transcriptional activator Gal4p and the repressor Gal80p, which binds and masks the activation domain of Gal4p under non-inducing conditions. Here we demonstrate that Gal80p dimerizes with high affinity and that this dimerization appears to stabilize the Gal4p-Gal80p interaction and also, indirectly, the Gal4p-DNA interaction in a (Gal4p)2(Gal80p)2DNA complex. In addition, Gal80 dimers transiently interact with each other to form higher order multimers. We provide evidence that adjacent Gal4p binding sites, when correctly spaced, greatly stabilize Gal80p dimer-dimer interactions and that this stabilization results in the complete repression of GAL genes with multiple Gal4p binding sites. In contrast, GAL genes under the control of a single Gal4p binding site do not stabilize Gal80p multimers, resulting in significant and biologically important transcriptional leakage. Cooperative binding experiments indicate that Gal80p dimer-dimer interaction probably does not lead to a stronger Gal4p-Gal80p interaction, but most likely to a more complete shielding of the Gal4p activation domain. PMID: 11179228 [PubMed - indexed for MEDLINE] 280: Biochemistry (Mosc) 2000 Dec;65(12):1362-6 Interaction of catalytic domains in cytochrome P450scc--adrenodoxin reductase--adrenodoxin fusion protein imported into yeast mitochondria. Novikova LA, Nazarov PA, Saveliev AS, Drutsa VL, Sergeev VN, Miller WL, Luzikov VN. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia. We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc--adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted. PMID: 11173506 [PubMed - indexed for MEDLINE] 281: Biochem Soc Trans 2000 Dec;28(6):615-6 Protein interactions of fatty acid synthase II. Honeyman G, Fawcett T. Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, UK. We have used a yeast two-hybrid approach to detect direct protein interactions between fatty acid synthase components. Enoyl-acyl carrier protein (ACP) reductase was found to interact with stearoyl-ACP desaturase and acyl-ACP thioesterase, but none of these proteins interacted with ACP in the yeast nucleus. PMID: 11171144 [PubMed - indexed for MEDLINE] 282: Biochemistry 2001 Jan 23;40(3):712-8 DNA topoisomerase II as the target for the anticancer drug TOP-53: mechanistic basis for drug action. Byl JA, Cline SD, Utsugi T, Kobunai T, Yamada Y, Osheroff N. Department of Biochemistry and Medicine (Hematology/Oncology), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA. TOP-53 is a promising anticancer agent that displays high activity against non-small cell lung cancer in animal tumor models [Utsugi, T., et al. (1996) Cancer Res. 56, 2809-2814]. Compared to its parent compound, etoposide, TOP-53 is considerably more toxic to non-small cell lung cancer cells, is more active at generating chromosomal breaks, and displays improved cellular uptake and pharmacokinetics in animal lung tissues. Despite the preclinical success of TOP-53, several questions remain regarding its cytotoxic mechanism. Therefore, this study characterized the basis for drug action. Results indicate that topoisomerase II is the primary cytotoxic target for TOP-53. Furthermore, the drug kills cells by acting as a topoisomerase II poison. TOP-53 exhibits a DNA cleavage site specificity that is identical to that of etoposide. Like its parent compound, the drug increases the number of enzyme-mediated DNA breaks by interfering with the DNA religation activity of the enzyme. TOP-53 is considerably more efficient than etoposide at enhancing topoisomerase II-mediated DNA cleavage and exhibits high activity against human topoisomerase IIalpha and IIbeta in vitro and in cultured cells. Therefore, at least in part, the enhanced cytotoxic activity of TOP-53 can be attributed to an enhanced activity against topoisomerase II. Finally, TOP-53 displays nearly wild-type activity against a mutant yeast type II enzyme that is highly resistant to etoposide. This finding suggests that TOP-53 can retain activity against systems that have developed resistance to etoposide, and indicates that substituents on the etoposide C-ring are important for topoisomerase II-drug interactions. PMID: 11170388 [PubMed - indexed for MEDLINE] 283: Genes Cells 2000 Dec;5(12):975-89 Interactions between Mcm10p and other replication factors are required for proper initiation and elongation of chromosomal DNA replication in Saccharomyces cerevisiae. Kawasaki Y, Hiraga S, Sugino A. Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Previous work showed that Mcm10p interacts with the Mcm2-7 protein complex that may be functioning as the replication-licensing factor. In addition, Mcm10p is required during origin activation and disassembly of the prereplicative complex, which allows smooth passage of replication forks. RESULTS: We show that an mcm10 mutation causes a slow progression of DNA synthesis and a loss of chromosome integrity during the S phase and prevents entry into mitosis, despite apparent completion of chromosomal DNA replication at nonpermissive temperatures. Furthermore, Mcm10p interacts genetically with the origin recognition complex (ORC) and various replication elongation factors, including a subunit of DNA polymerases epsilon and delta. Mcm10p is an abundant protein (approximately 4 x 10(4) copies per haploid cell) that is almost exclusively localized in the chromatin and/or nuclear matrix fractions during all phases of the cell cycle. When it is visualized by the chromosome-spreading method followed by immunostaining, Mcm10p forms punctate foci on chromatin throughout the cell cycle and these foci mostly overlap with those of Orc1p, a component of ORC. CONCLUSIONS: These results suggest that Mcm10p, like the Mcm2-7 proteins, is a critical component of the prereplication chromatin and acts together with ORC during the initiation of chromosomal DNA replication; in addition, Mcm10p plays an important role during the elongation of DNA replication. PMID: 11168584 [PubMed - indexed for MEDLINE] 284: Biochem Biophys Res Commun 2001 Jan 12;280(1):151-7 Imaging and mapping protein-binding sites on DNA regulatory regions with atomic force microscopy. Moreno-Herrero F, Herrero P, Colchero J, Baro AM, Moreno F. Departamento de Bioquimica y Biologia Molecular, Instituto Universitario de Biotecnologia de Asturias, 33006 Oviedo, Spain. Regulation of gene expression is fundamental in biological systems. A systematic search for protein binding sites in gene promoters has been done in recent years. Biochemical techniques are easy and reliable when analysing protein interactions with short pieces of DNA, but are difficult and tedious when long pieces of DNA have to be analysed. Here we propose AFM as a reliable and easy technique for identifying protein interaction sites in long DNA molecules like gene promoters. We support this idea using a well-known model: the interaction of the Pho4 protein with the PHO5 gene promoter. We have also applied the technique to demonstrate that Mig1 protein binds to two motifs in the promoter of HXK2 gene. Our results allow us to define Mig1p as a new factor probably contributing to the carbon source-dependent transcription regulation of HXK2 gene. Copyright 2001 Academic Press. PMID: 11162492 [PubMed - indexed for MEDLINE] 285: Nucleic Acids Res 2001 Feb 15;29(4):E18 A novel approach for the identification of protein-protein interaction with integral membrane proteins. Hubsman M, Yudkovsky G, Aronheim A. Department of Molecular Genetics and the Rappaport Family Institute for Research in the Medical Sciences and the B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Bat-Galim, Haifa 31096, Israel. Protein-protein interaction plays a major role in all biological processes. The currently available genetic methods such as the two-hybrid system and the protein recruitment system are relatively limited in their ability to identify interactions with integral membrane proteins. Here we describe the development of a reverse Ras recruitment system (reverse RRS), in which the bait used encodes a membrane protein. The bait is expressed in its natural environment, the membrane, whereas the protein partner (the prey) is fused to a cytoplasmic Ras mutant. Protein-protein interaction between the proteins encoded by the prey and the bait results in Ras membrane translocation and activation of a viability pathway in yeast. We devised the expression of the bait and prey proteins under the control of dual distinct inducible promoters, thus enabling a rapid selection of transformants in which growth is attributed solely to specific protein-protein interaction. The reverse RRS approach greatly extends the usefulness of the protein recruitment systems and the use of integral membrane proteins as baits. The system serves as an attractive approach to explore novel protein-protein interactions with high specificity and selectivity, where other methods fail. PMID: 11160938 [PubMed - indexed for MEDLINE] 286: Nucleic Acids Res 2001 Feb 1;29(3):629-37 Leucine zipper motif of chicken histone acetyltransferase-1 is essential for in vivo and in vitro interactions with the p48 subunit of chicken chromatin assembly factor-1. Ahmad A, Nagamatsu N, Kouriki H, Takami Y, Nakayama T. Department of Biochemistry, Miyazaki Medical College, 5200, Kihara, Kiyotake, Miyazaki 889-1692, Japan. We cloned cDNA encoding chicken cytoplasmic histone acetyltransferase-1, chHAT-1, comprising 408 amino acids including a putative initiation Met. It exhibits 80.4% identity to the human homolog and possesses a typical leucine zipper motif. The glutathione S:-transferase (GST) pull-down assay, involving truncated and missense mutants of the chicken chromatin assembly factor-1 (chCAF-1)p48, revealed not only that a region (comprising amino acids 376-405 of chCAF-1p48 and containing the seventh WD dipeptide motif) binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction. The GST pull-down assay, involving truncated and missense chHAT-1 mutants, established that a region, comprising amino acids 380-408 of chHAT-1 and containing the leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. In addition, mutation of each of four Leu residues in the leucine zipper motif prevents the in vitro interaction. The yeast two-hybrid assay revealed that all four Leu residues within the leucine zipper motif of chHAT-1 are necessary for its in vivo interaction with chCAF-1p48. These results indicate not only that the proper leucine zipper motif of chHAT-1 is essential for its interaction with chCAF-1p48, but also that the propeller structure of chCAF-1p48 expected to act as a platform for protein-protein interactions may not be necessary for this interaction of chHAT-1. PMID: 11160883 [PubMed - indexed for MEDLINE] 287: Mol Biol Cell 2001 Jan;12(1):37-51 Vps41p function in the alkaline phosphatase pathway requires homo-oligomerization and interaction with AP-3 through two distinct domains. Darsow T, Katzmann DJ, Cowles CR, Emr SD. Department of Cellular and Molecular Medicine and Division of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668, USA. Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein-dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3. PMID: 11160821 [PubMed - indexed for MEDLINE] 288: Biophys J 2001 Jan;80(1):427-34 Tryptophan fluorescence of yeast actin resolved via conserved mutations. Doyle TC, Hansen JE, Reisler E. Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA. Actin contains four tryptophan residues, W79, W86, W340, and W356, all located in subdomain 1 of the protein. Replacement of each of these residues with either tyrosine (W79Y and W356Y) or phenylalanine (W86F and W340F) generated viable proteins in the yeast Saccharomyces cerevisiae, which, when purified, allowed the analysis of the contribution of these residues to the overall tryptophan fluorescence of actin. The sum of the relative contributions of these tryptophans was found to account for the intrinsic fluorescence of wild-type actin, indicating that energy transfer between the tryptophans is not the main determinant of their quantum yield, and that these mutations induce little conformational change to the protein. This was borne out by virtually identical polymerization rates and similar myosin interactions of each of the mutants and the wild-type actin. In addition, these mutants allowed the dissection of the microenvironment of each tryptophan as actin undergoes conformational changes upon metal cation exchange and polymerization. Based on the relative tryptophan contributions determined from single mutants, a triple mutant of yeast actin (W79) was generated that showed small intrinsic fluorescence and should be useful for studies of actin interactions with actin-binding proteins. PMID: 11159413 [PubMed - indexed for MEDLINE] 289: Proc Natl Acad Sci U S A 2001 Jan 30;98(3):914-9 Subunit interactions influence the biochemical and biological properties of Hsp104. Schirmer EC, Ware DM, Queitsch C, Kowal AS, Lindquist SL. Department of Molecular Genetics and Cell Biology and Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637, USA. Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vitro, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization; analogous NBD2 mutations reduce ATPase activity and severely impair hexamerization. We report that high protein concentrations overcome the assembly defects of NBD2 mutants and increase ATP hydrolysis severalfold, changing V(max) with little effect on K(m). In a complementary fashion, the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate inhibits hexamerization of wild-type (WT) Hsp104, lowering V(max) with little effect on K(m). ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating that it is influenced by cooperative subunit interactions. To further analyze the effects of subunit interactions on Hsp104, we assessed the effects of mutant Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that hexamerizes but does not hydrolyze ATP reduces the ATPase activity of WT Hsp104 in vitro. In vivo, this mutant is not toxic but specifically inhibits the thermotolerance function of WT Hsp104. Thus, interactions between subunits influence the ATPase activity of Hsp104, play a vital role in its biological functions, and provide a mechanism for conditionally inactivating Hsp104 function in vivo. PMID: 11158570 [PubMed - indexed for MEDLINE] 290: Mol Endocrinol 2001 Feb;15(2):241-54 Two distinct nuclear receptor-interaction domains and CREB-binding protein-dependent transactivation function of activating signal cointegrator-2. Lee SK, Jung SY, Kim YS, Na SY, Lee YC, Lee JW. Center for Ligand and Transcription, Department of Biology, Chonnam National University, Kwangju 500-757, Korea. ASC-2 is a recently isolated transcriptional cointegrator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors, AP-1, nuclear factor kappaB (NFkappaB), serum response factor (SRF), and numerous other transcription factors. ASC-2 contained two nuclear receptor-interaction domains, both of which are dependent on the integrity of their core LXXLL sequences. Surprisingly, the C-terminal LXXLL motif specifically interacted with oxysterol receptor LXRss, whereas the N-terminal motif bound a broad range of nuclear receptors. These interactions appeared to be essential because a specific subregion of ASC-2 including the N- or C-terminal LXXLL motif acted as a potent dominant negative mutant with transactivation by appropriate nuclear receptors. In addition, the autonomous transactivation domain (AD) of ASC-2 was found to consist of three separable subregions; i.e. AD1, AD2, and AD3. In particular, AD2 and AD3 were binding sites for CREB binding protein (CBP), and CBP-neutralizing E1A repressed the autonomous transactivation function of ASC-2. Furthermore, the receptor transactivation was not enhanced by ASC-2 in the presence of E1A and significantly impaired by overexpressed AD2. From these results, we concluded that ASC-2 directly binds to nuclear receptors and recruits CBP to mediate the nuclear receptor transactivation in vivo. PMID: 11158331 [PubMed - indexed for MEDLINE] 291: J Cell Biol 2001 Feb 5;152(3):451-69 A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis. Song S, Lee KS. Laboratory of Metabolism, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. Coordination of mitotic exit with timely initiation of cytokinesis is critical to ensure completion of mitotic events before cell division. The Saccharomyces cerevisiae polo kinase Cdc5 functions in a pathway leading to the degradation of mitotic cyclin Clb2, thereby permitting mitotic exit. Here we provide evidence that Cdc5 also plays a role in regulating cytokinesis and that an intact polo-box, a conserved motif in the noncatalytic COOH-terminal domain of Cdc5, is required for this event. Depletion of Cdc5 function leads to an arrest in cytokinesis. Overexpression of the COOH-terminal domain of Cdc5 (cdc5DeltaN), but not the corresponding polo-box mutant, resulted in connected cells. These cells shared cytoplasms with incomplete septa, and possessed aberrant septin ring structures. Provision of additional copies of endogenous CDC5 remedied this phenotype, suggesting a dominant-negative inhibition of cytokinesis. The polo-box-dependent interactions between Cdc5 and septins (Cdc11 and Cdc12) and genetic interactions between the dominant-negative cdc5DeltaN and Cyk2/Hof1 or Myo1 suggest that direct interactions between cdc5DeltaN and septins resulted in inhibition of Cyk2/Hof1- and Myo1-mediated cytokinetic pathways. Thus, we propose that Cdc5 may coordinate mitotic exit with cytokinesis by participating in both anaphase promoting complex activation and a polo-box-dependent cytokinetic pathway. PMID: 11157974 [PubMed - indexed for MEDLINE] 292: Hum Mol Genet 2001 Feb 15;10(4):423-9 Direct interactions of the five known Fanconi anaemia proteins suggest a common functional pathway. Medhurst AL, Huber PA, Waisfisz Q, de Winter JP, Mathew CG. Division of Medical and Molecular Genetics, Guy's, King's and St Thomas' School of Medicine, 8th Floor, Guy's Tower, Guy's Hospital, London SE1 9RT, UK. Fanconi anaemia (FA) is an autosomal recessive inherited disorder associated with a progressive aplastic anaemia, diverse congenital abnormalities and cancer. The condition is genetically heterogeneous, with at least seven complementation groups (A-G) described. Cells from individuals who are homozygous for mutations in FA genes are characterized by chromosomal instability and hypersensitivity to DNA interstrand crosslinking agents. These features suggest a possible role for the encoded proteins in the recognition or repair of these lesions, but neither their function nor whether they operate in a concerted or discrete functional pathways is known. The recent cloning of the FANCF and FANCE genes has allowed us to investigate the interaction of the proteins encoded by five of the seven complementation groups of FA. We used the yeast two-hybrid system and co-immunoprecipitation analysis to test the 10 possible pairs of proteins for direct interaction. In addition to the previously described binding of FANCA to FANCG, we now demonstrate direct interaction of FANCF with FANCG, of FANCC with FANCE and a weaker interaction of FANCE with both FANCA and FANCG. These findings show that the newly identified FANCE protein is an integral part of the FA pathway, and support the concept of a functional link between all known proteins encoded by the genes that are mutated in this disorder. These proteins may act either as a multimeric complex or by sequential recruitment of subsets of the proteins in a common pathway that protects the genomic integrity of mammalian cells. PMID: 11157805 [PubMed - indexed for MEDLINE] 293: Genes Dev 2001 Jan 15;15(2):147-57 The Sir1 protein's association with a silenced chromosome domain. Gardner KA, Fox CA. Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA. Silencing of the cryptic mating-type locus HMR requires recognition of a small DNA sequence element, the HMR-E silencer, by the Sir1p, one of four Sir proteins required for the assembly of silenced chromatin domains in Saccharomyces cerevisiae. The Sir1p recognizes the silencer through interactions with the origin recognition complex (ORC), a protein complex that binds the silencer DNA directly. Sir1p was physically associated with HMR in chromatin, and this association required a Sir1p-ORC interaction, suggesting that it reflected the Sir1p silencer-recognition function required for silencing. Sir1p was not associated with nonsilencer replication origins that bind the ORC, indicating that a Sir1p-ORC interaction is confined to silencers. Significantly, the other SIR genes were required for Sir1p's association with HMR. Thus, multiple protein contacts required for and unique to silent chromatin may confine a Sir1p-ORC interaction to silencers. The Sir1p was present at extremely low concentrations in yeast cells yet was associated with HMR at all stages of the cell cycle examined. These data provide insights into the mechanisms that establish and restrict the assembly of silenced chromatin to only a few discrete chromosomal domains. PMID: 11157772 [PubMed - indexed for MEDLINE] 294: J Biol Chem 2000 Apr 14;275(15):11141-6 Interaction in vivo and in vitro of the metastasis-inducing S100 protein, S100A4 (p9Ka) with S100A1. Wang G, Rudland PS, White MR, Barraclough R. Cancer and Polio Research Fund Laboratories, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom. The calcium-binding protein S100A4 (p9Ka) has been shown to cause a metastatic phenotype in rodent mammary tumor cells and in transgenic mouse model systems. mRNA for S100A4 (p9Ka) is present at a generally higher level in breast carcinoma than in benign breast tumor specimens, and the presence of immunocytochemically detected S100A4 correlates strongly with a poor prognosis for breast cancer patients. Recombinant S100A4 (p9Ka) has been reported to interact in vitro with cytoskeletal components and to form oligomers, particularly homodimers in vitro. Using the yeast two-hybrid system, a strong interaction between S100A4 (p9Ka) and another S100 protein, S100A1, was detected. Site-directed mutagenesis of conserved amino acid residues involved in the dimerization of S100 proteins abolished the interactions. The interaction between S100A4 and S100A1 was also observed in vitro using affinity column chromatography and gel overlay techniques. Both S100A1 and S100A4 can occur in the same cultured mammary cells, suggesting that in cells containing both proteins, S100A1 might modulate the metastasis-inducing capability of S100A4. PMID: 10753920 [PubMed - indexed for MEDLINE] 295: J Biol Chem 2001 Mar 30;276(13):9846-54 The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is essential for its E1 activity and E1-E2 complex formation. Komatsu M, Tanida I, Ueno T, Ohsumi M, Ohsumi Y, Kominami E. Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan. Apg7p/Cvt2p, a protein-activating enzyme, is essential for both the Apg12p-Apg5p conjugation system and the Apg8p membrane targeting in autophagy and cytoplasm-to-vacuole targeting in the yeast Saccharomyces cerevisiae. Similar to the ubiquitin-conjugating system, both Apg12p and Apg8p are activated by Apg7p, an E1-like enzyme. Apg12p is then transferred to Apg10p, an E2-like enzyme, and conjugated with Apg5p, whereas Apg8p is transferred to Apg3p, another E2-like enzyme, followed by conjugation with phosphatidylethanolamine. Evidence is presented here that Apg7p forms a homodimer with two active-site cysteine residues via the C-terminal region. The dimerization of Apg7p is independent of the other Apg proteins and facilitated by overexpressed Apg12p. The C-terminal 123 amino acids of Apg7p (residues 508 to 630 out of 630 amino acids) are sufficient for its dimerization, where there is neither an ATP binding domain nor an active-site cysteine essential for its E1 activity. The deletion of its carboxyl 40 amino acids (residues 591-630 out of 630 amino acids) results in several defects of not only Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an active-site cysteine. Furthermore, the carboxyl 40 amino acids of Apg7p are also essential for the interaction of Apg7p with Apg3p to form the E1-E2 complex for Apg8p. These results suggest that Apg7p forms a homodimer via the C-terminal region and that the C-terminal region is essential for both the activity of the E1 enzyme for Apg12p and Apg8p as well as the formation of an E1-E2 complex for Apg8p. PMID: 11139573 [PubMed - indexed for MEDLINE] 296: Mol Cell Biol 2001 Feb;21(3):966-76 Molecular dissection of interactions between Rad51 and members of the recombination-repair group. Krejci L, Damborsky J, Thomsen B, Duno M, Bendixen C. Department of Analysis of Biologically Important Molecular Complexes, Masaryk University, 612 65 Brno, Czech Republic. Recombination is important for the repair of DNA damage and for chromosome segregation during meiosis; it has also been shown to participate in the regulation of cell proliferation. In the yeast Saccharomyces cerevisiae, recombination requires products of the RAD52 epistasis group. The Rad51 protein associates with the Rad51, Rad52, Rad54, and Rad55 proteins to form a dynamic complex. We describe a new strategy to screen for mutations which cause specific disruption of the interaction between certain proteins in the complex, leaving other interactions intact. This approach defines distinct protein interaction domains and protein relationships within the Rad51 complex. Alignment of the mutations onto the constructed three-dimensional model of the Rad51 protein reveal possible partially overlapping interfaces for the Rad51-Rad52 and the Rad51-Rad54 interactions. Rad51-Rad55 and Rad51-Rad51 interactions are affected by the same spectrum of mutations, indicating similarity between the two modes of binding. Finally, the detection of a subset of mutations within Rad51 which disrupt the interaction with mutant Rad52 protein but activate the interaction with Rad54 suggests that dynamic changes within the Rad51 protein may contribute to an ordered reaction process. PMID: 11154282 [PubMed - indexed for MEDLINE] 297: J Cell Biol 2001 Jan 8;152(1):197-212 Mitotic spindle integrity and kinetochore function linked by the Duo1p/Dam1p complex. Cheeseman IM, Enquist-Newman M, Muller-Reichert T, Drubin DG, Barnes G. Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA. Duo1p and Dam1p were previously identified as spindle proteins in the budding yeast, Saccharomyces cerevisiae. Here, analyses of a diverse collection of duo1 and dam1 alleles were used to develop a deeper understanding of the functions and interactions of Duo1p and Dam1p. Based on the similarity of mutant phenotypes, genetic interactions between duo1 and dam1 alleles, interdependent localization to the mitotic spindle, and Duo1p/Dam1p coimmunoprecipitation from yeast protein extracts, these analyses indicated that Duo1p and Dam1p perform a shared function in vivo as components of a protein complex. Duo1p and Dam1p are not required to assemble bipolar spindles, but they are required to maintain metaphase and anaphase spindle integrity. Immunofluorescence and electron microscopy of duo1 and dam1 mutant spindles revealed a diverse variety of spindle defects. Our results also indicate a second, previously unidentified, role for the Duo1p/Dam1p complex. duo1 and dam1 mutants show high rates of chromosome missegregation, premature anaphase events while arrested in metaphase, and genetic interactions with a subset of kinetochore components consistent with a role in kinetochore function. In addition, Duo1p and Dam1p localize to kinetochores in chromosome spreads, suggesting that this complex may serve as a link between the kinetochore and the mitotic spindle. PMID: 11149931 [PubMed - indexed for MEDLINE] 298: J Cell Biol 2001 Jan 8;152(1):51-64 Membrane recruitment of Aut7p in the autophagy and cytoplasm to vacuole targeting pathways requires Aut1p, Aut2p, and the autophagy conjugation complex. Kim J, Huang WP, Klionsky DJ. Department of Biology, University of Michigan, Ann Arbor, Michigan 48109, USA. Autophagy is a degradative pathway by which cells sequester nonessential, bulk cytosol into double-membrane vesicles (autophagosomes) and deliver them to the vacuole for recycling. Using this strategy, eukaryotic cells survive periods of nutritional starvation. Under nutrient-rich conditions, autophagy machinery is required for the delivery of a resident vacuolar hydrolase, aminopeptidase I, by the cytoplasm to vacuole targeting (Cvt) pathway. In both pathways, the vesicle formation process requires the function of the starvation-induced Aut7 protein, which is recruited from the cytosol to the forming Cvt vesicles and autophagosomes. The membrane binding of Aut7p represents an early step in vesicle formation. In this study, we identify several requirements for Aut7p membrane association. After synthesis in the cytosol, Aut7p is proteolytically cleaved in an Aut2p-dependent manner. While this novel processing event is essential for Aut7p membrane binding, Aut7p must undergo additional physical interactions with Aut1p and the autophagy (Apg) conjugation complex before recruitment to the membrane. Lack of these interactions results in a cytosolic distribution of Aut7p rather than localization to forming Cvt vesicles and autophagosomes. This study assigns a functional role for the Apg conjugation system as a mediator of Aut7p membrane recruitment. Further, we demonstrate that Aut1p, which physically interacts with components of the Apg conjugation complex and Aut7p, constitutes an additional factor required for Aut7p membrane recruitment. These findings define a series of steps that results in the modification of Aut7p and its subsequent binding to the sequestering transport vesicles of the autophagy and cytoplasm to vacuole targeting pathways. PMID: 11149920 [PubMed - indexed for MEDLINE] 299: Biochemistry 2001 Jan 16;40(2):422-8 Interactions between yeast iso-1-cytochrome c and its peroxidase. Pielak GJ, Wang X. Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA. gary_pielak@unc.edu Isothermal titration calorimetry was used to study the formation of 19 complexes involving yeast iso-1-ferricytochrome c (Cc) and ferricytochrome c peroxidase (CcP). The complexes comprised combinations of the wild-type proteins, six CcP variants, and three Cc variants. Sixteen protein combinations were designed to probe the crystallographically defined interface between Cc and CcP. The data show that the high-affinity sites on Cc and CcP coincide with the crystallographically defined sites. Changing charged residues to alanine increases the enthalpy of complex formation by a constant amount, but the decrease in stability depends on the location of the amino acid substitution. Deleting methyl groups has a small effect on the binding enthalpy and a larger deleterious effect on the binding free energy, consistent with model studies of the hydrophobic effect, and showing that nonpolar interactions also stabilize the complex. Double-mutant cycles were used to determine the coupling energies for nine Cc-CcP residue pairs. Comparing these energies to the crystal structure of the complex leads to the conclusion that many of the substitutions induce a rearrangement of the complex. PMID: 11148036 [PubMed - indexed for MEDLINE] 300: RNA 2000 Dec;6(12):1882-94 The bI4 group I intron binds directly to both its protein splicing partners, a tRNA synthetase and maturase, to facilitate RNA splicing activity. Rho SB, Martinis SA. Department of Biology and Biochemistry, University of Houston, Texas 77204-5513, USA. The imported mitochondrial leucyl-tRNA synthetase (NAM2p) and a mitochondrial-expressed intron-encoded maturase protein are required for splicing the fourth intron (bI4) of the yeast cob gene, which expresses an electron transfer protein that is essential to respiration. However, the role of the tRNA synthetase, as well as the function of the bI4 maturase, remain unclear. As a first step towards elucidating the mechanistic role of these protein splicing factors in this group I intron splicing reaction, we tested the hypothesis that both leucyl-tRNA synthetase and bI4 maturase interact directly with the bI4 intron. We developed a yeast three-hybrid system and determined that both the tRNA synthetase and bI4 maturase can bind directly and independently via RNA-protein interactions to the large bI4 group I intron. We also showed, using modified two-hybrid and three-hybrid assays, that the bI4 intron bridges interactions between the two protein splicing partners. In the presence of either the bI4 maturase or the Leu-tRNA synthetase, bI4 intron transcribed recombinantly with flanking exons in the yeast nucleus exhibited splicing activity. These data combined with previous genetic results are consistent with a novel model for a ternary splicing complex (two protein: one RNA) in which both protein splicing partners bind directly to the bI4 intron and facilitate its self-splicing activity. PMID: 11142386 [PubMed - indexed for MEDLINE] 301: Nucleic Acids Res 2001 Jan 15;29(2):536-44 Cnr interferes with dimerization of the replication protein alpha in phage-plasmid P4. Tocchetti A, Serina S, Oliva I, Deho G, Ghisotti D. Dipartimento di Genetica e di Biologia dei Microrganismi, Universita di Milano, Via Celoria 26, 20133 Milano, Italy. DNA replication of phage-plasmid P4 in its host Escherichia coli depends on its replication protein alpha. In the plasmid state, P4 copy number is controlled by the regulator protein Cnr (copy number regulation). Mutations in alpha (alpha(cr)) that prevent regulation by Cnr cause P4 over-replication and cell death. Using the two-hybrid system in Saccharomyces cerevisiae and a system based on lambda immunity in E.coli for in vivo detection of protein-protein interactions, we found that (i) alpha protein interacts with Cnr, whereas alpha(cr) proteins do not; (ii) both alpha-alpha and alpha(cr)-alpha(cr) interactions occur and the interaction domain is located within the C-terminal of alpha; (iii) Cnr-Cnr interaction also occurs. Using an in vivo competition assay, we found that Cnr interferes with both alpha-alpha and alpha(cr)-alpha(cr) dimerization. Our data suggest that Cnr and alpha interact in at least two ways, which may have different functional roles in P4 replication control. PMID: 11139624 [PubMed - indexed for MEDLINE] 302: Genetics 2001 Jan;157(1):91-101 WW domains of Rsp5p define different functions: determination of roles in fluid phase and uracil permease endocytosis in Saccharomyces cerevisiae. Gajewska B, Kaminska J, Jesionowska A, Martin NC, Hopper AK, Zoladek T. Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland. Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions. To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30 degrees. Thus, individual WW domains are not essential. Each rsp5-w mutation caused temperature-sensitive growth. Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is important for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis. Thus, various WW domains play different roles in the endocytosis of these two substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle. Altering WW domains does not change the location of Rsp5p. PMID: 11139494 [PubMed - indexed for MEDLINE] 303: Exp Cell Res 2001 Jan 15;262(2):75-83 The human histone deacetylase family. Gray SG, Ekstrom TJ. Laboratory for Molecular Development and Tumor Biology, Centre for Molecular Medicine (CMM), Stockholm, S-171 76, Sweden. Steven.Gray@vai.org Since the identification of the first histone deacetylase (Taunton et al., Science 272, 408-411), several new members have been isolated. They can loosely be separated into entities on the basis of their similarity to various yeast histone deacetylases. The first class is represented by its closeness to the yeast Rpd3-like proteins, and the second most recently discovered class has similarities to yeast Hda1-like proteins. However, due to the fact that several different research groups isolated the Hda1-like histone deacetylases independently, there have been various different nomenclatures used to describe the various members, which can lead to confusion in the interpretation of this family's functions and interactions. With the discovery of another novel murine histone deacetylase, homologous to yeast Sir2, the number of members of this family is set to increase, as 7 human homologues of this gene have been isolated. In the light of these recent discoveries, we have examined the literature data and conducted a database analysis of the isolated histone deacetylases and potential candidates. The results obtained suggest that the number of histone deacetylases within the human genome may be as high as 17 and are discussed in relation to their homology to the yeast histone deacetylases. Copyright 2001 Academic Press. Publication Types: Review Review, Tutorial PMID: 11139331 [PubMed - indexed for MEDLINE] 304: J Biol Chem 2001 Mar 23;276(12):8848-55 Mapping the DNA topoisomerase III binding domain of the Sgs1 DNA helicase. Fricke WM, Kaliraman V, Brill SJ. Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08855, USA. brill@mbcl.rutgers.edu Several members of the RecQ family of DNA helicases are known to interact with DNA topoisomerase III (Top3). Here we show that the Saccharomyces cerevisiae Sgs1 and Top3 proteins physically interact in cell extracts and bind directly in vitro. Sgs1 and Top3 proteins coimmunoprecipitate from cell extracts under stringent conditions, indicating that Sgs1 and Top3 are present in a stable complex. The domain of Sgs1 which interacts with Top3 was identified by expressing Sgs1 truncations in yeast. The results indicate that the NH(2)-terminal 158 amino acids of Sgs1 are sufficient for the high affinity interaction between Sgs1 and Top3. In vitro assays using purified Top3 and NH(2)-terminal Sgs1 fragments demonstrate that at least part of the interaction is through direct protein-protein interactions with these 158 amino acids. Consistent with these physical data, we find that mutant phenotypes caused by a point mutation or small deletions in the Sgs1 NH(2) terminus can be suppressed by Top3 overexpression. We conclude that Sgs1 and Top3 form a tight complex in vivo and that the first 158 amino acids of Sgs1 are necessary and sufficient for this interaction. Thus, a primary role of the Sgs1 amino terminus is to mediate the Top3 interaction. PMID: 11124263 [PubMed - indexed for MEDLINE] 305: Mol Gen Genet 2000 Nov;264(4):378-91 Allele-specific interactions between the yeast RFC1 and RFC5 genes suggest a basis for RFC subunit-subunit interactions. Beckwith W, McAlear MA. Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459-0175, USA. Replication factor C (RFC) is an essential, multi-subunit ATPase that functions in DNA replication, DNA repair, and DNA metabolism-related checkpoints. In order to investigate how the individual RFC subunits contribute to these functions in vivo, we undertook a genetic analysis of RFC genes from budding yeast. We isolated and characterized mutations in the RFC5 gene that could suppress the cold-sensitive phenotype of rfc1-1 mutants. Analysis of the RFC5 suppressors revealed that they could not suppress the elongated telomere phenotype, the sensitivity to DNA damaging agents, or the mutator phenotype of rfc1-1 mutants. Unlike the checkpoint-defective rfc5-1 mutation, the RFC5 suppressor mutations did not interfere with the methylmethane sulfonate- or hydroxyurea-induced phosphorylation of Rad53p. The Rfc5p suppressor substitutions mapped to amino acid positions in the conserved RFC box motifs IV-VII. Comparisons of the structures of related RFC box-containing proteins suggest that these RFC motifs may function to coordinate interactions between neighboring subunits of multi-subunit ATPases. PMID: 11129041 [PubMed - indexed for MEDLINE] 306: J Mol Biol 2001 Jan 12;305(2):219-30 Isolation of mutations that disrupt cooperative DNA binding by the Drosophila bicoid protein. Burz DS, Hanes SD. Molecular Genetics Program Wadsworth Center, New York State Department of Health, USA. Cooperative DNA binding is thought to contribute to the ability of the Drosophila melanogaster protein, Bicoid, to stimulate transcription of target genes in precise sub-domains within the embryo. As a first step toward testing this idea, we devised a genetic screen to isolate mutations in Bicoid that specifically disrupt cooperative interactions, but do not disrupt DNA recognition or transcription activation. The screen was carried out in Saccharomyces cerevisiae and 12 cooperativity mutants were identified. The mutations map across most of the Bicoid protein, with some located within the DNA-binding domain (homeodomain). Four homeodomain mutants were characterized in yeast and shown to activate a single-site reporter gene to levels comparable to that of wild-type, indicating that DNA binding per se is not affected. However, these mutants failed to show cooperative coupling between high and low-affinity sites, and showed reduced activation of a reporter gene carrying a natural Drosophila enhancer. Homology modeling indicated that none of the four mutations is in residues that contact DNA. Instead, these residues are likely to interact with other DNA-bound Bicoid monomers or other parts of the Bicoid protein. In vitro, the isolated homeodomains did not show strong cooperativity defects, supporting the idea that other regions of Bicoid are also important for cooperativity. This study describes the first systematic screen to identify cooperativity mutations in a eukaryotic DNA-binding protein. Copyright 2001 Academic Press. PMID: 11124901 [PubMed - indexed for MEDLINE] 307: J Biol Chem 2001 Apr 20;276(16):13034-8 A mutational epitope for cytochrome C binding to the apoptosis protease activation factor-1. Yu T, Wang X, Purring-Koch C, Wei Y, McLendon GL. Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA. Cytochrome c (Cc) binding to apoptosis protease activation factor-1 (Apaf-1) is a critical activation step in the execution phase of apoptosis. Here we report studies that help define the Cc:Apaf-1 binding surface. It is shown that a large number of Cc residues, including residues 7, 25, 39, 62-65, and 72, are involved in the Cc:Apaf-1 interaction. Mutation of residue 72 eliminated Cc activity whereas mutations of residues 7, 25, 39, and 62-65 showed reduced activity in an additive fashion. The implications of this binding model for both recognition and modulation of protein-protein interactions are briefly discussed. PMID: 11112785 [PubMed - indexed for MEDLINE] 308: J Mol Biol 2000 Dec 15;304(5):941-51 Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9. Liao H, Yuan C, Su MI, Yongkiettrakul S, Qin D, Li H, Byeon IJ, Pei D, Tsai MD. Departments of Chemistry and Biochemistry, The Ohio State Biochemistry Program, and Campus Chemical Instrument Center, The Ohio State University, Columbus, OH 43210, USA. Forkhead-associated (FHA) domains have been shown to recognize both pThr and pTyr-peptides. The solution structures of the FHA2 domain of Rad53 from Saccharomyces cerevisiae, and its complex with a pTyr peptide, have been reported recently. We now report the solution structure of the other FHA domain of Rad53, FHA1 (residues 14-164), and identification of binding sites of FHA1 and its target protein Rad9. The FHA1 structure consists of 11 beta-strands, which form two large twisted anti-parallel beta-sheets folding into a beta-sandwich. Three short alpha-helices were also identified. The beta-strands are linked by several loops and turns. These structural features of free FHA1 are similar to those of free FHA2, but there are significant differences in the loops. Screening of a peptide library [XXX(pT)XXX] against FHA1 revealed an absolute requirement for Asp at the +3 position and a preference for Ala at the +2 position. These two criteria are met by a pThr motif (192)TEAD(195) in Rad9. Surface plasmon resonance analysis showed that a pThr peptide containing this motif, (188)SLEV(pT)EADATFVQ(200) from Rad9, binds to FHA1 with a K(d) value of 0.36 microM. Other peptides containing pTXXD sequences also bound to FHA1, but less tightly (K(d)=4-70 microM). These results suggest that Thr192 of Rad9 is the likely phosphorylation site recognized by the FHA1 domain of Rad53. The tight-binding peptide was then used to identify residues of FHA1 involved in the interaction with the pThr peptide. The results are compared with the interactions between the FHA2 domain and a pTyr peptide derived from Rad9 reported previously. Copyright 2000 Academic Press. PMID: 11124038 [PubMed - indexed for MEDLINE] 309: Curr Opin Microbiol 2000 Dec;3(6):573-81 Pheromone response, mating and cell biology. Elion EA. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. eline_elion@hms.harvard.edu Saccharomyces cerevisiae responds to mating pheromones by activating a receptor-G-protein-coupled mitogen-activated protein kinase (MAPK) cascade that is also used by other signaling pathways. The activation of the MAPK cascade may involve conformational changes through prebound receptor and heterotrimeric G-protein. G beta may then recruit Cdc42-bound MAPKKKK Ste20 to MAPKKK Ste11 through direct interactions with Ste20 and the Ste5 scaffold. Ste20 activates Ste11 by derepressing an autoinhibitory domain. An underlying nuclear shuttling machinery may be required for proper recruitment of Ste5 to G beta. Subsequent polarized growth is mediated by a similar mechanism involving Far1, which binds G beta in addition to Cdc24 and Bem1. Far1 and Cdc24 also undergo nuclear shuttling and the nuclear pool of Far1 may temporally regulate access of Cdc24 to the cell cortex. Publication Types: Review Review, Tutorial PMID: 11121776 [PubMed - indexed for MEDLINE] 310: Biochim Biophys Acta 2000 Dec 11;1499(1-2):85-100 Mutations in SPC110, encoding the yeast spindle pole body calmodulin-binding protein, cause defects in cell integrity as well as spindle formation. Stirling DA, Stark MJ. Department of Biochemistry, University of Dundee, MSI/WTB Complex, DD1 5EH, Dundee, UK. d.a.stirling@dundee.ac.uk The 110 kDa spindle pole body component, Spc110p, is an essential target of calmodulin in budding yeast. Cells with mutations which reduce calmodulin binding to Spc110p are unable to form a mitotic spindle and die. Here we show that these effects can be overcome either directly by increasing extracellular calcium or calmodulin expression, which reverse the primary spindle defect, or indirectly through increased extracellular osmolarity or high dosage of MID2 or SLG1/HCS77/WSC1 which preserve viability. We propose that overcoming a cell integrity defect associated with the mitotic arrest enables the defective spindle pole bodies to provide sufficient function for proliferation of a large proportion of mutant cells. Our findings demonstrate a role for calcium in the Spc110p-calmodulin interaction in vivo and have important general implications for the interpretation of genetic interactions involving cell integrity genes. PMID: 11118641 [PubMed - indexed for MEDLINE] 311: Biochim Biophys Acta 2000 Dec 11;1499(1-2):63-73 Oligomerization properties of the acidic ribosomal P-proteins from Saccharomyces cerevisiae: effect of P1A protein phosphorylation on the formation of the P1A-P2B hetero-complex. Tchorzewski M, Boguszewska A, Dukowski P, Grankowski N. Maria Curie-Skllodowska University, Institute of Microbiology and Biotechnology, Department of Molecular Biology, Akademicka Street 19, 20-033, Lublin, Poland. Acidic ribosomal P-proteins form, in all eukaryotic cells, a lateral protuberance, the so-called 'stalk', which is directly involved in translational activity of the ribosomes. In Saccharomyces cerevisiae cells, there are four distinct P-proteins: P1A, P1B, P2A and P2B. In spite of the high level of their structural homology, they are not completely equivalent and may perform different functions. As yet, the protein-protein interactions between yeast P-proteins have not been fully defined. In this paper, the interplay between yeast P-proteins has been investigated by means of a two-hybrid system, chemical cross-linking and gel filtration. The data presented herein show that all P-proteins are able to form homo-oligomeric complexes. By analyzing hetero-interactions, we were able to detect strong interactions between P1A and P2B proteins. Additionally, the pair of P1B and P2A proteins is also able to form a hetero-complex, though at a very low efficiency. All P-proteins are phosphorylated by numerous protein kinases. Using the multifunctional protein kinase CK II, we have shown that incorporation of phosphate into P1A protein can exert its effect on the hetero-oligomerization process, namely by preventing the formation of the hetero-oligomer P1A-P/P2B. These findings are the first to show differences in the oligomerization behavior of the yeast P-proteins; moreover, they emphasize a significant impact of the phosphorylation on the formations of P-protein complex. PMID: 11118639 [PubMed - indexed for MEDLINE] 312: J Biol Chem 2001 Mar 16;276(11):8616-22 Nam1p, a protein involved in RNA processing and translation, is coupled to transcription through an interaction with yeast mitochondrial RNA polymerase. Rodeheffer MS, Boone BE, Bryan AC, Shadel GS. Department of Biochemistry, Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia 30322, USA. Alignment of three fungal mtRNA polymerases revealed conserved amino acid sequences in an amino-terminal region of the Saccharomyces cerevisiae enzyme implicated previously as harboring an important functional domain. Phenotypic analysis of deletion and point mutations, in conjunction with a yeast two-hybrid assay, revealed that Nam1p, a protein involved in RNA processing and translation in mitochondria, binds specifically to this domain. The significance of this interaction in vivo was demonstrated by the fact that the temperature-sensitive phenotype of a deletion mutation (rpo41Delta2), which impinges on this amino-terminal domain, is suppressed by overproducing Nam1p. In addition, mutations in the amino-terminal domain result specifically in decreased steady-state levels of mature mitochondrial CYTB and COXI transcripts, which is a primary defect observed in NAM1 null mutant yeast strains. Finally, one point mutation (R129D) did not abolish Nam1p binding, yet displayed an obvious COX1/CYTB transcript defect. This mutation exhibited the most severe mitochondrial phenotype, suggesting that mutations in the amino-terminal domain can perturb other critical interactions, in addition to Nam1p binding, that contribute to the observed phenotypes. These results implicate the amino-terminal domain of mtRNA polymerases in coupling additional factors and activities involved in mitochondrial gene expression directly to the transcription machinery. PMID: 11118450 [PubMed - indexed for MEDLINE] 313: Plant Physiol 2000 Dec;124(4):1844-53 Interaction specificity of Arabidopsis calcineurin B-like calcium sensors and their target kinases. Kim KN, Cheong YH, Gupta R, Luan S. Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA. Calcium is a critical component in a number of plant signal transduction pathways. A new family of calcium sensors called calcineurin B-like proteins (AtCBLs) have been recently identified from Arabidopsis. These calcium sensors have been shown to interact with a family of protein kinases (CIPKs). Here we report that each individual member of AtCBL family specifically interacts with a subset of CIPKs and present structural basis for the interaction and for the specificity underlying these interactions. Although the C-terminal region of CIPKs is responsible for interaction with AtCBLs, the N-terminal region of CIPKs is also involved in determining the specificity of such interaction. We have also shown that all three EF-hand motifs in AtCBL members are required for the interaction with CIPKs. Several AtCBL members failed to interact with any of the CIPKs presented in this study, suggesting that these AtCBL members either have other CIPKs as targets or they target distinct proteins other than CIPKs. These results may provide structural basis for the functional specificity of CBL family of calcium sensors and their targets. PMID: 11115898 [PubMed - indexed for MEDLINE] 314: Curr Biol 2000 Nov 30;10(23):1519-22 Yeast Eap1p, an eIF4E-associated protein, has a separate function involving genetic stability. Chial HJ, Stemm-Wolf AJ, McBratney S, Winey M. Present address: Department of Biology, St Olaf College, Northfield, Minnesota 55057-1098, USA. A rate-limiting step during translation initiation in eukaryotic cells involves binding of the initiation factor eIF4E to the 7-methylguanosine-containing cap of mRNAs. Overexpression of eIF4E leads to malignant transformation [1-3], and eIF4E is elevated in many human cancers [4-7]. In mammalian cells, three eIF4E-binding proteins each interact with eIF4E and inhibit its function [8-10]. In yeast, EAP1 encodes a protein that binds eIF4E and inhibits cap-dependent translation in vitro [11]. A point mutation in the canonical eIF4E-binding motif of Eap1p blocks its interaction with eIF4E [11]. Here, we characterized the genetic interactions between EAP1 and NDC1, a gene whose function is required for duplication of the spindle pole body (SPB) [12], the centrosome-equivalent organelle in yeast that functions as the centrosome. We found that the deletion of EAP1 is lethal when combined with the ndc1-1 mutation. Mutations in NDC1 or altered NDC1 gene dosage lead to genetic instability [13,14]. Yeast strains lacking EAP1 also exhibit genetic instability. We tested whether these phenotypes are due to loss of EAP1 function in regulating translation. We found that both the synthetic lethal phenotype and the genetic instability phenotypes are rescued by a mutant allele of EAP1 that is unable to bind eIF4E. Our findings suggest that Eap1p carries out an eIF4E-independent function to maintain genetic stability, most likely involving SPBs. PMID: 11114520 [PubMed - indexed for MEDLINE] 315: Proc Natl Acad Sci U S A 2000 Dec 19;97(26):14400-5 Activation of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14-3-3 to histone deacetylase 5. McKinsey TA, Zhang CL, Olson EN. Department of Molecular Biology, The University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA. Skeletal muscle differentiation is controlled by interactions between myocyte enhancer factor-2 (MEF2) and myogenic basic helix-loop-helix transcription factors. Association of MEF2 with histone deacetylases (HDAC) -4 and -5 results in repression of MEF2 target genes and inhibition of myogenesis. Calcium/calmodulin-dependent protein kinase (CaMK) signaling promotes myogenesis by disrupting MEF2-HDAC complexes and stimulating HDAC nuclear export. To further define the mechanisms that confer CaMK responsiveness to HDAC4 and -5, we performed yeast two-hybrid screens to identify HDAC-interacting factors. These screens revealed interactions between HDAC4 and members of the 14-3-3 family of proteins, which function as signal-dependent intracellular chaperones. HDAC4 binds constitutively to 14-3-3 in yeast and mammalian cells, whereas HDAC5 binding to 14-3-3 is largely dependent on CaMK signaling. CaMK phosphorylates serines -259 and -498 in HDAC5, which subsequently serve as docking sites for 14-3-3. Our studies suggest that 14-3-3 binding to HDAC5 is required for CaMK-dependent disruption of MEF2-HDAC complexes and nuclear export of HDAC5, and implicate 14-3-3 as a signal-dependent regulator of muscle cell differentiation. PMID: 11114197 [PubMed - indexed for MEDLINE] 316: Mol Cell Biol 2001 Jan;21(1):209-23 Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4). Serin G, Gersappe A, Black JD, Aronoff R, Maquat LE. Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. Nonsense-mediated mRNA decay (NMD), also called mRNA surveillance, is an important pathway used by all organisms that have been tested to degrade mRNAs that prematurely terminate translation and, as a consequence, eliminate the production of aberrant proteins that could be potentially harmful. In mammalian cells, NMD appears to involve splicing-dependent alterations to mRNA as well as ribosome-associated components of the translational apparatus. To date, human (h) Upf1 protein (p) (hUpf1p), a group 1 RNA helicase named after its Saccharomyces cerevisiae orthologue that functions in both translation termination and NMD, has been the only factor shown to be required for NMD in mammalian cells. Here, we describe human orthologues to S. cerevisiae Upf2p and S. cerevisiae Upf3p (Caenorhabditis elegans SMG-4) based on limited amino acid similarities. The existence of these orthologues provides evidence for a higher degree of evolutionary conservation of NMD than previously appreciated. Interestingly, human orthologues to S. cerevisiae Upf3p (C. elegans SMG-4) derive from two genes, one of which is X-linked and both of which generate multiple isoforms due to alternative pre-mRNA splicing. We demonstrate using immunoprecipitations of epitope-tagged proteins transiently produced in HeLa cells that hUpf2p interacts with hUpf1p, hUpf3p-X, and hUpf3p, and we define the domains required for the interactions. Furthermore, we find by using indirect immunofluorescence that hUpf1p is detected only in the cytoplasm, hUpf2p is detected primarily in the cytoplasm, and hUpf3p-X localizes primarily to nuclei. The finding that hUpf3p-X is a shuttling protein provides additional indication that NMD has both nuclear and cytoplasmic components. PMID: 11113196 [PubMed - indexed for MEDLINE] 317: Biochemistry 2000 Dec 19;39(50):15462-74 Synthesis and biophysical analysis of transmembrane domains of a Saccharomyces cerevisiae G protein-coupled receptor. Xie H, Ding FX, Schreiber D, Eng G, Liu SF, Arshava B, Arevalo E, Becker JM, Naider F. Department of Chemistry, The College of Staten Island Staten Island, New York 10314, USA. The Ste2p receptor for alpha-factor, a tridecapeptide mating pheromone of the yeast Saccharomyces cerevisiae, belongs to the G protein-coupled family of receptors. In this paper we report on the synthesis of peptides corresponding to five of the seven transmembrane domains (M1-M5) and two homologues of the sixth transmembrane domain corresponding to the wild-type sequence and a mutant sequence found in a constitutively active receptor. The secondary structures of all new transmembrane peptides and previously synthesized peptides corresponding to domains 6 and 7 were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol-water mixtures, sodium dodecyl sulfate micelles, and dimyristoyl phosphatidyl choline bilayers. Tryptophan fluorescence quenching experiments were used to assess the penetration of the membrane peptides into lipid bilayers. All peptides were predominantly (40-80%) helical in trifluoroethanol and most trifluoroethanol-water mixtures. In contrast, two of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVIFTGDNFKKKG) and M6-31 (KQFDSFHILLINleSAQSLLVPSIIFILAYSLK) formed stable beta-sheet structures in both sodium dodecyl sulfate micelles and DMPC bilayers. Polyacrylamide gel electrophoresis showed that these two peptides formed high molecular aggregates in the presence of SDS whereas all other peptides moved as monomeric species. The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS) corresponding to the sequence in the constitutive mutant was predominantly helical under a variety of conditions, whereas the homologous wild-type sequence (KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to form beta-structures. These results demonstrate a connection between a conformational shift in secondary structure, as detected by biophysical techniques, and receptor function. The aggregation of particular transmembrane domains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or multimers. PMID: 11112532 [PubMed - indexed for MEDLINE] 318: Biochim Biophys Acta 2000 Dec 15;1529(1-3):63-88 Sterol methyl transferase: enzymology and inhibition. Nes WD. Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA. u0nes@ttacs.ttu.edu Sterol C-methylations catalyzed by the (S)-adenosyl-L-methionine: Delta(24)-sterol methyl transferase (SMT) have provided the focus for study of electrophilic alkylations, a reaction type of functional importance in C-C bond formation of natural products. SMTs occur generally in nature, but do not occur in animal systems, suggesting that the difference in sterol synthetic pathways can be exploited therapeutically and in insect-plant interactions. The SMT genes from several plants and fungi have been cloned, sequenced and expressed in bacteria or yeast and bioengineered into tobacco or tomato plants. These enzymes share significant amino acid sequence similarity in the putative sterol and AdoMet binding sites. Investigations of the molecular recognition of sterol fitness and studies with stereospecifically labeled substrates as well as various sterol analogs assayed with native or mutant SMTs from fungi and plants have been carried out recently in our own and other laboratories. These analyses have led to an active-site model, referred to as the 'steric-electric plug' model, which is consistent with a non-covalent mechanism involving the intermediacy of a 24beta-methyl (or ethyl) sterol bound to the ternary complex. Despite the seeming differences between fungal and plant SMT activities the recent data indicate that a distinct SMT or family of SMTs exist in these organisms which bind and transform sterols according to a similar mechanistic plan. Vascular plants have been found to express different complements of C(1)/C(2)-activities in the form of at least three SMT isoforms. This enzyme multiplicity can be a target of regulatory control to affect phytosterol homeostasis in transgenic plants. The state of our current understanding of SMT enzymology and inhibition is presented. Publication Types: Review Review, Tutorial PMID: 11111078 [PubMed - indexed for MEDLINE] 319: J Comput Biol 2000;7(3-4):601-20 Using Bayesian networks to analyze expression data. Friedman N, Linial M, Nachman I, Pe'er D. School of Computer Science and Engineering, Hebrew University, Jerusalem, Israel. nir@cs.huji.ac.il DNA hybridization arrays simultaneously measure the expression level for thousands of genes. These measurements provide a "snapshot" of transcription levels within the cell. A major challenge in computational biology is to uncover, from such measurements, gene/protein interactions and key biological features of cellular systems. In this paper, we propose a new framework for discovering interactions between genes based on multiple expression measurements. This framework builds on the use of Bayesian networks for representing statistical dependencies. A Bayesian network is a graph-based model of joint multivariate probability distributions that captures properties of conditional independence between variables. Such models are attractive for their ability to describe complex stochastic processes and because they provide a clear methodology for learning from (noisy) observations. We start by showing how Bayesian networks can describe interactions between genes. We then describe a method for recovering gene interactions from microarray data using tools for learning Bayesian networks. Finally, we demonstrate this method on the S. cerevisiae cell-cycle measurements of Spellman et al. (1998). PMID: 11108481 [PubMed - indexed for MEDLINE] 320: Mol Cell 2000 Nov;6(5):1169-82 The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms. Durocher D, Taylor IA, Sarbassova D, Haire LF, Westcott SL, Jackson SP, Smerdon SJ, Yaffe MB. Wellcome Trust/Cancer Research Campaign Institute of Cancer and Developmental Biology and Department of Zoology University of Cambridge CB2 1QR, Cambridge, United Kingdom. Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, including those within a number of DNA damage checkpoint kinases, and determined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide, at 1.6 A resolution. The structure reveals a striking similarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain complexes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes. PMID: 11106755 [PubMed - indexed for MEDLINE] 321: Genetics 2000 Dec;156(4):1503-17 Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae. Ben-Yehuda S, Dix I, Russell CS, McGarvey M, Beggs JD, Kupiec M. Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel. The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression. PMID: 11102353 [PubMed - indexed for MEDLINE] 322: Nat Struct Biol 2000 Dec;7(12):1156-64 Crystal structures of ribosome anti-association factor IF6. Groft CM, Beckmann R, Sali A, Burley SK. Laboratories of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA. Ribosome anti-association factor eIF6 (originally named according to translation initiation terminology as eukaryotic initiation factor 6) binds to the large ribosomal subunit, thereby preventing inappropriate interactions with the small subunit during initiation of protein synthesis. We have determined the X-ray structures of two IF6 homologs, Methanococcus jannaschii archaeal aIF6 and Sacchromyces cerevisiae eIF6, revealing a phylogenetically conserved 25 kDa protein consisting of five quasi identical alpha/beta subdomains arrayed about a five-fold axis of pseudosymmetry. Yeast eIF6 prevents ribosomal subunit association. Comparative protein structure modeling with other known archaeal and eukaryotic homologs demonstrated the presence of two conserved surface regions, one or both of which may bind the large ribosomal subunit. PMID: 11101899 [PubMed - indexed for MEDLINE] 323: Nat Biotechnol 2000 Dec;18(12):1257-61 Comment in: Nat Biotechnol. 2000 Dec;18(12):1242-3. A network of protein-protein interactions in yeast. Schwikowski B, Uetz P, Fields S. The Institute for Systems Biology, 4225 Roosevelt Way NE, Suite 200, Seattle, WA 98105, USA. A global analysis of 2,709 published interactions between proteins of the yeast Saccharomyces cerevisiae has been performed, enabling the establishment of a single large network of 2,358 interactions among 1,548 proteins. Proteins of known function and cellular location tend to cluster together, with 63% of the interactions occurring between proteins with a common functional assignment and 76% occurring between proteins found in the same subcellular compartment. Possible functions can be assigned to a protein based on the known functions of its interacting partners. This approach correctly predicts a functional category for 72% of the 1,393 characterized proteins with at least one partner of known function, and has been applied to predict functions for 364 previously uncharacterized proteins. PMID: 11101803 [PubMed - indexed for MEDLINE] 324: Proc Natl Acad Sci U S A 2000 Dec 5;97(25):13732-7 A new screen for protein interactions reveals that the Saccharomyces cerevisiae high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter. Laser H, Bongards C, Schuller J, Heck S, Johnsson N, Lehming N. Max-Delbruck-Laboratorium in der Max-Planck-Gesellschaft, Carl-von-Linne-Weg 10, 50829 Cologne, Germany. The split-ubiquitin assay detects protein interactions in vivo. To identify proteins interacting with Gal4p and Tup1p, two transcriptional regulators, we converted the split-ubiquitin assay into a generally applicable screen for binding partners of specific proteins in vivo. A library of genomic Saccharomyces cerevisiae DNA fragments fused to the N-terminal half of ubiquitin was constructed and transformed into yeast strains carrying either Gal4p or Tup1p as a bait. Both proteins were C-terminally extended by the C-terminal half of ubiquitin followed by a modified Ura3p with an arginine in position 1, a destabilizing residue in the N-end rule pathway. The bait fusion protein alone is stable and enzymatically active. However, upon interaction with its prey, a native-like ubiquitin is reconstituted. RUra3p is then cleaved off by the ubiquitin-specific proteases and rapidly degraded by the N-end rule pathway. In both screens, Nhp6B was identified as a protein in close proximity to Gal4p as well as to Tup1p. Direct interaction between either protein and Nhp6B was confirmed by coprecipitation assays. Genetic analysis revealed that Nhp6B, a member of the HMG1 family of DNA-binding proteins, can influence transcriptional activation as well as repression at a specific locus in the chromosome of the yeast S. cerevisiae. PMID: 11095729 [PubMed - indexed for MEDLINE] 325: Nucleic Acids Res 2000 Dec 1;28(23):4665-73 Signaling through regulated transcription factor interaction: mapping of a regulatory interaction domain in the Myb-related Bas1p. Pinson B, Kongsrud TL, Ording E, Johansen L, Daignan-Fornier B, Gabrielsen OS. Department of Biochemistry, University of Oslo, PO Box 1041, Blindern, N-0316 Oslo 3, Norway. Gene activation in eukaryotes is inherently combinatorial depending on cooperation between different transcription factors. An example where this cooperation seems to be directly exploited for regulation is the Bas1p/Bas2p couple in yeast. Bas1p is a Myb-related transcription factor that acts together with the homeodomain-related Bas2p (Pho2p) to regulate purine and histidine biosynthesis genes in response to extracellular purine limitation. We show that fusion of the two factors abolished adenine repression, suggesting that what is regulated by adenine is the Bas1p-Bas2p interaction. Analysis of Bas1p deletions revealed a critical domain (Bas1p interaction and regulatory domain, BIRD) acting in two-hybrid assays as an adenine-dependent Bas1p-Bas2p interaction domain. BIRD had a dual function, as an internal repressor of a centrally located Bas1p transactivation domain on the ADE1 promoter and as a Bas2p-dependent activator on the HIS4 promoter. This promoter-dependent behavior reflected a differential binding to the two promoters in vivo. On ADE1 Bas1p bound the promoter efficiently by itself, but required adenine limitation and Bas2p interaction through BIRD for derepression. On HIS4 efficient promoter binding and derepression required both factors and adenine limitation. We propose a promoter-dependent model for adenine regulation in yeast based on controlled Bas1p-Bas2p interactions through BIRD and exploited differentially by the two promoters. PMID: 11095676 [PubMed - indexed for MEDLINE] 326: Proc Natl Acad Sci U S A 2000 Nov 21;97(24):13203-8 Comment in: Proc Natl Acad Sci U S A. 2000 Nov 21;97(24):12935-6. A computationally directed screen identifying interacting coiled coils from Saccharomyces cerevisiae. Newman JR, Wolf E, Kim PS. Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142, USA. Computational methods can frequently identify protein-interaction motifs in otherwise uncharacterized open reading frames. However, the identification of candidate ligands for these motifs (e.g., so that partnering can be determined experimentally in a directed manner) is often beyond the scope of current computational capabilities. One exception is provided by the coiled-coil interaction motif, which consists of two or more alpha helices that wrap around each other: the ligands for coiled-coil sequences are generally other coiled-coil sequences, thereby greatly simplifying the motif/ligand recognition problem. Here, we describe a two-step approach to identifying protein-protein interactions mediated by two-stranded coiled coils that occur in Saccharomyces cerevisiae. Coiled coils from the yeast genome are first predicted computationally, by using the multicoil program, and associations between coiled coils are then determined experimentally by using the yeast two-hybrid assay. We report 213 unique interactions between 162 putative coiled-coil sequences. We evaluate the resulting interactions, focusing on associations identified between components of the spindle pole body (the yeast centrosome). PMID: 11087867 [PubMed - indexed for MEDLINE] 327: Proc Natl Acad Sci U S A 2000 Nov 21;97(24):13080-5 Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase. Caruthers JM, Johnson ER, McKay DB. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA. The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 A resolution; it has a parallel alpha-beta topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related helicases. Using data to 2.8 A resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a "dumbbell" structure consisting of two compact domains connected by an extended linker. By using the structures of other helicases as a template, compact structures can be modeled for eIF4A that suggest (i) helicase motif IV binds RNA; (ii) Arg-298, which is conserved in the DEA(D/H)-box RNA helicase family but is absent from many other helicases, also binds RNA; and (iii) motifs V and VI "link" the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding. PMID: 11087862 [PubMed - indexed for MEDLINE] 328: Biochemistry 2000 Nov 21;39(46):14103-12 Biochemical and structural analysis of the interaction between the UBA(2) domain of the DNA repair protein HHR23A and HIV-1 Vpr. Withers-Ward ES, Mueller TD, Chen IS, Feigon J. Department of Microbiology, University of California at Los Angeles, Los Angeles, California 90095, USA. The DNA repair protein HHR23A is a highly conserved protein that functions in nucleotide excision repair. HHR23A contains two ubiquitin associated domains (UBA) that are conserved in a number of proteins with diverse functions involved in ubiquitination, UV excision repair, and signaling pathways via protein kinases. The cellular binding partners of UBA domains remain unclear; however, we previously found that the HHR23A UBA(2) domain interacts specifically with the HIV-1 Vpr protein. Analysis of the low resolution solution structure of HHR23A UBA(2) revealed a hydrophobic loop region of the UBA(2) domain that we predicted was the interface for protein/protein interactions. Here we present results of in vitro binding studies that demonstrate the requirement of this hydrophobic loop region for interaction with human immunodeficiency virus (HIV-1) Vpr. A single point mutation of the Pro at residue 333 to a Glu totally abolishes the binding of HIV-1 Vpr to UBA(2). High resolution NMR structures of the binding deficient UBA(2) mutant P333E as well as of the wild-type UBA(2) domain were determined to compare the effect of this mutation on the structure. Small but significant differences are observed only locally at the site of the mutation. The biochemical and structural analysis confirms the function of the HHR23A UBA(2) GFP-loop as the protein/protein interacting domain. PMID: 11087358 [PubMed - indexed for MEDLINE] 329: Curr Biol 2000 Nov 2;10(21):1375-8 The spindle checkpoint of Saccharomyces cerevisiae responds to separable microtubule-dependent events. Daum JR, Gomez-Ospina N, Winey M, Burke DJ. Department of Biochemistry and Molecular Genetics, University of Virginia Medical Center, Charlottesville, Virginia 22908-0733, USA. The spindle checkpoint regulates microtubule-based chromosome segregation and helps to maintain genomic stability [1,2]. Mutational inactivation of spindle checkpoint genes has been implicated in the progression of several types of human cancer. Recent evidence from budding yeast suggests that the spindle checkpoint is complex. Order-of-function experiments have defined two separable pathways within the checkpoint. One pathway, defined by MAD2, controls the metaphase-to-anaphase transition and the other, defined by BUB2, controls the exit from mitosis [3-6]. The relationships between the separate branches of the checkpoint, and especially the events that trigger the pathways, have not been defined. We localized a Bub2p-GFP fusion protein to the cytoplasmic side of the spindle pole body and used a kar9 mutant to show that cells with misoriented spindles are arrested in anaphase of mitosis. We used a kar9 bub2 double mutant to show that the arrest is BUB2 dependent. We conclude that the separate pathways of the spindle checkpoint respond to different classes of microtubules. The MAD2 branch of the pathway responds to kinetochore microtubule interactions and the BUB2 branch of the pathway operates within the cytoplasm, responding to spindle misorientation. PMID: 11084338 [PubMed - indexed for MEDLINE] 330: Anal Chem 2000 Nov 1;72(21):5151-7 A fluorescent indicator for detecting protein-protein interactions in vivo based on protein splicing. Ozawa T, Nogami S, Sato M, Ohya Y, Umezawa Y. Department of Chemistry, School of Science, University of Tokyo, Japan. We describe a new method with general applicability for monitoring any protein-protein interaction in vivo. The principle is based on a protein splicing system, which involves a self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to formation of a new protein in which the exteins are linked directly by a peptide bond. As the exteins, split N- and C-terminal halves of enhanced green fluorescent protein (EGFP) were used. When a single peptide consisting of an intein derived from Saccharomyces cerevisiae intervening the split EGFP was expressed in Escherichia coli, the two external regions of EGFP were ligated, thereby forming the EGFP corresponding fluorophore. Genetic alteration of the intein, which involved large deletion of the central region encoding 104 amino acids, was performed. In the expression of the residual N- and C-terminal intein fragments each fused to the split EGFP exteins, the splicing in trans did not proceed. However, upon coexpression of calmodulin and its target peptide M13, each connected to the N- and C-terminal inteins, fluorescence of EGFP was observed. These results demonstrate that interaction of calmodulin and M13 triggers the refolding of intein, which induces the protein splicing, thereby folding the ligated extein correctly for yielding the EGFP fluorophore. This method opens a new way not only to screen protein-protein interactions but also to visualize the interaction in vivo in transgenic animals. PMID: 11080857 [PubMed - indexed for MEDLINE] 331: EMBO J 2000 Nov 15;19(22):6141-9 The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p. Owen DJ, Ornaghi P, Yang JC, Lowe N, Evans PR, Ballario P, Neuhaus D, Filetici P, Travers AA. MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK and Centro di studio per gli Acidi Nucleici, CNR, c/o Dipartimento di Genetica e Biologia Molecolare, Universita 'La Sapienza', P.le A.Moro 5, 00185 Roma, Italy. The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 A resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N:-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N:-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed. PMID: 11080160 [PubMed - indexed for MEDLINE] 332: Methods Enzymol 2000;328:297-321 Using the yeast three-hybrid system to detect and analyze RNA-protein interactions. Kraemer B, Zhang B, SenGupta D, Fields S, Wickens M. Department of Biochemistry, University of Wisconsin, Madison 53706, USA. PMID: 11075352 [PubMed - indexed for MEDLINE] 333: Methods Enzymol 2000;328:111-27 The yeast tribid system: cDNA expression cloning of protein interactions dependent on posttranslational modifications. Kochan JP, Volpers C, Osborne MA. Department of Metabolic Diseases, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110, USA. PMID: 11075342 [PubMed - indexed for MEDLINE] 334: Methods Enzymol 2000;328:89-103 Yeast three-hybrid system for detecting ligand-receptor interactions. Griffith EC, Licitra EJ, Liu JO. Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA. PMID: 11075340 [PubMed - indexed for MEDLINE] 335: Methods Enzymol 2000;328:47-59 Analysis and identification of protein-protein interactions using protein recruitment systems. Aronheim A, Karin M. Department of Molecular Genetics, Technion Israel Institute of Technology, Haifa, Israel. PMID: 11075337 [PubMed - indexed for MEDLINE] 336: Methods Enzymol 2000;328:3-14 High-throughput screening for protein-protein interactions using two-hybrid assay. Cagney G, Uetz P, Fields S. Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada. PMID: 11075334 [PubMed - indexed for MEDLINE] 337: Mol Cell Biol 2000 Dec;20(23):8944-57 Novel Upf2p orthologues suggest a functional link between translation initiation and nonsense surveillance complexes. Mendell JT, Medghalchi SM, Lake RG, Noensie EN, Dietz HC. Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. Transcripts harboring premature signals for translation termination are recognized and rapidly degraded by eukaryotic cells through a pathway known as nonsense-mediated mRNA decay (NMD). In addition to protecting cells by preventing the translation of potentially deleterious truncated peptides, studies have suggested that NMD plays a broader role in the regulation of the steady-state levels of physiologic transcripts. In Saccharomyces cerevisiae, three trans-acting factors (Upf1p to Upf3p) are required for NMD. Orthologues of Upf1p have been identified in numerous species, showing that the NMD machinery, at least in part, is conserved through evolution. In this study, we demonstrate additional functional conservation of the NMD pathway through the identification of Upf2p homologues in Schizosaccharomyces pombe and humans (rent2). Disruption of S. pombe UPF2 established that this gene is required for NMD in fission yeast. rent2 was demonstrated to interact directly with rent1, a known trans-effector of NMD in mammalian cells. Additionally, fragments of rent2 were shown to possess nuclear targeting activity, although the native protein localizes to the cytoplasmic compartment. Finally, novel functional domains of Upf2p and rent2 with homology to eukaryotic initiation factor 4G (eIF4G) and other translational regulatory proteins were identified. Directed mutations within these so-called eIF4G homology (4GH) domains were sufficient to abolish the function of S. pombe Upf2p. Furthermore, using the two-hybrid system, we obtained evidence for direct interaction between rent2 and human eIF4AI and Sui1, both components of the translation initiation complex. Based on these findings, a novel model in which Upf2p and rent2 effects decreased translation and accelerated decay of nonsense transcripts through competitive interactions with eIF4G-binding partners is proposed. PMID: 11073994 [PubMed - indexed for MEDLINE] 338: Mol Cell Biol 2000 Dec;20(23):8879-88 A specificity and targeting subunit of a human SWI/SNF family-related chromatin-remodeling complex. Nie Z, Xue Y, Yang D, Zhou S, Deroo BJ, Archer TK, Wang W. Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA. The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc from Saccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the beta-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions. PMID: 11073988 [PubMed - indexed for MEDLINE] 339: Mol Cell Biol 2000 Dec;20(23):8709-19 In vivo requirement of activator-specific binding targets of mediator. Park JM, Kim HS, Han SJ, Hwang MS, Lee YC, Kim YJ. Genome Regulation Center, Creative Research Initiative, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea. There has been no unequivocal demonstration that the activator binding targets identified in vitro play a key role in transcriptional activation in vivo. To examine whether activator-Mediator interactions are required for gene transcription under physiological conditions, we performed functional analyses with Mediator components that interact specifically with natural yeast activators. Different activators interact with Mediator via distinct binding targets. Deletion of a distinct activator binding region of Mediator completely compromised gene activation in vivo by some, but not all, transcriptional activators. These demonstrate that the activator-specific targets in Mediator are essential for transcriptional activation in living cells, but their requirement was affected by the nature of the activator-DNA interaction and the existence of a postrecruitment activation process. PMID: 11073972 [PubMed - indexed for MEDLINE] 340: J Bacteriol 2000 Dec;182(23):6638-44 InvB is a type III secretion chaperone specific for SspA. Bronstein PA, Miao EA, Miller SI. Department of Microbiology, University of Washington, Seattle, Washington 98195, USA. A wide variety of gram-negative bacteria utilize a specialized apparatus called the type III secretion system (TTSS) to translocate virulence factors directly into the cytoplasm of eukaryotic cells. These translocated effectors contribute to the pathogen's ability to infect and replicate within plant and animal hosts. The amino terminus of effector proteins contains sequences that are necessary and sufficient for both secretion and translocation by TTSS. Portions of these sequences contain binding sites for type III chaperones, which facilitate efficient secretion and translocation of specific effectors through TTSS. In this study, we have utilized the yeast two-hybrid assay to identify protein-protein interactions between effector and chaperone proteins encoded within Salmonella pathogenicity island 1 (SPI-1). Several interactions were identified including a novel interaction between the effector protein, SspA (SipA), and a putative chaperone, InvB. InvB was demonstrated to bind to the amino terminus of SspA in the bacterial cytoplasm. Furthermore, InvB acts as a type III chaperone for the efficient secretion and translocation of SspA by SPI-1. InvB also permitted translocation of SspA through the SPI-2 TTSS, indicating that it is an important regulator in the recognition of SspA as a target of TTSS. Finally, it was determined that InvB does not alter the transcription of sspA but that its absence results in reduced SspA protein levels in Salmonella enterica serovar Typhimurium. PMID: 11073906 [PubMed - indexed for MEDLINE] 341: Nucleic Acids Res 2000 Nov 15;28(22):4523-30 Predicting regulons and their cis-regulatory motifs by comparative genomics. Manson McGuire A, Church GM. Department of Genetics, Warren Alpert Building, Room 513, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. We have combined and compared three techniques for predicting functional interactions based on comparative genomics (methods based on conserved operons, protein fusions and correlated evolution) and optimized these methods to predict coregulated sets of genes in 24 complete genomes, including Saccharomyces cerevisiae, Caenorhabditis elegans and 22 prokaryotes. The method based on conserved operons was the most useful for this purpose. Upstream regions of the genes comprising these predicted regulons were then used to search for regulatory motifs in 22 prokaryotic genomes using the motif-discovery program AlignACE. Many significant upstream motifs, including five known Escherichia coli regulatory motifs, were identified in this manner. The presence of a significant regulatory motif was used to refine the members of the predicted regulons to generate a final set of predicted regulons that share significant regulatory elements. PMID: 11071941 [PubMed - indexed for MEDLINE] 342: Nucleic Acids Res 2000 Nov 15;28(22):4460-6 A network of yeast basic helix-loop-helix interactions. Robinson KA, Koepke JI, Kharodawala M, Lopes JM. Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Detroit, MI 48202, USA. The Ino4 protein belongs to the basic helix-loop-helix (bHLH) family of proteins. It is known to form a dimer with Ino2p, which regulates phospholipid biosynthetic genes. Mammalian bHLH proteins have been shown to form multiple dimer combinations. However, this flexibility in dimerization had not been documented for yeast bHLH proteins. Using the yeast two-hybrid assay and a biochemical assay we show that Ino4p dimerizes with the Pho4p, Rtg1p, Rtg3p and Sgc1p bHLH proteins. Screening a yeast cDNA library identified three additional proteins that interact with Ino4p: Bck2p, YLR422W and YNR064C. The interaction with Bck2p prompted us to examine if any of the Bck2p-associated functions affect expression of phospholipid biosynthetic genes. We found that hyperosmotic growth conditions altered the growth phase regulation of a phospholipid biosynthetic gene, CHO1. There are two recent reports of initial whole genome yeast two-hybrid interactions. Interestingly, one of these reports identified five proteins that interact with Ino4p: Ino2p, Hcs1p, Apl2p, YMR317W and YNL279W. Ino2p is the only protein in common with the data presented here. Our finding that Ino4p interacts with five bHLH proteins suggests that Ino4p is likely to be a central player in the coordination of multiple biological processes. PMID: 11071933 [PubMed - indexed for MEDLINE] 343: Mol Biol Cell 2000 Nov;11(11):3859-71 Sec62p, a component of the endoplasmic reticulum protein translocation machinery, contains multiple binding sites for the Sec-complex. Wittke S, Dunnwald M, Johnsson N. Max-Delbruck-Laboratorium, D-50829 Koln, Germany. SEC62 encodes an essential component of the Sec-complex that is responsible for posttranslational protein translocation across the membrane of the endoplasmic reticulum in Saccharomyces cerevisiae. The specific role of Sec62p in translocation was not known and difficult to identify because it is part of an oligomeric protein complex in the endoplasmic reticulum membrane. An in vivo competition assay allowed us to characterize and dissect physical and functional interactions between Sec62p and components of the Sec-complex. We could show that Sec62p binds via its cytosolic N- and C-terminal domains to the Sec-complex. The N-terminal domain, which harbors the major interaction site, binds directly to the last 14 residues of Sec63p. The C-terminal binding site of Sec62p is less important for complex stability, but adjoins the region in Sec62p that might be involved in signal sequence recognition. PMID: 11071912 [PubMed - indexed for MEDLINE] 344: Mol Biol Cell 2000 Nov;11(11):3689-702 Bud6 directs sequential microtubule interactions with the bud tip and bud neck during spindle morphogenesis in Saccharomyces cerevisiae. Segal M, Bloom K, Reed SI. Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA. In budding yeast, spindle polarity relies on a precise temporal program of cytoplasmic microtubule-cortex interactions throughout spindle assembly. Loss of Clb5-dependent kinase activity under conditions of attenuated Cdc28 function disrupts this program, resulting in diploid-specific lethality. Here we show that polarity loss is tolerated by haploids due to a more prominent contribution of microtubule-neck interactions to spindle orientation inherent to haploids. These differences are mediated by the relative partition of Bud6 between the bud tip and bud neck, distinguishing haploids from diploids. Bud6 localizes initially to the bud tip and accumulates at the neck concomitant with spindle assembly. bud6Delta mutant phenotypes are consistent with Bud6's role as a cortical cue for cytoplasmic microtubule capture. Moreover, mutations that affect Bud6 localization and partitioning disrupt the sequential program of microtubule-cortex interactions accordingly. These data support a model whereby Bud6 sequentially cues microtubule capture events at the bud tip followed by capture events at the bud neck, necessary for correct spindle morphogenesis and polarity. PMID: 11071900 [PubMed - indexed for MEDLINE] 345: Proc Natl Acad Sci U S A 2000 Nov 7;97(23):12583-8 Interaction of yeast kinetochore proteins with centromere-protein/transcription factor Cbf1. Hemmerich P, Stoyan T, Wieland G, Koch M, Lechner J, Diekmann S. Institut fuer Molekulare Biotechnologie, Abteilung Molekularbiologie, Beutenbergstrasse 11, 07745 Jena, Germany. phemmer@imb-jena.de The centromere-kinetochore complex of Saccharomyces cerevisiae is a specialized chromosomal substructure that mediates attachment of duplicated chromosomes to the mitotic spindle by a regulated network of protein-DNA and protein-protein interactions. We have used in vitro assays to analyze putative molecular interactions between components of the yeast centromerekinetochore complex. Glutathione S-transferase pull-down experiments showed the direct interaction of in vitro translated p110, p64, and p58 of the essential CBF3 kinetochore protein complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip) that specifically binds to the CDEI region on the centromere DNA. Furthermore, recombinant p64 and p23 each stimulated the in vitro DNA binding activity of Cbf1p. The N-terminal 70 amino acids of p23 were sufficient to mediate this effect. P64 could also promote the multimerization activity of Cbf1p in the presence of centromere DNA in vitro. These results show the direct physical interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components can promote the binding of Cbf1p to its binding site in the yeast kinetochore. A functional comparison of the centromere binding proteins with transcription factors binding at MET16 promoters reveals the strong analogy between centromeres and the MET16 promoter. PMID: 11070082 [PubMed - indexed for MEDLINE] 346: Genes Dev 2000 Nov 1;14(21):2737-44 Ssn6-Tup1 interacts with class I histone deacetylases required for repression. Watson AD, Edmondson DG, Bone JR, Mukai Y, Yu Y, Du W, Stillman DJ, Roth SY. Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA. Ssn6-Tup1 regulates multiple genes in yeast, providing a paradigm for corepressor functions. Tup1 interacts directly with histones H3 and H4, and mutation of these histones synergistically compromises Ssn6-Tup1-mediated repression. In vitro, Tup1 interacts preferentially with underacetylated isoforms of H3 and H4, suggesting that histone acetylation may modulate Tup1 functions in vivo. Here we report that histone hyperacetylation caused by combined mutations in genes encoding the histone deacetylases (HDACs) Rpd3, Hos1, and Hos2 abolishes Ssn6-Tup1 repression. Unlike HDAC mutations that do not affect repression, this combination of mutations causes concomitant hyperacetylation of both H3 and H4. Strikingly, two of these class I HDACs interact physically with Ssn6-Tup1. These findings suggest that Ssn6-Tup1 actively recruits deacetylase activities to deacetylate adjacent nucleosomes and promote Tup1-histone interactions. PMID: 11069890 [PubMed - indexed for MEDLINE] 347: J Cell Sci 2000 Dec;113 Pt 23:4143-9 The FHA domain mediates phosphoprotein interactions. Li J, Lee GI, Van Doren SR, Walker JC. Division of Biological Sciences and Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, USA. The forkhead-associated (FHA) domain is a phosphopeptide-binding domain first identified in a group of forkhead transcription factors but is present in a wide variety of proteins from both prokaryotes and eukaryotes. In yeast and human, many proteins containing an FHA domain are found in the nucleus and involved in DNA repair, cell cycle arrest, or pre-mRNA processing. In plants, the FHA domain is part of a protein that is localized to the plasma membrane and participates in the regulation of receptor-like protein kinase signaling pathways. Recent studies show that a functional FHA domain consists of 120-140 amino acid residues, which is significantly larger than the sequence motif first described. Although FHA domains do not exhibit extensive sequence similarity, they share similar secondary and tertiary structures, featuring a sandwich of two anti-parallel (beta)-sheets. One intriguing finding is that FHA domains may bind phosphothreonine, phosphoserine and sometimes phosphotyrosine, distinguishing them from other well-studied phosphoprotein-binding domains. The diversity of proteins containing FHA domains and potential differences in binding specificities suggest the FHA domain is involved in coordinating diverse cellular processes. Publication Types: Review Review Literature PMID: 11069759 [PubMed - indexed for MEDLINE] 348: Arch Biochem Biophys 2000 Oct 15;382(2):262-74 Interaction of insulin-like growth factor binding protein-4, Miz-1, leptin, lipocalin-type prostaglandin D synthase, and granulin precursor with the N-terminal half of type III hexokinase. Sui D, Wilson JE. Department of Biochemistry, Michigan State University, East Lansing 48824, USA. Insulin-like growth factor binding protein-4, Miz-1, leptin, prostaglandin D synthase, and granulin precursor were identified as proteins interacting with the N-terminal half of mammalian Type III hexokinase (HKIII) in the yeast two-hybrid method. These interactions were confirmed by in vitro binding studies. All five of these proteins, and their mRNAs, were present in PC12 cells, as shown by immunoblotting and RT-PCR, respectively. All were coimmunoprecipitated from PC12 extracts with an antibody against HKIII, but not with anti-Type I hexokinase. Moreover, all of these proteins were coimmunoprecipitated using antileptin as precipitating antibody, indicating the existence of a macromolecular complex including these five proteins and HKIII. Transfection of M+R 42 cells with HKIII-green fluorescent protein (GFP) reporter constructs gave a diffuse intracellular fluorescence. Cotransfection with leptin or Miz-1 resulted in distinctly different localization of the HKIII-GFP fusion protein, at intracellular sites coincident with localization of leptin-GFP or Miz-1-GFP reporter constructs. PMID: 11068878 [PubMed - indexed for MEDLINE] 349: Genetics 2000 Nov;156(3):973-81 CSE4 genetically interacts with the Saccharomyces cerevisiae centromere DNA elements CDE I and CDE II but not CDE III. Implications for the path of the centromere dna around a cse4p variant nucleosome. Keith KC, Fitzgerald-Hayes M. Department of Biochemistry and Molecular Biology, Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA. Each Saccharomyces cerevisiae chromosome contains a single centromere composed of three conserved DNA elements, CDE I, II, and III. The histone H3 variant, Cse4p, is an essential component of the S. cerevisiae centromere and is thought to replace H3 in specialized nucleosomes at the yeast centromere. To investigate the genetic interactions between Cse4p and centromere DNA, we measured the chromosome loss rates exhibited by cse4 cen3 double-mutant cells that express mutant Cse4 proteins and carry chromosomes containing mutant centromere DNA (cen3). When compared to loss rates for cells carrying the same cen3 DNA mutants but expressing wild-type Cse4p, we found that mutations throughout the Cse4p histone-fold domain caused surprisingly large increases in the loss of chromosomes carrying CDE I or CDE II mutant centromeres, but had no effect on chromosomes with CDE III mutant centromeres. Our genetic evidence is consistent with direct interactions between Cse4p and the CDE I-CDE II region of the centromere DNA. On the basis of these and other results from genetic, biochemical, and structural studies, we propose a model that best describes the path of the centromere DNA around a specialized Cse4p-nucleosome. PMID: 11063678 [PubMed - indexed for MEDLINE] 350: Genetics 2000 Nov;156(3):943-51 Synthetic interactions of the post-Golgi sec mutations of Saccharomyces cerevisiae. Finger FP, Novick P. Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002, USA. In the budding yeast Saccharomyces cerevisiae, synthetic lethality has been extensively used both to characterize interactions between genes previously identified as likely to be involved in similar processes as well as to uncover new interactions. We have performed a large study of the synthetic lethal interactions of the post-Golgi sec mutations. Included in this study are the interactions of the post-Golgi sec mutations with each other, with mutations affecting earlier stages of the secretory pathway, with selected mutations affecting the actin cytoskeleton, and with selected cell division cycle (cdc) mutations affecting processes thought to be important for or involving secretion, such as polarity establishment and cytokinesis. Synthetic negative interactions of the post-Golgi sec mutations appear (as predicted) to be largely stage specific, although there are some notable exceptions. The significance of these results is discussed in the context of both secretory pathway function and the utility of synthetic lethality studies and their interpretation. PMID: 11063675 [PubMed - indexed for MEDLINE] 351: Virology 2000 Nov 10;277(1):127-35 The human T-cell leukemia virus type I (HTLV-I) X region encoded protein p13(II) interacts with cellular proteins. Hou X, Foley S, Cueto M, Robinson MA. Laboratory of Immunogenetics, Twinbrook II Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, Maryland, 20852, USA. Interactions between the Human T-cell leukemia virus type I (HTLV-I) gene product p13(II) and cellular proteins were investigated using the yeast two-hybrid system. Variant forms of p13(II) were derived from two HTLV-I molecular clones, K30p and K34p, that differ in both virus production and in vivo and in vitro infectivity. Two nucleotide differences between the p13 from K30p (p13K30) and K34p (p13K34) result in a Trp-Arg substitution at amino acid 17 and the truncation of the 25 carboxyl-terminal residues of p13K34. A cDNA library from an HTLV-I-infected rabbit T-cell line was screened with p13K30 and p13K34 as bait. Products of two cDNA clones, C44 and C254, interacted with p13K34 but not with p13K30. Interactions were further confirmed using the GST-fusion protein coprecipitation assay. Sequence analysis of C44 and C254 cDNA clones revealed similarities to members of the nucleoside monophosphate kinase superfamily and actin-binding protein 280, respectively. Further analysis of the function of these two proteins and the consequence of their interaction with p13 may help elucidate a role for p13 in virus production, infectivity, or the pathogenesis of HTLV-I. Copyright 2000 Academic Press. PMID: 11062043 [PubMed - indexed for MEDLINE] 352: Biochem Biophys Res Commun 2000 Nov 2;277(3):589-93 Combined transformation and genetic technique verification of protein-protein interactions in the yeast two-hybrid system. Tyagi S, Lal SK. Virology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, 1100069, India. The yeast two-hybrid system is frequently used to identify protein-protein interactions. The assay is based on the functional reconstitution of a transcriptional activator. Since an indirect phenotype of the positive clones is the basis for selection of positive interacting clones, the two-hybrid screens are vulnerable to false positives. Here we report a screening protocol based on the sequential use of the cotransformation approach followed by the genetic method for verifying true two-hybrid interactions. Using this procedure, we have screened a cDNA library and have been able to isolate true positives from the yeast two-hybrid screen. Copyright 2000 Academic Press. PMID: 11061998 [PubMed - indexed for MEDLINE] 353: J Biol Chem 2001 Jan 26;276(4):2608-15 A hybrid between Na+,K+-ATPase and H+,K+-ATPase is sensitive to palytoxin, ouabain, and SCH 28080. Farley RA, Schreiber S, Wang SG, Scheiner-Bobis G. Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles 90033, USA. rfarley@hsc.usc.edu Na(+),K(+)-ATPase is inhibited by cardiac glycosides such as ouabain, and palytoxin, which do not inhibit gastric H(+),K(+)-ATPase. Gastric H(+),K(+)-ATPase is inhibited by SCH28080, which has no effect on Na(+),K(+)-ATPase. The goal of the current study was to identify amino acid sequences of the gastric proton-potassium pump that are involved in recognition of the pump-specific inhibitor SCH 28080. A chimeric polypeptide consisting of the rat sodium pump alpha3 subunit with the peptide Gln(905)-Val(930) of the gastric proton pump alpha subunit substituted in place of the original Asn(886)-Ala(911) sequence was expressed together with the gastric beta subunit in the yeast Saccharomyces cerevisiae. Yeast cells that express this subunit combination are sensitive to palytoxin, which interacts specifically with the sodium pump, and lose intracellular K(+) ions. The palytoxin-induced K(+) efflux is inhibited by the sodium pump-specific inhibitor ouabain and also by the gastric proton pump-specific inhibitor SCH 28080. The IC(50) for SCH 28080 inhibition of palytoxin-induced K(+) efflux is 14.3 +/- 2.4 microm, which is similar to the K(i) for SCH 28080 inhibition of ATP hydrolysis by the gastric H(+),K(+)-ATPase. In contrast, palytoxin-induced K(+) efflux from cells expressing either the native alpha3 and beta1 subunits of the sodium pump or the alpha3 subunit of the sodium pump together with the beta subunit of the gastric proton pump is inhibited by ouabain but not by SCH 28080. The acquisition of SCH 28080 sensitivity by the chimera indicates that the Gln(905)-Val(930) peptide of the gastric proton pump is likely to be involved in the interactions of the gastric proton-potassium pump with SCH 28080. PMID: 11054424 [PubMed - indexed for MEDLINE] 354: Biophys J 2000 Nov;79(5):2624-31 PMP1 18-38, a yeast plasma membrane protein fragment, binds phosphatidylserine from bilayer mixtures with phosphatidylcholine: a (2)H-NMR study. Roux M, Beswick V, Coic YM, Huynh-Dinh T, Sanson A, Neumann JM. Departement de Biologie Cellulaire et Moleculaire, Section de Biophysique des Proteines et des Membranes, CEA and URA CNRS 2096, Centre d'Etudes de Saclay, 91191 Gif sur Yvette Cedex, France. roux@dsvidf.cea.fr PMP1 is a 38-residue plasma membrane protein of the yeast Saccharomyces cerevisiae that regulates the activity of the H(+)-ATPase. The cytoplasmic domain conformation results in a specific interfacial distribution of five basic side chains, thought to strongly interact with anionic phospholipids. We have used the PMP1 18-38 fragment to carry out a deuterium nuclear magnetic resonance ((2)H-NMR) study for investigating the interactions between the PMP1 cytoplasmic domain and phosphatidylserines. For this purpose, mixed bilayers of 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphoserine (POPS) were used as model membranes (POPC/POPS 5:1, m/m). Spectra of headgroup- and chain-deuterated POPC and POPS phospholipids, POPC-d4, POPC-d31, POPS-d3, and POPS-d31, were recorded at different temperatures and for various concentrations of the PMP1 fragment. Data obtained from POPS deuterons revealed the formation of specific peptide-POPS complexes giving rise to a slow exchange between free and bound PS lipids, scarcely observed in solid-state NMR studies of lipid-peptide/protein interactions. The stoichiometry of the complex (8 POPS per peptide) was determined and its significance is discussed. The data obtained with headgroup-deuterated POPC were rationalized with a model that integrates the electrostatic perturbation induced by the cationic peptide on the negatively charged membrane interface, and a "spacer" effect due to the intercalation of POPS/PMP1f complexes between choline headgroups. PMID: 11053135 [PubMed - indexed for MEDLINE] 355: Endocrine 2000 Aug;13(1):55-62 c-Jun targets amino terminus of androgen receptor in regulating androgen-responsive transcription. Bubulya A, Zhou XF, Shen XQ, Fisher CJ, Shemshedini L. Department of Biology, University of Toledo, OH 43606, USA. The human androgen receptor (hAR) is a member of the nuclear receptor superfamily and functions as a ligand-inducible transcription factor. We have previously proposed that c-Jun mediates the transcriptional activity of this receptor. The modular nature of hAR was used in this study to generate several fusions with the heterologous DNA-binding domain of the yeast transcription factor GAL4 in an attempt to identify the c-Jun-responsive domains within the receptor. Our results suggest that the target of c-Jun action is the amino terminus (AB region) of the receptor and that hAR amino acids 502-521 are critical for the c-Jun response. Additionally, amino acids 503-555 were shown to harbor an autonomous transactivation that is stimulated by c-Jun. Furthermore, we demonstrated that transcription intermediary factor-2 (TIF-2), a coactivator that acts on the activation function-2, stimulates the full-length hAR. These results suggest that c-Jun and TIF-2 can work together as coactivators on the hAR by targeting distinct portions of the receptor. PMID: 11051047 [PubMed - indexed for MEDLINE] 356: Mol Cell Biol 2000 Nov;20(22):8548-59 Saccharomyces cerevisiae cdc42p GTPase is involved in preventing the recurrence of bud emergence during the cell cycle. Richman TJ, Johnson DI. Department of Microbiology and Molecular Genetics and Markey Center for Molecular Genetics, University of Vermont Burlington, Vermont 05405, USA. The Saccharomyces cerevisiae Cdc42p GTPase interacts with multiple regulators and downstream effectors through an approximately 25-amino-acid effector domain. Four effector domain mutations, Y32K, F37A, D38E, and Y40C, were introduced into Cdc42p and characterized for their effects on these interactions. Each mutant protein showed differential interactions with a number of downstream effectors and regulators and various levels of functionality. Specifically, Cdc42(D38E)p showed reduced interactions with the Cla4p p21-activated protein kinase and the Bem3p GTPase-activating protein and cdc42(D38E) was the only mutant allele able to complement the Deltacdc42 null mutant. However, the mutant protein was only partially functional, as indicated by a temperature-dependent multibudded phenotype seen in conjunction with defects in both septin ring localization and activation of the Swe1p-dependent morphogenetic checkpoint. Further analysis of this mutant suggested that the multiple buds emerged consecutively with a premature termination of bud enlargement preceding the appearance of the next bud. Cortical actin, the septin ring, Cla4p-green fluorescent protein (GFP), and GFP-Cdc24p all predominantly localized to one bud at a time per multibudded cell. These data suggest that Cdc42(D38E)p triggers a morphogenetic defect post-bud emergence, leading to cessation of bud growth and reorganization of the budding machinery to another random budding site, indicating that Cdc42p is involved in prevention of the initiation of supernumerary buds during the cell cycle. PMID: 11046150 [PubMed - indexed for MEDLINE] 357: Mol Cell Biol 2000 Nov;20(22):8343-51 Functional interaction between Ssu72 and the Rpb2 subunit of RNA polymerase II in Saccharomyces cerevisiae. Pappas DL Jr, Hampsey M. Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA. SSU72 is an essential gene encoding a phylogenetically conserved protein of unknown function that interacts with the general transcription factor TFIIB. A recessive ssu72-1 allele was identified as a synthetic enhancer of a TFIIB (sua7-1) defect, resulting in a heat-sensitive (Ts(-)) phenotype and a dramatic downstream shift in transcription start site selection. Here we describe a new allele, ssu72-2, that confers a Ts(-) phenotype in a SUA7 wild-type background. In an effort to further define Ssu72, we isolated suppressors of the ssu72-2 mutation. One suppressor is allelic to RPB2, the gene encoding the second-largest subunit of RNA polymerase II (RNAP II). Sequence analysis of the rpb2-100 suppressor defined a cysteine replacement of the phylogenetically invariant arginine residue at position 512 (R512C), located within homology block D of Rpb2. The ssu72-2 and rpb2-100 mutations adversely affected noninduced gene expression, with no apparent effects on activated transcription in vivo. Although isolated as a suppressor of the ssu72-2 Ts(-) defect, rpb2-100 enhanced the transcriptional defects associated with ssu72-2. The Ssu72 protein interacts directly with purified RNAP II in a coimmunoprecipitation assay, suggesting that the genetic interactions between ssu72-2 and rpb2-100 are a consequence of physical interactions. These results define Ssu72 as a highly conserved factor that physically and functionally interacts with the RNAP II core machinery during transcription initiation. PMID: 11046131 [PubMed - indexed for MEDLINE] 358: J Biol Chem 2001 Jan 12;276(2):1051-6 Biochemical analysis of the eIF2beta gamma complex reveals a structural function for eIF2alpha in catalyzed nucleotide exchange. Nika J, Rippel S, Hannig EM. Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75083, USA. Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-dependent manner. Initiation requires hydrolysis of eIF2-bound GTP, which releases an eIF2.GDP complex that is recycled to the GTP form by the nucleotide exchange factor eIF2B. The alpha-subunit of eIF2 plays a critical role in regulating nucleotide exchange via phosphorylation at serine 51, which converts eIF2 into a competitive inhibitor of the eIF2B-catalyzed exchange reaction. We purified a form of eIF2 (eIF2betagamma) completely devoid of the alpha-subunit to further study the role of eIF2alpha in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2alpha structural gene (SUI2). Removal of the alpha-subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRNA(i)(Met) ligands by eIF2 in vitro. Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2betagamma and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the alpha-subunit increased K(m) for eIF2betagamma.GDP by an order of magnitude, with a smaller increase in V(max). These data indicate that eIF2alpha is required for structural interactions between eIF2 and eIF2B that promote wild-type rates of nucleotide exchange. We suggest that this function contributes to the ability of the alpha-subunit to control the rate of nucleotide exchange through reversible phosphorylation. PMID: 11042214 [PubMed - indexed for MEDLINE] 359: J Biol Chem 2001 Jan 12;276(2):1204-10 Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase. Panisko EA, McAlister-Henn L. Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA. Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase is an octamer composed of four each of two nonidentical but related subunits designated IDH1 and IDH2. IDH2 was previously shown to contain the catalytic site, whereas IDH1 contributes regulatory properties including cooperativity with respect to isocitrate and allosteric activation by AMP. In this study, interactions between IDH1 and IDH2 were detected using the yeast two-hybrid system, but interactions between identical subunit polypeptides were not detected with this or other methods. A model for heterodimeric interactions between the subunits is therefore proposed for this enzyme. A corollary of this model, based on the three-dimensional structure of the homologous enzyme from Escherichia coli, is that some interactions between subunits occur at isocitrate binding sites. Based on this model, two residues (Lys-183 and Asp-217) in the regulatory IDH1 subunit were predicted to be important in the catalytic site of IDH2. We found that individually replacing these residues with alanine results in mutant enzymes that exhibit a drastic reduction in catalysis both in vitro and in vivo. Also based on this model, the two analogous residues (Lys-189 and Asp-222) of the catalytic IDH2 subunit were predicted to contribute to the regulatory site of IDH1. A K189A substitution in IDH2 was found to produce a decrease in activation of the enzyme by AMP and a loss of cooperativity with respect to isocitrate. A D222A substitution in IDH2 produces similar regulatory defects and a substantial reduction in V(max) in the absence of AMP. Collectively, these results suggest that the basic structural/functional unit of yeast isocitrate dehydrogenase is a heterodimer of IDH1 and IDH2 subunits and that each subunit contributes to the isocitrate binding site of the other. PMID: 11042198 [PubMed - indexed for MEDLINE] 360: J Biol Chem 2001 Jan 19;276(3):2023-30 The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae. Phylip LH, Lees WE, Brownsey BG, Bur D, Dunn BM, Winther JR, Gustchina A, Li M, Copeland T, Wlodawer A, Kay J. School of Biosciences, Cardiff University, P. O. Box 911, Cardiff CF10 3US, Wales, United Kingdom. The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar sequences/structures from a wide variety of other species. Systematic truncation and mutagenesis of the IA3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence. Crystal structures of different forms of IA3 complexed with proteinase A showed that residues in the N-terminal half of the IA3 sequence became ordered and formed an almost perfect alpha-helix in the active site of the enzyme. This potent, specific interaction was directed primarily by hydrophobic interactions made by three key features in the inhibitory sequence. Whereas IA3 was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme. PMID: 11042188 [PubMed - indexed for MEDLINE] 361: J Biol Chem 2001 Jan 19;276(3):2122-31 Plant initiation factor 3 subunit composition resembles mammalian initiation factor 3 and has a novel subunit. Burks EA, Bezerra PP, Le H, Gallie DR, Browning KS. Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712, USA. Eukaryotic initiation factor 3 (eIF3) is a multisubunit complex that is required for binding of mRNA to 40 S ribosomal subunits, stabilization of ternary complex binding to 40 S subunits, and dissociation of 40 and 60 S subunits. These functions and the complex nature of eIF3 suggest multiple interactions with many components of the translational machinery. Recently, the subunits of mammalian and Saccharomyces cerevisiae eIF3 were identified, and substantial differences in the subunit composition of mammalian and S. cerevisiae were observed. Mammalian eIF3 consists of 11 nonidentical subunits, whereas S. cerevisiae eIF3 consists of up to eight nonidentical subunits. Only five of the subunits of mammalian and S. cerevisiae are shared in common, and these five subunits comprise a "core" complex in S. cerevisiae. eIF3 from wheat consists of at least 10 subunits, but their relationship to either the mammalian or S. cerevisiae eIF3 subunits is unknown. Peptide sequences derived from purified wheat eIF3 subunits were used to correlate each subunit with mammalian and/or S. cerevisiae subunits. The peptide sequences were also used to identify Arabidopsis thaliana cDNAs for each of the eIF3 subunits. We report seven new cDNAs for A. thaliana eIF3 subunits. A. thaliana eIF3 was purified and characterized to confirm that the subunit composition and activity of wheat and A. thaliana eIF3 were similar. We report that plant eIF3 closely resembles the subunit composition of mammalian eIF3, having 10 out of 11 subunits in common. Further, we find a novel subunit in the plant eIF3 complex not present in either mammalian or S. cerevisiae eIF3. These results suggest that plant and mammalian eIF3 evolved similarly, whereas S. cerevisiae has diverged. PMID: 11042177 [PubMed - indexed for MEDLINE] 362: J Biotechnol 2001 Nov 17;84(1):87-91 Improved resistance to transition metals of a cobalt-substituted alcohol dehydrogenase 1 from Saccharomyces cerevisiae. Cavaletto M, Pessione E, Vanni A, Giunta C. Dipartimento di Biologia Animale e dell'Uomo, Universita di Torino, Via Accademia Albertina 13, 10123, Torino, Italy. maria.calvetto@unito.it Cobalt-substituted alcohol dehydrogenase 1 was purified from a yeast culture of Saccharomyces cerevisiae. Its reactivity towards different transition metals was tested and compared with the native zinc enzyme. The cobalt enzyme displayed a catalytic efficiency 100-fold higher than that of the zinc enzyme. Copper, nickel and cadmium exerted a mixed-type inhibition, with a scale of inhibition efficiency: Cu(2+)>Ni(2+)>Cd(2+). In general, a higher resistance of the modified protein to the inhibitory action of transition metals was observed, with two orders of magnitude for copper I(50). The presence of nickel in the complexes enzyme-coenzyme-inhibitor-substrate resulted in a decrease of the ampholytic nature of the catalytic site. On the contrary, cadmium and copper exerted an enhancement of this parameter. Electrostatic or other types of interactions may be involved in conferring a good resistance in the basic pH range, making cobalt enzyme very suitable for biotechnological processes. PMID: 11035192 [PubMed - indexed for MEDLINE] 363: J Biol Chem 2001 Jan 5;276(1):395-405 Mutations in the TATA-binding protein, affecting transcriptional activation, show synthetic lethality with the TAF145 gene lacking the TAF N-terminal domain in Saccharomyces cerevisiae. Kobayashi A, Miyake T, Ohyama Y, Kawaichi M, Kokubo T. Division of Gene Function in Animals, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. The general transcription factor TFIID, which is composed of the TATA box-binding protein (TBP) and a set of TBP-associated factors (TAFs), is crucial for both basal and regulated transcription by RNA polymerase II. The N-terminal small segment of yeast TAF145 (yTAF145) binds to TBP and thereby inhibits TBP function. To understand the physiological role of this inhibitory domain, which is designated as TAND (TAF N-terminal domain), we screened mutations, synthetically lethal with the TAF145 gene lacking TAND (taf145 Delta TAND), in Saccharomyces cerevisiae by exploiting a red/white colony-sectoring assay. Our screen yielded several recessive nsl (Delta TAND synthetic lethal) mutations, two of which, nsl1-1 and nsl1-2, define the same complementation group. The NSL1 gene was found to be identical to the SPT15 gene encoding TBP. Interestingly, both temperature-sensitive nsl1/spt15 alleles, which harbor the single amino acid substitutions, S118L and P65S, respectively, were defective in transcriptional activation in vivo. Several other previously characterized activation-deficient spt15 alleles also displayed synthetic lethal interactions with taf145 Delta TAND, indicating that TAND and TBP carry an overlapping but as yet unidentified function that is specifically required for transcriptional regulation. PMID: 11035037 [PubMed - indexed for MEDLINE] 364: Mol Biol Cell 2000 Oct;11(10):3629-43 Yeast exocytic v-SNAREs confer endocytosis. Gurunathan S, Chapman-Shimshoni D, Trajkovic S, Gerst JE. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel. In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface. As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1 (SNC1(ala43)), are deficient in the endocytic uptake of components from the cell surface. We found that both SNC and temperature-shifted SNC1(ala43) yeast are deficient in their ability to deliver the soluble dye FM4-64 to the vacuole. Under conditions in which vesicles accumulate, FM4-64 stained primarily the cytoplasm as well as fragmented vacuoles. In addition, alpha-factor-stimulated endocytosis of the alpha-factor receptor, Ste2, was fully blocked, as evidenced using a Ste2-green fluorescent protein fusion protein as well as metabolic labeling studies. This suggests a direct role for Snc v-SNAREs in the retrieval of membrane proteins from the cell surface. Moreover, this idea is supported by genetic and physical data that demonstrate functional interactions with t-SNAREs that confer endosomal transport (e.g., Tlg1,2). Notably, Snc1(ala43) was found to be nonfunctional in cells lacking Tlg1 or Tlg2. Thus, we propose that synaptobrevin/VAMP family members are engaged in anterograde and retrograde protein sorting steps between the Golgi and the plasma membrane. PMID: 11029060 [PubMed - indexed for MEDLINE] 365: Mol Biol Cell 2000 Oct;11(10):3381-96 Identification of a new vertebrate nucleoporin, Nup188, with the use of a novel organelle trap assay. Miller BR, Powers M, Park M, Fischer W, Forbes DJ. Department of Biology, University of California at San Diego, La Jolla, California 92093, USA. The study of the nuclear pore in vertebrates would benefit from a strategy to directly identify new nucleoporins and interactions between those nucleoporins. We have developed a novel two-step "organelle trap" assay involving affinity selection and in vitro pore assembly. In the first step, soluble proteins derived from Xenopus egg extracts are applied to a column containing a ligand of interest. The bound proteins are then tagged by biotinylation and eluted. In the second step, potential nucleoporins are selected for by virtue of their ability to assemble into annulate lamellae, a cytoplasmic mimic of nuclear pores. The incorporated proteins are then recognized by their biotin tag. Here we use the lectin wheat germ agglutinin (WGA) as ligand; WGA inhibits nuclear transport and has been shown to directly bind three known nucleoporins from Xenopus extract, Nup62, Nup98, and Nup214, all of which contain N-acetylglucosamine residues. Under reduced-stringency conditions, three additional proteins bind to WGA-Sepharose and are revealed by the organelle trap assay. We identified all three as partner nucleoporins. Two were discovered to be Xenopus Nup93 and Nup205. The third is a novel vertebrate nucleoporin, Nup188. This new vertebrate protein, Xenopus Nup188, exists in a complex with xNup93 and xNup205. The Nup93-Nup188-Nup205 complex does not bind directly to WGA but binds indirectly via the N-acetylglucosamine-modified nucleoporins. A gene encoding human Nup188 was also identified. The discovery of vertebrate Nup188, related to a yeast nucleoporin, and its novel protein-protein interactions illustrates the power of the two-step organelle trap assay and identifies new building blocks for constructing the nuclear pore. PMID: 11029043 [PubMed - indexed for MEDLINE] 366: Nucleic Acids Res 2000 Oct 15;28(20):3897-903 Localisation of the DmCdc45 DNA replication factor in the mitotic cycle and during chorion gene amplification. Loebel D, Huikeshoven H, Cotterill S. Department of Biochemistry and Immunology, St Georges Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK. The cdc45 protein was originally identified in Saccharomyces cerevisiae and shown to be essential for initiation of eukaryotic DNA replication. Subsequent isolation and characterisation of the corresponding genes from fission yeast, Xenopus and mammals also support a replication role for the protein in these species. They further suggest that during the course of its function cdc45 interacts with a number of other replication proteins, including minichromosome maintenance proteins, the origin recognition complex and DNA polymerase alpha. We have cloned the gene coding for cdc45 protein from Drosophila melanogaster. We have analysed the expression pattern of the cdc45 protein throughout the cell cycle and the life cycle using a combination of indirect immunofluorescence and subcellular fractionation. Our data show that cellular localisation and developmental regulation of the protein is consistent with a role in DNA replication. DmCdc45 is predominantly expressed in proliferating cells. In addition, its subcellular location is nuclear during interphase and the protein shows association with chromatin. The chromatin-associated form of the protein shows a post-translational modification, which may be involved in control of the action of the protein. DmCdc45 shows interactions with mcm proteins, however, the interactions detected show some specificity, perhaps suggesting a preferential association with particular mcm proteins. In addition we show that a stoichiometric mcm interaction may not be obligatory for the function of cdc45 in follicle cell replication, because, unlike the mcm proteins, DmCdc45 localises to the chorion amplification foci in the follicle cells of the ovary. PMID: 11024168 [PubMed - indexed for MEDLINE] 367: J Cell Biol 2000 Oct 2;151(1):167-78 Spontaneous release of cytosolic proteins from posttranslational substrates before their transport into the endoplasmic reticulum. Plath K, Rapoport TA. Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. In posttranslational translocation in yeast, completed protein substrates are transported across the endoplasmic reticulum membrane through a translocation channel formed by the Sec complex. We have used photo-cross-linking to investigate interactions of cytosolic proteins with a substrate synthesized in a reticulocyte lysate system, before its posttranslational translocation through the channel in the yeast membrane. Upon termination of translation, the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC) are released from the polypeptide chain, and the full-length substrate interacts with several different cytosolic proteins. At least two distinct complexes exist that contain among other proteins either 70-kD heat shock protein (Hsp70) or tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1 (TRiC/CCT), which keep the substrate competent for translocation. None of the cytosolic factors appear to interact specifically with the signal sequence. Dissociation of the cytosolic proteins from the substrate is accelerated to the same extent by the Sec complex and an unspecific GroEL trap, indicating that release occurs spontaneously without the Sec complex playing an active role. Once bound to the Sec complex, the substrate is stripped of all cytosolic proteins, allowing it to subsequently be transported through the membrane channel without the interference of cytosolic binding partners. PMID: 11018062 [PubMed - indexed for MEDLINE] 368: J Cell Biol 2000 Oct 2;151(1):15-28 Tea2p is a kinesin-like protein required to generate polarized growth in fission yeast. Browning H, Hayles J, Mata J, Aveline L, Nurse P, McIntosh JR. Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA. browninh@icrf.icnet.uk Cytoplasmic microtubules are critical for establishing and maintaining cell shape and polarity. Our investigations of kinesin-like proteins (klps) and morphological mutants in the fission yeast Schizosaccharomyces pombe have identified a kinesin-like gene, tea2(+), that is required for cells to generate proper polarized growth. Cells deleted for this gene are often bent during exponential growth and initiate growth from improper sites as they exit stationary phase. They have a reduced cytoplasmic microtubule network and display severe morphological defects in genetic backgrounds that produce long cells. The tip-specific marker, Tea1p, is mislocalized in both tea2-1 and tea2Delta cells, indicating that Tea2p function is necessary for proper localization of Tea1p. Tea2p is localized to the tips of the cell and in a punctate pattern within the cell, often coincident with the ends of cytoplasmic microtubules. These results suggest that this kinesin promotes microtubule growth, possibly through interactions with the microtubule end, and that it is important for establishing and maintaining polarized growth along the long axis of the cell. PMID: 11018050 [PubMed - indexed for MEDLINE] 369: J Biol Chem 2001 Jan 5;276(1):488-94 Identification and characterization of two novel components of the Prp19p-associated complex, Ntc30p and Ntc20p. Chen CH, Tsai WY, Chen HR, Wang CH, Cheng SC. Institute of Microbiology and Immunology, National Yang-Ming University, Shih-Pai 112, Taiwan, Republic of China. The yeast Saccharomyces cerevisiae Prp19p protein is an essential splicing factor and a spliceosomal component. It is not tightly associated with small nuclear RNAs (snRNAs) but is associated with a protein complex consisting of at least eight proteins. We have identified two novel components of the Prp19p-associated complex, Ntc30p and Ntc20p. Like other identified components of the complex, both Ntc30p and Ntc20p are associated with the spliceosome in the same manner as Prp19p immediately after or concurrently with dissociation of U4, indicating that the entire complex may bind to the spliceosome as an intact form. Neither Ntc30p nor Ntc20p directly interacts with Prp19p, but both interact with another component of the complex, Ntc85p. Immunoprecipitation analysis revealed an ordered interactions of these components in formation of the Prp19p-associated complex. Although null mutants of NTC30 or NTC20 showed no obvious growth phenotype, deletion of both genes impaired yeast growth resulting in accumulation of precursor mRNA. Extracts prepared from such a strain were defective in pre-mRNA splicing in vitro, but the splicing activity could be restored upon addition of the purified Prp19p-associated complex. These results indicate that Ntc30p and Ntc20p are auxiliary splicing factors the functions of which may be modulating the function of the Prp19p-associated complex. PMID: 11018040 [PubMed - indexed for MEDLINE] 370: Nat Struct Biol 2000 Oct;7(10):894-902 Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly. Munson M, Chen X, Cocina AE, Schultz SM, Hughson FM. Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA. In the eukaryotic secretory and endocytic pathways, transport vesicles shuttle cargo among intracellular organelles and to and from the plasma membrane. Cargo delivery entails fusion of the transport vesicle with its target, a process thought to be mediated by membrane bridging SNARE protein complexes. Temporal and spatial control of intracellular trafficking depends in part on regulating the assembly of these complexes. In vitro, SNARE assembly is inhibited by the closed conformation adopted by the syntaxin family of SNAREs. To visualize this closed conformation directly, the X-ray crystal structure of a yeast syntaxin, Sso1p, has been determined and refined to 2.1 A resolution. Mutants designed to destabilize the closed conformation exhibit accelerated rates of SNARE assembly. Our results provide insight into the mechanism of SNARE assembly and its intramolecular and intermolecular regulation. PMID: 11017200 [PubMed - indexed for MEDLINE] 371: Nat Genet 2000 Oct;26(2):141-2 Prediction of protein interactions: metabolic enzymes are frequently involved in gene fusion. Tsoka S, Ouzounis CA. Computational Genomics Group, Research Programme, The European Bioinformatics Institute, EMBL Cambridge Outstation, Cambridge, UK. PMID: 11017064 [PubMed - indexed for MEDLINE] 372: Nat Biotechnol 2000 Oct;18(10):1075-9 Erratum in: Nat Biotechnol 2000 Dec;18(12):1318 Use of G-protein fusions to monitor integral membrane protein-protein interactions in yeast. Ehrhard KN, Jacoby JJ, Fu XY, Jahn R, Dohlman HG. Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06536, USA. The control of protein-protein interactions is a fundamental aspect of cell regulation. Here we describe a new approach to detect the interaction of two proteins in vivo. By this method, one binding partner is an integral membrane protein whereas the other is soluble but fused to a G-protein gamma-subunit. If the binding partners interact, G-protein signaling is disrupted. We demonstrate interaction between known binding partners, syntaxin 1a with neuronal Sec1 (nSec1), and the fibroblast-derived growth factor receptor 3 (FGFR3) with SNT-1. In addition, we describe a genetic screen to identify nSec1 mutants that are expressed normally, but are no longer able to bind to syntaxin 1a. This provides a convenient method to study interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. PMID: 11017046 [PubMed - indexed for MEDLINE] 373: Mol Gen Genet 2000 Sep;264(1-2):89-97 Genetic analysis of the Saccharomyces cerevisiae Sgs1 helicase defines an essential function for the Sgs1-Top3 complex in the absence of SRS2 or TOP1. Duno M, Thomsen B, Westergaard O, Krejci L, Bendixen C. Section for Molecular Genetics, Danish Institute of Agricultural Sciences, Tjele. The Saccharomyces cerevisiae gene SGS1 encodes a DNA helicase that shows homology to the Escherichia coli protein RecQ and the products of the BLM and WRN genes in humans, which are defective in Bloom's and Werner's syndrome, respectively. Recently, it has been proposed that this helicase is involved in maintaining the integrity of the rDNA and that loss of Sgs1 function leads to accelerated aging. Sgs1 has been isolated on the basis of its genetic interaction with both topoisomerase I and topoisomerase III, as well as in a two-hybrid screen for proteins that interact with the C-terminal portion of topoisomerase II. We have defined the minimal structural elements of Sgs1 required for its interactions with the three topoisomerases, and demonstrate that the complex phenotypes associated with sgs1 mutants are a consequence of a dysfunctional Sgs1-Top3 complex. We also report that the synthetic relationship between mutations in SGS1 and SRS2, which encodes another helicase implicated in recombinational repair, likewise result from a dysfunctional Sgs1-Top3 interaction. Our findings indicate that Sgs1 may act on different DNA structures depending on the activity of topoisomerase I, Srs2 and topoisomerase III. PMID: 11016837 [PubMed - indexed for MEDLINE] 374: Genetics 2000 Oct;156(2):535-47 Synthetic lethal interactions suggest a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation. Costa PJ, Arndt KM. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA. Strong evidence indicates that transcription elongation by RNA polymerase II (pol II) is a highly regulated process. Here we present genetic results that indicate a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation. A screen for synthetic lethal mutations was carried out with an rtf1 deletion mutation to identify factors that interact with Rtf1 or regulate the same process as Rtf1. The screen uncovered mutations in SRB5, CTK1, FCP1, and POB3. These genes encode an Srb/mediator component, a CTD kinase, a CTD phosphatase, and a protein involved in the regulation of transcription by chromatin structure, respectively. All of these gene products have been directly or indirectly implicated in transcription elongation, indicating that Rtf1 may also regulate this process. In support of this view, we show that RTF1 functionally interacts with genes that encode known elongation factors, including SPT4, SPT5, SPT16, and PPR2. We also show that a deletion of RTF1 causes sensitivity to 6-azauracil and mycophenolic acid, phenotypes correlated with a transcription elongation defect. Collectively, our results suggest that Rtf1 may function as a novel transcription elongation factor in yeast. PMID: 11014804 [PubMed - indexed for MEDLINE] 375: Genetics 2000 Oct;156(2):523-34 Suppressors of mdm20 in yeast identify new alleles of ACT1 and TPM1 predicted to enhance actin-tropomyosin interactions. Singer JM, Hermann GJ, Shaw JM. Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA. The actin cytoskeleton is required for many aspects of cell division in yeast, including mitochondrial partitioning into growing buds (mitochondrial inheritance). Yeast cells lacking MDM20 function display defects in both mitochondrial inheritance and actin organization, specifically, a lack of visible actin cables and enhanced sensitivity to Latrunculin A. mdm20 mutants also exhibit a temperature-sensitive growth phenotype, which we exploited to isolate second-site suppressor mutations. Nine dominant suppressors selected in an mdm20/mdm20 background rescue temperature-sensitive growth defects and mitochondrial inheritance defects and partially restore actin cables in haploid and diploid mdm20 strains. The suppressor mutations define new alleles of ACT1 and TPM1, which encode actin and the major form of tropomyosin in yeast, respectively. The ACT1 mutations cluster in a region of the actin protein predicted to contact tropomyosin, suggesting that they stabilize actin cables by enhancing actin-tropomyosin interactions. The characteristics of the mutant ACT1 and TPM1 alleles and their potential effects on protein structure and binding are discussed. PMID: 11014803 [PubMed - indexed for MEDLINE] 376: Biochem Pharmacol 2000 Oct 15;60(8):1009-13 Protein recruitment systems for the analysis of protein-protein interactions. Aronheim A. Department of Molecular Genetics, the B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel. aronheim@tx.technion.ac.il Following the completion of genome projects in a number of organisms, it is becoming evident that a relatively large proportion of the genes identified encode for proteins that have no sequence homology with known proteins. One possible approach towards understanding protein function is to identify the proteins with which a particular protein associates. Although very powerful, the most commonly used genetic method, the two-hybrid system, is limited in its ability to detect all possible protein-protein interactions. The development of novel approaches, such as the protein recruitment systems, provides attractive alternatives towards identification of protein-protein interactions where other methods have failed to function. Publication Types: Review Review, Tutorial PMID: 11007935 [PubMed - indexed for MEDLINE] 377: J Biol Chem 2000 Nov 24;275(47):36498-501 Functional interaction of proliferating cell nuclear antigen with MSH2-MSH6 and MSH2-MSH3 complexes. Clark AB, Valle F, Drotschmann K, Gary RK, Kunkel TA. Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA. Eukaryotic DNA mismatch repair requires the concerted action of several proteins, including proliferating cell nuclear antigen (PCNA) and heterodimers of MSH2 complexed with either MSH3 or MSH6. Here we report that MSH3 and MSH6, but not MSH2, contain N-terminal sequence motifs characteristic of proteins that bind to PCNA. MSH3 and MSH6 peptides containing these motifs bound PCNA, as did the intact Msh2-Msh6 complex. This binding was strongly reduced when alanine was substituted for conserved residues in the motif. Yeast strains containing alanine substitutions in the PCNA binding motif of Msh6 or Msh3 had elevated mutation rates, indicating that these interactions are important for genome stability. When human MSH3 or MSH6 peptides containing the PCNA binding motif were added to a human cell extract, mismatch repair activity was inhibited at a step preceding DNA resynthesis. Thus, MSH3 and MSH6 interactions with PCNA may facilitate early steps in DNA mismatch repair and may also be important for other roles of these eukaryotic MutS homologs. PMID: 11005803 [PubMed - indexed for MEDLINE] 378: Biochim Biophys Acta 2000 Aug 15;1459(2-3):316-24 Haem-polypeptide interactions during cytochrome c maturation. Thony-Meyer L. Institute of Microbiology, ETH Zurich, Schmelzbergstrasse 7, CH-8092, Zurich, Switzerland. lthoeny@micro.biol.ethz.ch Cytochrome c maturation involves the translocation of a polypeptide, the apocytochrome, and its cofactor, haem, through a membrane, before the two molecules are ligated covalently. This review article focuses on the current knowledge on the journey of haem during this process, which is known best in the Gram-negative bacterium Escherichia coli. As haem always occurs bound to protein, its passage across the cytoplasmic membrane and incorporation into the apocytochrome appears to be mediated by a set of proteinaceous maturation factors, the Ccm (cytochrome c maturation) proteins. At least three of them, CcmC, CcmE and CcmF, are thought to interact directly with haem. CcmE binds haem covalently, thus representing an intermediate of the haem trafficking pathway. CcmC is required for binding of haem to CcmE, and CcmF for releasing it from CcmE and transferring it onto the apocytochrome. The mechanism by which haem crosses the cytoplasmic membrane is currently unknown. Publication Types: Review Review, Tutorial PMID: 11004446 [PubMed - indexed for MEDLINE] 379: Mol Cell Biol 2000 Oct;20(20):7438-49 A motif shared by TFIIF and TFIIB mediates their interaction with the RNA polymerase II carboxy-terminal domain phosphatase Fcp1p in Saccharomyces cerevisiae. Kobor MS, Simon LD, Omichinski J, Zhong G, Archambault J, Greenblatt J. Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada. Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo. PMID: 11003641 [PubMed - indexed for MEDLINE] 380: RNA 2000 Sep;6(9):1289-305 The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function. Lindsey-Boltz LA, Chawla G, Srinivasan N, Vijayraghavan U, Garcia-Blanco MA. Program in Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA. In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160-455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the beta-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between beta strands in the WD repeats, we have identified four amino acids, 235TETG238, that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing. PMID: 10999606 [PubMed - indexed for MEDLINE] 381: Structure Fold Des 2000 Aug 15;8(8):841-50 Crystal structure and mutational analysis of the Saccharomyces cerevisiae cell cycle regulatory protein Cks1: implications for domain swapping, anion binding and protein interactions. Bourne Y, Watson MH, Arvai AS, Bernstein SL, Reed SI, Tainer JA. Centre National de la Recherche Scientifique, Marseille, France. yves@afmb.cnrs-mrs.fr BACKGROUND: The Saccharomyces cerevisiae protein Cks1 (cyclin-dependent kinase subunit 1) is essential for cell-cycle progression. The biological function of Cks1 can be modulated by a switch between two distinct molecular assemblies: the single domain fold, which results from the closing of a beta-hinge motif, and the intersubunit beta-strand interchanged dimer, which arises from the opening of the beta-hinge motif. The crystal structure of a cyclin-dependent kinase (Cdk) in complex with the human Cks homolog CksHs1 single-domain fold revealed the importance of conserved hydrophobic residues and charged residues within the beta-hinge motif. RESULTS: The 3.0 A resolution Cks1 structure reveals the strict structural conservation of the Cks alpha/beta-core fold and the beta-hinge motif. The beta hinge identified in the Cks1 structure includes a novel pivot and exposes a cluster of conserved tyrosine residues that are involved in Cdk binding but are sequestered in the beta-interchanged Cks homolog suc1 dimer structure. This Cks1 structure confirms the conservation of the Cks anion-binding site, which interacts with sidechain residues from the C-terminal alpha helix of another subunit in the crystal. CONCLUSIONS: The Cks1 structure exemplifies the conservation of the beta-interchanged dimer and the anion-binding site in evolutionarily distant yeast and human Cks homologs. Mutational analyses including in vivo rescue of CKS1 disruption support the dual functional roles of the beta-hinge residue Glu94, which participates in Cdk binding, and of the anion-binding pocket that is located 22 A away and on an opposite face to Glu94. The Cks1 structure suggests a biological role for the beta-interchanged dimer and the anion-binding site in targeting Cdks to specific phosphoproteins during cell-cycle progression. PMID: 10997903 [PubMed - indexed for MEDLINE] 382: Yeast 2000 Sep 30;16(13):1229-41 A network of proteins around Rvs167p and Rvs161p, two proteins related to the yeast actin cytoskeleton. Bon E, Recordon-Navarro P, Durrens P, Iwase M, Toh-E A, Aigle M. Laboratoire de Biologie Cellulaire de la Levure, IBGC, 1 rue Camille Saint-Saens, 33077 Bordeaux, France. The Rvs161p and Rvs167p proteins of Saccharomyces cerevisiae, homologues of higher eukaryotes' amphiphysins, associate with actin and appear to be involved in several functions related to the actin cytoskeleton. In order to identify partners of the Rvsp proteins, yeast libraries constructed in two-hybrid vectors were screened using either Rvs167p or Rvs161p as a bait. The selected candidates, representing 34 ORFs, were then tested against both Rvsp proteins, as well as domains of Rvs167p or Rvs161p. Among the most significant ones, 24 ORFs were specific preys of Rvs167p only and two gave interactions with Rvs161p only. Interestingly, five ORFs were preys of both Rvs161p and Rvs167p (RVS167, LAS17, YNL094w, YMR192w and YPL249c). Analysis of putative functions of the candidates confirm involvement of the Rvsp in endocytosis/vesicle traffic, but also opens possible new fields, such as nuclear functions. Copyright 2000 John Wiley & Sons, Ltd. PMID: 10992286 [PubMed - indexed for MEDLINE] 383: J Neurochem 2000 Oct;75(4):1335-51 Regulators of G protein signaling: a bestiary of modular protein binding domains. Burchett SA. Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut, USA. Members of the newly discovered regulator of G protein signaling (RGS) families of proteins have a common RGS domain. This RGS domain is necessary for conferring upon RGS proteins the capacity to regulate negatively a variety of Galpha protein subunits. However, RGS proteins are more than simply negative regulators of signaling. RGS proteins can function as effector antagonists, and recent evidence suggests that RGS proteins can have positive effects on signaling as well. Many RGS proteins possess additional C- and N-terminal modular protein-binding domains and motifs. The presence of these additional modules within the RGS proteins provides for multiple novel regulatory interactions performed by these molecules. These regions are involved in conferring regulatory selectivity to specific Galpha-coupled signaling pathways, enhancing the efficacy of the RGS domain, and the translocation or targeting of RGS proteins to intracellular membranes. In other instances, these domains are involved in cross-talk between different Galpha-coupled signaling pathways and, in some cases, likely serve to integrate small GTPases with these G protein signaling pathways. This review discusses these C- and N-terminal domains and their roles in the biology of the brain-enriched RGS proteins. Methods that can be used to investigate the function of these domains are also discussed. Publication Types: Review Review, Tutorial PMID: 10987813 [PubMed - indexed for MEDLINE] 384: FEBS Lett 2000 Sep 8;481(1):13-8 Caspase-3 and inhibitor of apoptosis protein(s) interactions in Saccharomyces cerevisiae and mammalian cells. Wright ME, Han DK, Hockenbery DM. Molecular and Cellular Biology Program, Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA. Using a heterologous yeast expression assay, we show that inhibitor of apoptosis proteins (IAPs) suppress caspase-3-mediated cytotoxicity in the order of XIAP>c-IAP2>c-IAP1>survivin. The same ordering of IAP activities was demonstrated in mammalian cells expressing an auto-activating caspase-3. The relative anti-apoptotic activities of each IAP depended on the particular death stimulus. For IAP-expressing cells treated with camptothecin, survival correlated with their intrinsic anti-caspase-3 activity. However, c-IAP1-transfected cells were disproportionately resistant to tumor necrosis factor-alpha, suggesting that its anti-apoptotic activities extend beyond caspase-3 or -7 inhibition. Yeast-based caspase assays provide rapid, reliable information on specificity and activity of the IAPs and aid in identifying critical targets in mammalian apoptotic pathways. PMID: 10984607 [PubMed - indexed for MEDLINE] 385: Mol Biol Cell 2000 Sep;11(9):2949-59 Bim1p/Yeb1p mediates the Kar9p-dependent cortical attachment of cytoplasmic microtubules. Miller RK, Cheng SC, Rose MD. Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08544, USA. In Saccharomyces cerevisiae, positioning of the mitotic spindle depends on the interaction of cytoplasmic microtubules with the cell cortex. In this process, cortical Kar9p in the bud acts as a link between the actin and microtubule cytoskeletons. To identify Kar9p-interacting proteins, a two-hybrid screen was conducted with the use of full-length Kar9p as bait, and three genes were identified: BIM1, STU2, and KAR9 itself. STU2 encodes a component of the spindle pole body. Bim1p is the yeast homologue of the human microtubule-binding protein EB1, which is a binding partner to the adenomatous polyposis coli protein involved in colon cancer. Eighty-nine amino acids within the third quarter of Bim1p was sufficient to confer interaction with Kar9p. The two-hybrid interactions were confirmed with the use of coimmunoprecipitation experiments. Genetic analysis placed Bim1p in the Kar9p pathway for nuclear migration. Bim1p was not required for Kar9p's cortical or spindle pole body localization. However, deletion of BIM1 eliminated Kar9p localization along cytoplasmic microtubules. Furthermore, in the bim1 mutants, the cytoplasmic microtubules no longer intersected the cortical dot of Green Fluorescent Protein-Kar9p. These experiments demonstrate that the interaction of cytoplasmic microtubules with the Kar9p cortical attachment site requires the microtubule-binding protein Bim1p. PMID: 10982392 [PubMed - indexed for MEDLINE] 386: J Virol 2000 Oct;74(19):9167-74 Yeast three-hybrid screening of rous sarcoma virus mutants with randomly mutagenized minimal packaging signals reveals regions important for gag interactions. Lee EG, Linial ML. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. We previously showed that the yeast three-hybrid system provides a genetic assay of both RNA and protein components for avian retroviral RNA encapsidation. In the current study, we used this assay to precisely define cis-acting determinants involved in avian leukosis sarcoma virus packaging RNA binding to Gag protein. In vivo screening of Rous sarcoma virus mutants was performed with randomly mutated minimal packaging sequences (MPsi) made using PCR amplification after cotransformation with GagDeltaPR protein into yeast cells. Colonies with low beta-galactosidase activity were analyzed to locate mutations in MPsi sequences affecting binding to Gag proteins. This genetic assay delineated secondary structural elements that are important for efficient RNA binding, including a single-stranded small bulge containing the initiation codon for uORF3, as well as adjacent stem structures. This implies a possible tertiary structure favoring the high-affinity binding sites for Gag. In most cases, results from the three-hybrid assay were well correlated with those from the viral RNA packaging assays. The results from random mutagenesis using the rapid three-hybrid binding assay are consistent with those from site-directed mutagenesis using in vivo packaging assays. PMID: 10982363 [PubMed - indexed for MEDLINE] 387: J Biol Chem 2000 Dec 8;275(49):38206-12 Mapping of a minimal apolipoprotein(a) interaction motif conserved in fibrin(ogen) beta - and gamma -chains. Klose R, Fresser F, Kochl S, Parson W, Kapetanopoulos A, Fruchart-Najib J, Baier G, Utermann G. Institute for Medical Biology and Human Genetics, Universitat Innsbruck, 6020 Innsbruck, Austria. Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a) as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of the blood clotting cascade has been demonstrated in vitro, but the domains in fibrin(ogen) critical for interaction are undefined. We used apo(a) kringle IV subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid interaction trap system. Among positive clones that emerged from the screen, clones were identified as fibrinogen beta- and gamma-chains. Peptide-based pull-down experiments confirmed that the emerging peptide motif, conserved in the carboxyl-terminal globular domains of the fibrinogen beta and gamma modules specifically interacts with apo(a)/Lp(a) in human plasma as well as in cell culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and of lysine binding sites in apo(a) for the interaction was evaluated by binding experiments with apo(a) mutants and a mutated fibrin(ogen) peptid. This confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major fibrin(ogen) binding site but also demonstrated lysine-independent interactions. PMID: 10980194 [PubMed - indexed for MEDLINE] 388: Genetics 2000 Sep;156(1):21-9 Important role for phylogenetically invariant PP2Acalpha active site and C-terminal residues revealed by mutational analysis in Saccharomyces cerevisiae. Evans DR, Hemmings BA. Friedrich Miescher Institute, Basel 4058 Switzerland. drhevans@usa.net PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acalpha functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acalpha Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acalpha catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acalpha C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acalpha catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo. PMID: 10978272 [PubMed - indexed for MEDLINE] 389: J Biol Chem 2000 Nov 24;275(47):37251-6 Functional connections between mediator components and general transcription factors of Saccharomyces cerevisiae. Sakurai H, Fukasawa T. School of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 920-0942, Japan. sakurai@kenroku.kanazawa-u.ac.jp The yeast Gal11 protein is an important component of the Mediator complex in RNA polymerase II-directed transcription. Gal11 and the general transcription factor (TF) IIE are involved in regulation of the protein kinase activity of TFIIH that phosphorylates the carboxyl-terminal domain of RNA polymerase II. We have previously shown that Gal11 binds the small and large subunits of TFIIE at two Gal11 domains, A and B, respectively, which are important for normal function of Gal11 in vivo. Here we demonstrate that Gal11 binds directly to TFIIH through domain A in vitro. A null mutation in GAL11 caused lethality of cells when combined with temperature-sensitive mutations in the genes encoding TFIIE or the carboxyl-terminal domain kinase, indicating the presence of genetic interactions between Gal11 and these proteins. Mutational depletion of Gal11 or TFIIE caused inefficient opening of the transcription initiation region, but had no significant effect on TATA-binding protein occupancy of the TATA sequence in vivo. These results suggest that the functions of Gal11 and TFIIE are necessary after recruitment of TATA-binding protein to the TATA box presumably at the step of stable preinitiation complex formation and/or promoter melting. We illustrate genetic interactions between Gal11 and other Mediator components such as Med2 and Pgd1/Hrs1/Med3. PMID: 10973956 [PubMed - indexed for MEDLINE] 390: Genes Dev 2000 Sep 1;14(17):2206-15 Promotion of Rad51-dependent D-loop formation by yeast recombination factor Rdh54/Tid1. Petukhova G, Sung P, Klein H. Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245-3207, USA. The first DNA joint formed in homologous recombination processes is a D-loop. Saccharomyces cerevisiae RDH54/TID1-encoded product, a Swi2/Snf2-like factor involved in recombination, is shown here to promote D-loop formation with Rad51 recombinase. Physical interaction between Rdh54 and Rad51 is functionally important because Rdh54 does not enhance the recombinase activity of the Escherichia coli RecA protein. Robust dsDNA-activated ATPase activity in Rdh54 generates unconstrained negative and positive supercoils in DNA. Efficient D-loop formation occurs with even topologically relaxed DNA, suggesting that via specific protein-protein interactions, the negative supercoils produced by Rdh54 are used by Rad51 for making DNA joints. PMID: 10970884 [PubMed - indexed for MEDLINE] 391: FEBS Lett 2000 Aug 25;480(1):37-41 Four years of post-genomic life with 6,000 yeast genes. Goffeau A. Unite de Biochimie Physiologique, Universite Catholique de Louvain, Louvain-la-Neuve, Belgium. goffeau@fysa.ucl.ac.be Four years after disclosure of the full yeast genome sequence, a series of resources including tens of thousands of mutant strains, plasmids bearing isolated genes and disruption cassettes are becoming publicly available. Deletions of each of the 6,000 putative yeast genes are being screened systematically for dozens of phenotypic traits. In addition, new global approaches such as DNA hybridization arrays, quantitative proteomics and two-hybrid interactions are being steadily improved. They progressively build up an immense computation network of billions of data points which will, within the next decade, characterize all molecular interactions occurring in a simple eukaryotic cell. In this process of acquisition of new basic knowledge, an international community of over 1,000 laboratories cooperates with a remarkable willingness to share projects and results. Publication Types: Review Review, Tutorial PMID: 10967326 [PubMed - indexed for MEDLINE] 392: J Mol Biol 2000 Sep 1;301(5):1097-112 The strength of acidic activation domains correlates with their affinity for both transcriptional and non-transcriptional proteins. Melcher K. Departments of Internal Medicine and Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75235-8573, USA. K.Melcher@em.uni-frankfurt.de Activation domains (ADs) appear to work by making specific protein-protein contacts with the transcriptional machinery. However, ADs show no apparent sequence conservation, they can be functionally replaced by a number of random peptides and unrelated proteins, and their function does not depend on sustaining a complex tertiary structure. To gain a broader perspective on the nature of interactions between acidic ADs and several of their proposed targets, the in vivo strengths of viral, human, yeast, and artificial activation domains were determined under physiological conditions, and mutant ADs with increased in vivo potencies were selected. The affinities between ADs and proposed targets were determined in vitro and all interactions were found to be of low-level affinity with dissociation constants above 10(-7)M. However, in vivo potencies of all ADs correlated nearly perfectly with their affinities for transcriptional proteins. Surprisingly, the weak interactions of the different ADs with at least two non-transcriptional proteins show the same rank order of binding and AD mutants selected for increased in vivo strength also have increased affinities to non-transcriptional proteins. Based on these results, isolated acidic ADs can bind with relatively low-level specificity and affinity to many different proteins and the strength of these semi-specific interactions determine the strength of an AD. I suggest that ADs expose flexible hydrophobic elements in an aqueous environment to contact hydrophobic patches over short distances, shifting specificity of activators largely to the DNA colocalization of arrays of ADs and targets. Copyright 2000 Academic Press. PMID: 10966808 [PubMed - indexed for MEDLINE] 393: Annu Rev Biochem 2000;69:829-80 Regulation of chromosome replication. Kelly TJ, Brown GW. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. tkelly@jhmi.edu The initiation of DNA replication in eukaryotic cells is tightly controlled to ensure that the genome is faithfully duplicated once each cell cycle. Genetic and biochemical studies in several model systems indicate that initiation is mediated by a common set of proteins, present in all eukaryotic species, and that the activities of these proteins are regulated during the cell cycle by specific protein kinases. Here we review the properties of the initiation proteins, their interactions with each other, and with origins of DNA replication. We also describe recent advances in understanding how the regulatory protein kinases control the progress of the initiation reaction. Finally, we describe the checkpoint mechanisms that function to preserve the integrity of the genome when the normal course of genome duplication is perturbed by factors that damage the DNA or inhibit DNA synthesis. Publication Types: Review Review, Academic PMID: 10966477 [PubMed - indexed for MEDLINE] 394: Annu Rev Biochem 2000;69:571-95 Mechanisms and control of mRNA decapping in Saccharomyces cerevisiae. Tucker M, Parker R. Department of Molecular and Cellular Biology and Howard Hughes Medical Institute, University of Arizona, Tucson, Arizona 85721, USA. The process of mRNA turnover is a critical component of the regulation of gene expression. In the past few years a discrete set of pathways for the degradation of polyadenylated mRNAs in eukaryotic cells have been described. A major pathway of mRNA degradation in yeast occurs by deadenylation of the mRNA, which leads to a decapping reaction, thereby exposing the mRNA to rapid 5' to 3' exonucleolytic degradation. A critical step in this pathway is decapping, since it effectively terminates the existence of the mRNA and is the site of numerous control inputs. In this review, we discuss the properties of the decapping enzyme and how its activity is regulated to give rise to differential mRNA turnover. A key point is that decapping appears to be controlled by access of the enzyme to the cap structure in a competition with the translation initiation complex. Strikingly, several proteins required for mRNA decapping show interactions with the translation machinery and suggest possible mechanisms for the triggering of mRNA decapping. Publication Types: Review Review, Academic PMID: 10966469 [PubMed - indexed for MEDLINE] 395: J Neurosci 2000 Sep 1;20(17):6333-9 Cbln3, a novel member of the precerebellin family that binds specifically to Cbln1. Pang Z, Zuo J, Morgan JI. Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. Precerebellin (Cbln1) is the precursor of the brain-specific hexadecapeptide cerebellin. Although cerebellin has properties of a conventional neuropeptide, its function is controversial because Cbln1 has structural features characteristic of circulating atypical collagens. Cbln1 is related to the three subunits of the complement C1q complex. Therefore, we hypothesized that Cbln1 participated in analogous heteromeric complexes with precerebellin-related proteins. Using LexA-Cbln1 as bait in a yeast two-hybrid screen, we isolated a cDNA encoding a novel Cbln1-related protein, designated Cbln3. The gene encoding cbln3 had the same intron-exon structure as cbln1 but mapped to a different mouse chromosome (14). The deduced amino acid sequence of Cbln3 was 55% identical to Cbln1 and also contained a C1q signature domain and signal sequence for secretion. In addition to binding avidly to Cbln3, Cbln1 also formed homomeric complexes. In contrast, Cbln3 homomeric association was weak. These interactions exhibited specificity because C1qB bound to neither Cbln1 nor Cbln3. Like cbln1, cbln3 was expressed in the cerebellum and dorsal cochlear nucleus in which it was detected in granule neurons. Because Cbln1 and Cbln3 are coexpressed in the brain and interact avidly, they may function as a secreted heteromeric complex in vivo. PMID: 10964938 [PubMed - indexed for MEDLINE] 396: Biochem Soc Trans 2000;28(4):410-4 Recruitment of chromatin remodelling factors during gene activation via the glucocorticoid receptor N-terminal domain. Wallberg AE, Flinn EM, Gustafsson JA, Wright AP. Department of Biosciences, Karolinska Institutet, Novum, SE-141 57 Huddinge, Sweden. We have shown that yeast mutants with defects in the Ada adaptor proteins are defective in hormone-dependent gene activation by ectopically expressed human glucocorticoid receptor (GR). Others have shown that the Ada2 protein is required for physical interactions between some activation domains and TBP (TATA-binding protein), whereas the Gcn5 (Ada4) protein has a histone acetyltransferase (HAT) activity. Although all HAT enzymes are able to acetylate histone substrates, some also acetylate non-histone proteins. Taken together, these observations suggest that the Ada proteins have the ability to effect different steps in the process of gene activation. It has recently been shown that the Ada proteins are present in two distinct protein complexes, the Ada complex and a larger SAGA complex. Our recent work has focused on determining (1) which of the Ada-containing complexes mediates gene activation by GR, (2) whether the HAT activity encoded by GCN5 is required for GR-dependent gene activation, (3) whether the Ada proteins contribute to GR-mediated activation at the level of chromatin remodelling and (4) how the role of these HAT complexes is integrated with other chromatin remodelling activities during GR-mediated gene activation. Our results suggest a model in which GR recruits the SAGA complex and that this contributes to chromatin remodelling via a mechanism involving the acetylation of histones. Furthermore, recruitment of the SWI/SNF remodelling complex also has a role in GR-mediated activation that is independent of the role of SAGA. These complexes are similar to analogous mammalian complexes and therefore these results are likely to be relevant to the human system. Publication Types: Review Review, Tutorial PMID: 10961930 [PubMed - indexed for MEDLINE] 397: J Bacteriol 2000 Sep;182(18):5262-6 Lantibiotic biosynthesis: interactions between prelacticin 481 and its putative modification enzyme, LctM. Uguen P, Le Pennec JP, Dufour A. Laboratoire de Biologie et Chimie Moleculaires, Universite de Bretagne Sud, Vannes, France. Class AII and AIII lantibiotics and mersacidin are antibacterial peptides containing unusual residues obtained by posttranslational modifications of prepeptides, presumably catalyzed by LanM. LctM, the LanM for lacticin 481, is essential for the production of this class AII lantibiotic. Using the yeast two-hybrid system, we showed direct contact between the prelacticin 481 and LctM, supporting the proposed LctM function. Sixteen domains are conserved between the 10 known LanM proteins, whereas three additional domains were found only in class AII LanM proteins and in MrsM, the LanM for mersacidin. All the truncated LctM proteins that we tested presented impaired LctA-binding activity. PMID: 10960114 [PubMed - indexed for MEDLINE] 398: J Biol Chem 2000 Nov 10;275(45):34837-40 Interactions of Cdk7 and Kin28 with Hint/PKCI-1 and Hnt1 histidine triad proteins. Korsisaari N, Makela TP. Haartman Institute & Biocentrum Helsinki, P. O. Box 21, University of Helsinki, 00014 Helsinki, Finland. Cyclin-dependent kinase 7 (Cdk7) forms a trimeric complex with cyclin H and Mat1 to form the mammalian Cdk-activating kinase, CAK, as well as a part of the basal transcription factor TFIIH, where Cdk7 phosphorylates the C-terminal domain (CTD) of the large subunit of RNA polymerase II. Here, we report a novel interaction between Cdk7 and a histidine triad (HIT) family protein, Hint/PKCI-1. This interaction was initially observed in a yeast two-hybrid study and subsequently verified by co-immunoprecipitation and subcellular localization studies, where overexpression of Cdk7 leads to partial relocalization of Hint to the nucleus. The physical association is independent of cyclin H binding or Cdk7 kinase activity and is conserved between the related Sacharomyces cerevisiae CTD kinase Kin28 and the HIT protein Hnt1. Furthermore, combination of a disruption of HNT1 and a KIN28 temperature-sensitive allele in S. cerevisiae led to highly elongated cell morphology and reduced colony formation, indicating a genetic interaction between KIN28 and HNT1. The physical and genetic interactions of Hint and Hnt1 with Cdk7 and Kin28 suggest a role for this class of histidine triad proteins in the regulation of Cdk7 and Kin28 functions. PMID: 10958787 [PubMed - indexed for MEDLINE] 399: Mol Cell Biol 2000 Sep;20(18):7037-48 The N terminus of the centromere H3-like protein Cse4p performs an essential function distinct from that of the histone fold domain. Chen Y, Baker RE, Keith KC, Harris K, Stoler S, Fitzgerald-Hayes M. Department of Biochemistry and Molecular Biology, University of Massachusetts at Amherst, 01003, USA. Cse4p is an evolutionarily conserved histone H3-like protein that is thought to replace H3 in a specialized nucleosome at the yeast (Saccharomyces cerevisiae) centromere. All known yeast, worm, fly, and human centromere H3-like proteins have highly conserved C-terminal histone fold domains (HFD) but very different N termini. We have carried out a comprehensive and systematic mutagenesis of the Cse4p N terminus to analyze its function. Surprisingly, only a 33-amino-acid domain within the 130-amino-acid-long N terminus is required for Cse4p N-terminal function. The spacing of the essential N-terminal domain (END) relative to the HFD can be changed significantly without an apparent effect on Cse4p function. The END appears to be important for interactions between Cse4p and known kinetochore components, including the Ctf19p/Mcm21p/Okp1p complex. Genetic and biochemical evidence shows that Cse4p proteins interact with each other in vivo and that nonfunctional cse4 END and HFD mutant proteins can form functional mixed complexes. These results support different roles for the Cse4p N terminus and the HFD in centromere function and are consistent with the proposed Cse4p nucleosome model. The structure-function characteristics of the Cse4p N terminus are relevant to understanding how other H3-like proteins, such as the human homolog CENP-A, function in kinetochore assembly and chromosome segregation. PMID: 10958698 [PubMed - indexed for MEDLINE] 400: Mol Pharmacol 2000 Sep;58(3):560-8 Probing the interaction of the cytotoxic bisdioxopiperazine ICRF-193 with the closed enzyme clamp of human topoisomerase IIalpha. Patel S, Jazrawi E, Creighton AM, Austin CA, Fisher LM. Molecular Genetics Group, Department of Biochemistry and Immunology, St. George's Hospital Medical School, University of London, London, United Kingdom. Topoisomerase II is an ATP-operated protein clamp that captures a DNA helix and transports it through another DNA duplex, allowing chromosome segregation at mitosis. A number of cytotoxic bisdioxopiperazines such as ICRF-193 target topoisomerase II by binding and trapping the closed enzyme clamp. To investigate this unusual mode of action, we have used yeast to select plasmid-borne human topoisomerase IIalpha alleles resistant to ICRF-193. Mutations in topoisomerase IIalpha of Leu-169 to Phe (L169F) (in the N-terminal ATPase domain) and Ala-648 to Pro (A648P) (in the core domain) were identified as conferring >50-fold and 5-fold resistance to ICRF-193 in vivo, respectively. The L169F mutation, located next to the Walker A box ATP-binding sequence, resulted in a mutant enzyme displaying ICRF-193-resistant topoisomerase and ATPase activities and whose closed clamp was refractory to ICRF-193-mediated trapping as an annulus on closed circular DNA. These data imply that the mutation interferes directly with ICRF-193 binding to the N-terminal ATPase gate. In contrast, the A648P enzyme displayed topoisomerase activities exhibiting wild-type sensitivity to ICRF-193. We suggest that the inefficient trapping of the A648P closed clamp results either from the observed increased ATP requirement, or more likely, from lowered salt stability, perhaps involving destabilization of ICRF-193 interactions with the B'-B' interface in the core domain. These results provide evidence for at least two different phenotypic classes of ICRF-193 resistance mutations and suggest that bisdioxopiperazine action involves the interplay of both the ATPase and core domains of topoisomerase IIalpha. PMID: 10953049 [PubMed - indexed for MEDLINE] 401: Biotechniques 2000 Aug;29(2):278-9, 282-4, 286-8 Streamlined yeast colorimetric reporter activity assays using scanners and plate readers. Serebriiskii IG, Toby GG, Golemis EA. Fox Chase Cancer Center, Philadelphia, PA, USA. ig_serebriiskii@fccc.edu Two-hybrid systems have become favored tools for detection and analysis of protein interactions because of their low cost and ease of use compared to biochemical or biophysical interaction technologies. It is possible to augment the utility of two-hybrid systems and derivative systems such as dual-bait two-hybrid systems by adapting strategies that speed the analysis of the relative strength of a series of protein-protein associations. This report describes two simple techniques that employ either a flatbed scanner or a plate reader to quantitate the activity of colorimetric reporters such as LacZ or GusA commonly used in two-hybrid approaches. Publication Types: Technical Report PMID: 10948429 [PubMed - indexed for MEDLINE] 402: DNA Cell Biol 2000 Jul;19(7):447-57 Loss control of Mcm5 interaction with chromatin in cdc6-1 mutated in CDC-NTP motif. Feng L, Hu Y, Wang B, Wu L, Jong A. Division of Hematology/Oncology, Childrens Hospital Los Angeles, and University of Southern California, School of Medicine, 90027, USA. Saccharomyces cerevisiae Cdc6 plays an essential role in establishing and maintaining the prereplicative complex (pre-RC) by interacting with the origin recognition complex (ORC) and associating with chromatin origins. These interactions are required to load minichromosome maintenance proteins (MCMs) and other initiator proteins onto replication origins. Although the temperature-sensitive cdc6 mutant, cdc6-1, has been widely used for these studies, the molecular mechanism of the cdc6-1 mutation has been unclear. In this study, we have identified a base substitution at Gly260-->Asp, near the CDC-NTP motif. Using a chromatin immunoprecipitation assay (CHIP), we found that cdc6-1 fails to load Mcm5 onto the replication origins. Chromatin fractions were used to study Mcm5 binding in both the wildtype and mutant background. These studies indicated that Cdc6 is also involved in unloading Mcm5 from chromatin. Specifically, the cdc6-1 mutation protein, cdc6(G260D), which failed to load Mcm5 onto replication origins, also failed to unload the Mcm5 protein. Furthermore, the overexpression of wildtype CDC6 accelerated the unloading of Mcm5 from chromatin fractions. In the absence of functional Cdc6, the Mcm5 protein showed nonorigin binding to chromatin with the cell cycle arrested at the G1S phase transition. Our results suggested that the cdc6(G260D) mutant protein fails to assemble an operational replicative complex and that wildtype Cdc6 plays a role in preventing re-replication by controlling the unloading the MCMs from chromatin origins. PMID: 10945234 [PubMed - indexed for MEDLINE] 403: EMBO J 2000 Aug 15;19(16):4372-82 The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally with the eIF4F translation initiation complex. Vilela C, Velasco C, Ptushkina M, McCarthy JE. Posttranscriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology PO Box 88, Manchester M60 1QD, UK. Dcp1 plays a key role in the mRNA decay process in Saccharomyces cerevisiae, cleaving off the 5' cap to leave an end susceptible to exonucleolytic degradation. The eukaryotic initiation factor complex eIF4F, which in yeast contains the core components eIF4E and eIF4G, uses the cap as a binding site, serving as an initial point of assembly for the translation apparatus, and also binds the poly(A) binding protein Pab1. We show that Dcp1 binds to eIF4G and Pab1 as free proteins, as well as to the complex eIF4E-eIF4G-Pab1. Dcp1 interacts with the N-terminal region of eIF4G but does not compete significantly with eIF4E or Pab1 for binding to eIF4G. Most importantly, eIF4G acts as a function-enhancing recruitment factor for Dcp1. However, eIF4E blocks this effect as a component of the high affinity cap-binding complex eIF4E-eIF4G. Indeed, cooperative enhancement of the eIF4E-cap interaction stabilizes yeast mRNAs in vivo. These data on interactions at the interface between translation and mRNA decay suggest how events at the 5' cap and 3' poly(A) tail might be coupled. PMID: 10944120 [PubMed - indexed for MEDLINE] 404: J Biol Chem 2000 Nov 24;275(47):36541-9 Structural basis for the species-specific activity of TFIIS. Shimasaki NB, Kane CM. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA. Many proteins involved in eukaryotic transcription are similar in function and in sequence between organisms. Despite the sequence similarities, there are many factors that do not function across species. For example, transcript elongation factor TFIIS is highly conserved among eukaryotes, and yet the TFIIS protein from Saccharomyces cerevisiae cannot function with mammalian RNA polymerase II and vice versa. To determine the reason for this species specificity, chimeras were constructed linking three structurally independent regions of the TFIIS proteins from yeast and human cells. Two independently folding domains, II and III, have been examined previously using NMR (). Yeast domain II alone is able to bind yeast RNA polymerase II with the same affinity as the full-length TFIIS protein, and this domain was expected to confer the species selectivity. Domain III has previously been shown to be readily exchanged between mammalian and yeast factors. However, the results presented here indicate that domain II is insufficient to confer species selectivity, and a primary determinant lies in a 30-amino acid highly conserved linker region connecting domain II with domain III. These 30 amino acids may physically orient domains II and III to support functional interactions between TFIIS and RNA polymerase II. PMID: 10940308 [PubMed - indexed for MEDLINE] 405: Mol Cell Biol 2000 Sep;20(17):6435-48 Cell cycle-dependent binding of yeast heat shock factor to nucleosomes. Venturi CB, Erkine AM, Gross DS. Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130, USA. In the nucleus, transcription factors must contend with the presence of chromatin in order to gain access to their cognate regulatory sequences. As most nuclear DNA is assembled into nucleosomes, activators must either invade a stable, preassembled nucleosome or preempt the formation of nucleosomes on newly replicated DNA, which is transiently free of histones. We have investigated the mechanism by which heat shock factor (HSF) binds to target nucleosomal heat shock elements (HSEs), using as our model a dinucleosomal heat shock promoter (hsp82-DeltaHSE1). We find that activated HSF cannot bind a stable, sequence-positioned nucleosome in G(1)-arrested cells. It can do so readily, however, following release from G(1) arrest or after the imposition of either an early S- or late G(2)-phase arrest. Surprisingly, despite the S-phase requirement, HSF nucleosomal binding activity is restored in the absence of hsp82 replication. These results contrast with the prevailing paradigm for activator-nucleosome interactions and implicate a nonreplicative, S-phase-specific event as a prerequisite for HSF binding to nucleosomal sites in vivo. PMID: 10938121 [PubMed - indexed for MEDLINE] 406: Mol Cell Biol 2000 Sep;20(17):6426-34 Protein kinase A and mitogen-activated protein kinase pathways antagonistically regulate fission yeast fbp1 transcription by employing different modes of action at two upstream activation sites. Neely LA, Hoffman CS. Department of Biology, Boston College, Massachusetts 02467, USA. A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of the Schizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized two cis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation of fbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulate fbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site. PMID: 10938120 [PubMed - indexed for MEDLINE] 407: Mol Cell Biol 2000 Sep;20(17):6244-58 Gic2p may link activated Cdc42p to components involved in actin polarization, including Bni1p and Bud6p (Aip3p). Jaquenoud M, Peter M. Swiss Institute for Experimental Cancer Research, 1066 Epalinges/VD, Switzerland. Gic2p is a Cdc42p effector which functions during cytoskeletal organization at bud emergence and in response to pheromones, but it is not understood how Gic2p interacts with the actin cytoskeleton. Here we show that Gic2p displayed multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting that Gic2p may regulate their function in vivo. In support of this idea, Gic2p cofractionated with Bud6p and Spa2p and interacted with Bud6p by coimmunoprecipitation and two-hybrid analysis. Importantly, localization of Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the Gic proteins but did not require an intact actin cytoskeleton. We identified a conserved domain in Gic2p which was necessary for its polarization function but dispensable for binding to Cdc42p-GTP and its localization to the site of polarization. Expression of a mutant Gic2p harboring a single-amino-acid substitution in this domain (Gic2p(W23A)) interfered with polarized growth in a dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the incipient bud site. We propose that at bud emergence, Gic2p functions as an adaptor which may link activated Cdc42p to components involved in actin organization and polarized growth, including Bni1p, Spa2p, and Bud6p. PMID: 10938101 [PubMed - indexed for MEDLINE] 408: Biochemistry 2000 Aug 15;39(32):9909-16 Effects of 5' leader and 3' trailer structures on pre-tRNA processing by nuclear RNase P. Ziehler WA, Day JJ, Fierke CA, Engelke DR. Department of Biological Chemistry and Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA. Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader preceding the aminoacyl acceptor stem and a 3' trailer extending beyond this stem. An early step in pre-tRNA maturation is removal of the 5' leader by the endoribonuclease, RNase P. Extensive pairing between leader and trailer sequences has previously been demonstrated to block RNase P cleavage, suggesting that the 5' leader and 3' trailer sequences might need to be separated for the substrate to be recognized by the eukaryotic holoenzyme. To address whether the nuclear RNase P holoenzyme recognizes the 5' leader and 3' trailer sequences independently, interactions of RNase P with pre-tRNA(Tyr) containing either the 5' leader, the 3' trailer, or both were examined. Kinetic analysis revealed little effect of the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage using the various pre-tRNA derivatives. However, the presence of a 3' trailer that pairs with the 5' leader increases the K(m) of pre-tRNA slightly, in agreement with previous results. Similarly, competition studies demonstrate that removal of a complementary 3' trailer lowers the apparent K(I), consistent with the structure between these two sequences interfering with their interaction with the enzyme. Deletion of both the 5' and 3' extensions to give mature termini resulted in the least effective competitor. Further studies showed that the nuclear holoenzyme, but not the B. subtilis holoenzyme, had a high affinity for single-stranded RNA in the absence of attached tRNA structure. The data suggest that yeast nuclear RNase P contains a minimum of two binding sites involved in substrate recognition, one that interacts with tRNA and one that interacts with the 3' trailer. Furthermore, base pairing between the 5' leader and 3' trailer hinders recognition. PMID: 10933810 [PubMed - indexed for MEDLINE] 409: Eur J Biochem 2000 Aug;267(16):5156-67 Flavin-protein interactions in flavocytochrome b2 as studied by NMR after reconstitution of the enzyme with 13C- and 15N-labelled flavin. Fleischmann G, Lederer F, Muller F, Bacher A, Ruterjans H. Institut fur Biophysikalische Chemie, J.W. Goethe-Universitat, Biozentrum N230, Frankfurt, Germany. A new procedure was devised for reversibly removing the flavin from flavocytochrome b2. It allowed reconstitution with selectively enriched 13C- and 15N-labelled FMN for an NMR analysis of the chemical shifts of the enriched positions as well as that of 31P. From these measurements, it was possible to deduce information about the hydrogen-bonding pattern of FMN in the protein, the hybridization states of the nitrogen atoms and (in part) the pi-electron distribution. The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1) and N(5) form hydrogen bonds to the apoenzyme in both redox states. Nevertheless, according to 15N-chemical shifts, the bond from the protein to N(3) is very weak in both redox states, whereas that to N(5) is strong for the oxidized state, and is weakened upon flavin reduction. On the other hand, the 13C-NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger hydrogen bonds with the enzyme than most other flavoproteins in both redox states. From coupling constant measurements it is shown that the N(3) proton is not solvent accessible. Although no N-H coupling constant could be measured for N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated in flavocytochrome b2 as in all other known flavoproteins. With respect to N(10), it is more sp3-hybridized in the oxidized state than in free FMN, whereas the other nitrogen atoms show a nearly planar structure. In the reduced state, N(5) and N(10) in bound FMN are both more sp3-hybridized than in free FMN, but N(5) exhibits a higher degree of sp3-hybridization than N(10), which is only slightly shifted out of the isoalloxazine plane. In addition, two-electron reduction of the enzyme leads to anion formation on N(1), as indicated by its 15N-chemical shift of N(1) and characteristic upfield shifts of the resonances of C(2), C(4) and C(4a) compared to the oxidized state, as observed for most flavoproteins. 31P-NMR measurements show that the phosphate geometry has changed in enzyme bound FMN compared to the free flavin in water, indicating a strong interaction of the phosphate group with the apoenzyme. PMID: 10931200 [PubMed - indexed for MEDLINE] 410: Biochim Biophys Acta 2000 Jul 31;1467(1):207-18 The yeast mitochondrial transport proteins: new sequences and consensus residues, lack of direct relation between consensus residues and transmembrane helices, expression patterns of the transport protein genes, and protein-protein interactions with other proteins. Belenkiy R, Haefele A, Eisen MB, Wohlrab H. Boston Biomedical Research Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Watertown, MA 02472, USA. Mitochondrial transport proteins (MTP) typically are homodimeric with a 30-kDa subunit with six transmembrane helices. The subunit possesses a sequence motif highly similar to Pro X Asp/Glu X X Lys/Arg X Arg within each of its three similar 10-kDa segments. Four (YNL083W, YFR045W, YPR021C, YDR470C) of the 35 yeast (S. cerevisiae) MTP genes were resequenced since the masses of their proteins deviate significantly from the typical 30 kDa. We now find these four proteins to have 545, 285, 902, and 502 residues, respectively. Together with only four other MTPs, the sequences of YPR021C and YDR470C show substitutions of some of the five residues that are absolutely conserved among the 12 MTPs with identified transport function and 17 other MTPs. We do now find these five consensus residues also in the new sequences of YNL083W and YFR045W. Additional analyses of the 35 yeast MTPs show that the location of transmembrane helix sequences do not correlate with the general consensus residues of the MTP family; protein segments connecting the six transmembrane helices and facing the intermembrane space are not uniformly short (about 20 residues) or long (about 40 residues) when facing the matrix; most MTPs have at least one transmembrane helix for which the sum of the negative hydropathy values of all residues yields a very small negative value, suggesting a membrane location bordering polar faces of other transmembrane helices or a non-transmembrane location. The extra residues of the three large MTPs are hydrophilic and at the N-terminal. The 200-residue N-terminal segment of YNL083W has four putative Ca2+-binding sites. The 500-residue N-terminal segment of YPR021C shows sequence similarity to enzymes of nucleic acid metabolism. cDNA microarray data show that YNL083W is expressed solely during sporulation, while the expressions of YFR045W, YPR021C, and YDR470C are induced by various stress situations. These results also show that the 35 MTP genes are expressed under a rather diverse set of metabolic conditions that may help identify the function of the proteins. Interestingly, yeast two-hybrid screens, that will also be useful in identifying the function of MTPs, indicate that MIR1, AAC3, YOR100C, and YPR011C do interact with non-MTPs. PMID: 10930523 [PubMed - indexed for MEDLINE] 411: J Biol Chem 2000 Oct 20;275(42):33158-66 Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells. Chekanova JA, Shaw RJ, Wills MA, Belostotsky DA. Department of Biological Sciences and the Center for Molecular Genetics, State University of New York at Albany, Albany, New York 12222, USA. Eukaryotic 3'-->5' exonucleolytic activities are essential for a wide variety of reactions of RNA maturation and metabolism, including processing of rRNA, small nuclear RNA, and small nucleolar RNA, and mRNA decay. Two related but distinct forms of a complex containing 10 3'-->5' exonucleases, the exosome, are found in yeast nucleus and cytoplasm, respectively, and related complexes exist in human cells. Here we report on the characterization of the AtRrp41p, an Arabidopsis thaliana homolog of the Saccharomyces cerevisiae exosome subunit Rrp41p (Ski6p). Purified recombinant AtRrp41p displays a processive phosphorolytic exonuclease activity and requires a single-stranded poly(A) tail on a substrate RNA as a "loading pad." The expression of the Arabidopsis RRP41 cDNA in yeast rescues the 5.8 S rRNA processing and 3'-->5' mRNA degradation defects of the yeast ski6-100 mutant. However, neither of these defects can explain the conditional lethal phenotype of the ski6-100 strain. Importantly, AtRrp41p shares additional function(s) with the yeast Rrp41p which are essential for cell viability because it also rescues the rrp41 (ski6) null mutant. AtRrp41p is found predominantly in a high molecular mass complex in Arabidopsis and in yeast cells, and it interacts in vitro with the yeast Rrp44p and Rrp4p exosome subunits, suggesting that it can participate in evolutionarily conserved interactions that could be essential for the integrity of the exosome complex. PMID: 10930416 [PubMed - indexed for MEDLINE] 412: Genetics 2000 Aug;155(4):1667-82 Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in the splicing factor Prp8 that influence spliceosome activation. Kuhn AN, Brow DA. Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706-1532, USA. The highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24. PMID: 10924465 [PubMed - indexed for MEDLINE] 413: Genetics 2000 Aug;155(4):1593-606 POB3 is required for both transcription and replication in the yeast Saccharomyces cerevisiae. Schlesinger MB, Formosa T. Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA. Spt16 and Pob3 form stable heterodimers in Saccharomyces cerevisiae, and homologous proteins have also been purified as complexes from diverse eukaryotes. This conserved factor has been implicated in both transcription and replication and may affect both by altering the characteristics of chromatin. Here we describe the isolation and properties of a set of pob3 mutants and confirm that they have defects in both replication and transcription. Mutation of POB3 caused the Spt(-) phenotype, spt16 and pob3 alleles displayed severe synthetic defects, and elevated levels of Pob3 suppressed some spt16 phenotypes. These results are consistent with previous reports that Spt16 and Pob3 act in a complex that modulates transcription. Additional genetic interactions were observed between pob3 mutations and the genes encoding several DNA replication factors, including POL1, CTF4, DNA2, and CHL12. pob3 alleles caused sensitivity to the ribonucleotide reductase inhibitor hydroxyurea, indicating a defect in a process requiring rapid dNTP synthesis. Mutation of the S phase checkpoint gene MEC1 caused pob3 mutants to lose viability rapidly under restrictive conditions, revealing defects in a process monitored by Mec1. Direct examination of DNA contents by flow cytometry showed that S phase onset and progression were delayed when POB3 was mutated. We conclude that Pob3 is required for normal replication as well as for transcription. PMID: 10924459 [PubMed - indexed for MEDLINE] 414: Genetics 2000 Aug;155(4):1543-59 Functional interaction between the PKC1 pathway and CDC31 network of SPB duplication genes. Khalfan W, Ivanovska I, Rose MD. Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA. The earliest known step in yeast spindle pole body (SPB) duplication requires Cdc31p and Kar1p, two physically interacting SPB components, and Dsk2p and Rad23p, a pair of ubiquitin-like proteins. Components of the PKC1 pathway were found to interact with these SPB duplication genes in two independent genetic screens. Initially, SLG1 and PKC1 were obtained as high-copy suppressors of dsk2Delta rad23Delta and a mutation in MPK1 was synthetically lethal with kar1-Delta17. Subsequently, we demonstrated extensive genetic interactions between the PKC1 pathway and the SPB duplication mutants that affect Cdc31p function. The genetic interactions are unlikely to be related to the cell-wall integrity function of the PKC1 pathway because the SPB mutants did not exhibit cell-wall defects. Overexpression of multiple PKC1 pathway components suppressed the G2/M arrest of the SPB duplication mutants and mutations in MPK1 exacerbated the cell cycle arrest of kar1-Delta17, suggesting a role for the PKC1 pathway in SPB duplication. We also found that mutations in SPC110, which encodes a major SPB component, showed genetic interactions with both CDC31 and the PKC1 pathway. In support of the model that the PKC1 pathway regulates SPB duplication, one of the phosphorylated forms of Spc110p was absent in pkc1 and mpk1Delta mutants. PMID: 10924456 [PubMed - indexed for MEDLINE] 415: J Biol Chem 2000 Nov 3;275(44):34068-72 Interactions between Spc2p and other components of the endoplasmic reticulum translocation sites of the yeast Saccharomyces cerevisiae. Antonin W, Meyer HA, Hartmann E. Abteilung Biochemie II, Zentrum Biochemie und Molekulare Zellbiologie, Universitat Gottingen, Heinrich-Duker Weg 12, Gottingen 37073, Germany. In yeast, the endoplasmic reticulum membrane proteins Sec11p and Spc3p are essential for the cleavage of signal peptides of nascent polypeptide chains during their passage through translocation sites. Genetic and biochemical experiments demonstrate that Sec11p and Spc3p are tightly associated with two other proteins, Spc1p and Spc2p, whose functions are largely unknown. Using anti-Spc2p antibodies, we show here that this heterotetrameric complex associates with Sbh1p and Sbh2p, the beta-subunits of the Sec61p complex and the Ssh1p complex, respectively. Depletion of Spc2p decreased the enzymatic activity of the SPC in vitro, led to a loss of Spc1p, and led to a down-regulation of the amount of Sec11p and Spc3p in the endoplasmic reticulum. Moreover, the deletion of Spc2p also decreased the expression level of Sbh2p. These data implicate that Spc2p not only enhances the enzymatic activity of the SPC but also facilitates the interactions between different components of the translocation site. PMID: 10921929 [PubMed - indexed for MEDLINE] 416: EMBO J 2000 Aug 1;19(15):4164-74 Elongation arrest is a physiologically important function of signal recognition particle. Mason N, Ciufo LF, Brown JD. Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK. Signal recognition particle (SRP) targets proteins for co-translational insertion through or into the endoplasmic reticulum membrane. Mammalian SRP slows nascent chain elongation by the ribosome during targeting in vitro. This 'elongation arrest' activity requires the SRP9/14 subunit of the particle and interactions of the C-terminus of SRP14. We have purified SRP from Saccharomyces cerevisiae and demonstrated that it too has elongation arrest activity. A yeast SRP containing Srp14p truncated at its C-terminus (delta C29) did not maintain elongation arrest, was substantially deficient in promoting translocation and interfered with targeting by wild-type SRP. In vivo, this mutation conferred a constitutive defect in the coupling of protein translation and translocation and temperature-sensitive growth, but only a slight defect in protein translocation. In combination, these data indicate that the primary defect in SRP delta C29 is in elongation arrest, and that this is a physiologically important and conserved function of eukaryotic SRP. PMID: 10921896 [PubMed - indexed for MEDLINE] 417: Mutat Res 2000 Jun 30;451(1-2):151-67 Mismatch repair proteins and mitotic genome stability. Harfe BD, Jinks-Robertson S. Department of Biology, Emory University, 1510 Clifton Road, Atlanta, GA 30322, USA. Mismatch repair (MMR) proteins play a critical role in maintaining the mitotic stability of eukaryotic genomes. MMR proteins repair errors made during DNA replication and in their absence, mutations accumulate at elevated rates. In addition, MMR proteins inhibit recombination between non-identical DNA sequences, and hence prevent genome rearrangements resulting from interactions between repetitive elements. This review provides an overview of the anti-mutator and anti-recombination functions of MMR proteins in the yeast Saccharomyces cerevisiae. Publication Types: Review Review, Tutorial PMID: 10915870 [PubMed - indexed for MEDLINE] 418: Mutat Res 2000 Jun 30;451(1-2):71-89 Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae. Lewis LK, Resnick MA. Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, PO Box 12233, 111 Alexander Drive, NIH, Research Triangle Park, NC 27709, USA. The ends of chromosomal DNA double-strand breaks (DSBs) can be accurately rejoined by at least two discrete pathways, homologous recombination and nonhomologous end-joining (NHEJ). The NHEJ pathway is essential for repair of specific classes of DSB termini in cells of the budding yeast Saccharomyces cerevisiae. Endonuclease-induced DSBs retaining complementary single-stranded DNA overhangs are repaired efficiently by end-joining. In contrast, damaged DSB ends (e.g., termini produced by ionizing radiation) are poor substrates for this pathway. NHEJ repair involves the functions of at least 10 genes, including YKU70, YKU80, DNL4, LIF1, SIR2, SIR3, SIR4, RAD50, MRE11, and XRS2. Most or all of these genes are required for efficient recombination-independent recircularization of linearized plasmids and for rejoining of EcoRI endonuclease-induced chromosomal DSBs in vivo. Several NHEJ mutants also display aberrant processing and rejoining of DSBs that are generated by HO endonuclease or formed spontaneously in dicentric plasmids. In addition, all NHEJ genes except DNL4 and LIF1 are required for stabilization of telomeric repeat sequences. Each of the proteins involved in NHEJ appears to bind, directly or through protein associations, with the ends of linear DNA. Enzymatic and/or structural roles in the rejoining of DSB termini have been postulated for several proteins within the group. Most yeast NHEJ genes have homologues in human cells and many biochemical activities and protein:protein interactions have been conserved in higher eucaryotes. Similarities and differences between NHEJ repair in yeast and mammalian cells are discussed. Publication Types: Review Review, Tutorial PMID: 10915866 [PubMed - indexed for MEDLINE] 419: Methods Enzymol 2000;322:297-322 Exploiting the utility of yeast in the context of programmed cell death. Torgler CN, Brown R, Meldrum E. Glaxo Wellcome Medicines Research Centre, Cell Biology Unit, Stevenage, United Kingdom. Many researchers have explored the extent to which yeast can be used to dissect the mechanisms of programmed cell death in higher cells. Yeast has been used as a system to analyze protein-protein interactions and structure-function relationships, and as a cloning tool to identify novel higher eukaryote regulators of apoptosis. In addition, classic genetic strategies in yeast have been used to analyze the mechanisms of action of core pathway members. The purpose of this chapter is to describe the strategies pursued and act as a source for the technical details necessary to exploit the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe in the context of programmed cell death. PMID: 10914027 [PubMed - indexed for MEDLINE] 420: Mol Cell Biol 2000 Aug;20(16):5960-73 Genetic interactions between TFIIS and the Swi-Snf chromatin-remodeling complex. Davie JK, Kane CM. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA. The eukaryotic transcript elongation factor TFIIS enables RNA polymerase II to read through blocks to elongation in vitro and interacts genetically with a variety of components of the transcription machinery in vivo. In Saccharomyces cerevisiae, the gene encoding TFIIS (PPR2) is not essential, and disruption strains exhibit only mild phenotypes and an increased sensitivity to 6-azauracil. The nonessential nature of TFIIS encouraged the use of a synthetic lethal screen to elucidate the in vivo roles of TFIIS as well as provide more information on other factors involved in the regulation of transcript elongation. Several genes were identified that are necessary for either cell survival or robust growth when the gene encoding TFIIS has been disrupted. These include UBP3, KEX2, STT4, and SWI2/SNF2. SWI1 and SNF5 disruptions were also synthetically lethal with ppr2Delta, suggesting that the reduced ability to remodel chromatin confers the synthetic phenotype. The synthetic phenotypes show marked osmosensitivity and cytoskeletal defects, including a terminal hyperelongated bud phenotype with the Swi-Snf complex. These results suggest that genes important in osmoregulation, cell membrane synthesis and integrity, and cell division may require the Swi-Snf complex and TFIIS for efficient transcription. The detection of these genetic interactions provides another functional link between the Swi-Snf complex and the elongation machinery. PMID: 10913179 [PubMed - indexed for MEDLINE] 421: Glycobiology 2000 Jul;10(7):737-44 Evidence for interaction of yeast protein kinase C with several subunits of oligosaccharyl transferase. Park H, Lennarz WJ. Department of Biochemistry and Cell Biology, and Institute for Cell and Developmental Biology, SUNY at Stony Brook, 11794, USA. Oligosaccharyltransferase (OT) in Saccharomyces cerevisiae is an enzyme complex consisting of 8 transmembrane proteins located in the endoplasmic reticulum (ER). Studies on potential protein-protein interactions in OT using a two-hybrid library screen revealed that protein kinase C (Pkc1p) interacted with the lumenal domains of several OT subunits. Additional genetic experiments revealed that overexpression of two OT subunits rescued the growth defect caused by overexpression of a Pkc1 active site mutant, implying that there are specific genetic interactions between PKC1 and OT. These in vivo findings were complemented by in vitro studies that showed that several of the OT subunits bound to a fusion protein consisting of glutathione S-transferase linked via its C-terminus to Pkc1p. Assays of OT activity, in which glycosylation of a simple acceptor peptide was assayed in microsomes from wild-type and a pkc1 null revealed a 50% reduction in activity in the microsomes from the null strain. In contrast, strains containing null mutations of two other genes known to be downstream of Pkc1p in the PKC1-MAP kinase pathway had a level of OT activity comparable to that of wild-type cells. These in vivo and in vitro experiments suggest that in yeast cells Pkc1p may be involved in regulation of the N-glycosylation of proteins. PMID: 10910977 [PubMed - indexed for MEDLINE] 422: Structure Fold Des 2000 Jul 15;8(7):751-62 X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with FMN at 1.8 A resolution. Safo MK, Mathews I, Musayev FN, di Salvo ML, Thiel DJ, Abraham DJ, Schirch V. Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23219, USA. msafo@hsc.vcu.edu BACKGROUND: Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP), a cofactor used by many enzymes involved in amino acid metabolism. The enzyme oxidizes either the 4'-hydroxyl group of pyridoxine 5'-phosphate (PNP) or the 4'-primary amine of pyridoxamine 5'-phosphate (PMP) to an aldehyde. PNPOx is a homodimeric enzyme with one flavin mononucleotide (FMN) molecule non-covalently bound to each subunit. A high degree of sequence homology among the 15 known members of the PNPOx family suggests that all members of this group have similar three-dimensional folds. RESULTS: The crystal structure of PNPOx from E. coli has been determined to 1.8 A resolution. The monomeric subunit folds into an eight-stranded beta sheet surrounded by five alpha-helical structures. Two monomers related by a twofold axis interact extensively along one-half of each monomer to form the dimer. There are two clefts at the dimer interface that are symmetry-related and extend from the top to the bottom of the dimer. An FMN cofactor that makes interactions with both subunits is located in each of these two clefts. CONCLUSIONS: The structure is quite similar to the recently deposited 2.7 A structure of Saccharomyces cerevisiae PNPOx and also, remarkably, shares a common structural fold with the FMN-binding protein from Desulfovibrio vulgaris and a domain of chymotrypsin. This high-resolution E. coli PNPOx structure permits predictions to be made about residues involved in substrate binding and catalysis. These predictions provide testable hypotheses, which can be answered by making site-directed mutants. PMID: 10903950 [PubMed - indexed for MEDLINE] 423: Nucleic Acids Res 2000 Jul 15;28(14):2634-42 The analysis of chimeric human/rainbow trout estrogen receptors reveals amino acid residues outside of P- and D-boxes important for the transactivation function. Petit FG, Valotaire Y, Pakdel F. Equipe d'Endocrinologie Moleculaire de la Reproduction, UPRES-A CNRS 6026, Universite de Rennes I, 35042 Rennes cedex, France. The amino acid sequence of rainbow trout estrogen receptor (rtER) is highly conserved in the C domain but presents few similarities in the A/B and E domains with human estrogen receptor alpha (hER) [NR3A1]. A previous study has shown that rtER and hER have differential functional activities in yeast Saccharomyces cerevisiae. To determine the domain(s) responsible for these differences, chimeric human/rainbow trout estrogen receptors were constructed. The A/B, C/D or E/F regions of rtER were replaced by corresponding regions of hER and expressed in yeast cells. Ligand-binding and transcription activation abilities of these hybrid receptors were compared with those of wild-type rtER or hER. Surprisingly, our data revealed that the human C/D domains play an important role in the magnitude of transactivation of ER. Two other chimeric ERs carrying either a C or D domain of hER showed that the C domain was responsible for this effect whereas the D domain did not affect hybrid receptor activities. Moreover, a chimeric hER carrying the C domain of rtER showed maximal transcriptional activity similar to that observed with rtER. Gel shift assays showed that, whereas rtER and hER present a similar binding affinity to an estrogen response element (ERE) element, the rtER C domain is responsible for a weaker DNA binding stability compared to those of hER. In addition, the human C domain allows approximately 2 times faster association of ER to an ERE. Utilization of reporter genes containing one or three EREs confirms that rtER requires protein-protein interactions for its stabilization on DNA and that the C domain is involved in this stabilization. Moreover, AF-1 may be implicated in this synergistic effect of EREs. Interestingly, although E domains of these two receptors are much less conserved, replacement of this domain in rtER by its human counterpart resulted in higher estradiol sensitivity but no increase in the magnitude of transactivation. Data from the chimeric receptors, rtER(hC) and hER(rtC), demonstrated that rtER AF-1 and AF-2 activation domains activated transcription in the presence of estradiol similar to both AF-1 and AF-2 hER. This implies that these domains, which show poor sequence homology, may interact with similar basal transcription factors. PMID: 10908317 [PubMed - indexed for MEDLINE] 424: Yeast 2000 Jun 30;17(2):95-110 Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins. Fromont-Racine M, Mayes AE, Brunet-Simon A, Rain JC, Colley A, Dix I, Decourty L, Joly N, Ricard F, Beggs JD, Legrain P. Genetique des Interactions Macromoleculaires, CNRS (URA 1300), Institut Pasteur, Paris Cedex 15, France. A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein-protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale. Copyright 2000 John Wiley & Sons, Ltd. PMID: 10900456 [PubMed - indexed for MEDLINE] 425: Yeast 2000 Jun 30;17(2):88-94 Yeast two-hybrid systems and protein interaction mapping projects for yeast and worm. Walhout AJ, Boulton SJ, Vidal M. Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. The availability of complete genome sequences necessitates the development of standardized functional assays to analyse the tens of thousands of predicted gene products in high-throughput experimental settings. Such approaches are collectively referred to as 'functional genomics'. One approach to investigate the properties of a proteome of interest is by systematic analysis of protein-protein interactions. So far, the yeast two-hybrid system is the most commonly used method for large-scale, high-throughput identification of potential protein-protein interactions. Here, we discuss several technical features of variants of the two-hybrid systems in light of data recently obtained from different protein interaction mapping projects for the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. Copyright 2000 John Wiley & Sons, Ltd. Publication Types: Review Review, Tutorial PMID: 10900455 [PubMed - indexed for MEDLINE] 426: Proc Natl Acad Sci U S A 2000 Aug 1;97(16):8967-72 The spliceosomal snRNP core complex of Trypanosoma brucei: cloning and functional analysis reveals seven Sm protein constituents. Palfi Z, Lucke S, Lahm HW, Lane WS, Kruft V, Bragado-Nilsson E, Seraphin B, Bindereif A. Institut fur Biochemie, Justus-Liebig-Universitat Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. Each of the trypanosome small nuclear ribonucleoproteins (snRNPs) U2, U4/U6, and U5, as well as the spliced leader (SL) RNP, contains a core of common proteins, which we have previously identified. This core is unusual because it is not recognized by anti-Sm Abs and it associates with an Sm-related sequence in the trypanosome small nuclear RNAs (snRNAs). Using peptide sequences derived from affinity-purified U2 snRNP proteins, we have cloned cDNAs for five common proteins of 8.5, 10, 12.5, 14, and 15 kDa of Trypanosoma brucei and identified them as Sm proteins SmF (8.5 kDa), -E (10 kDa), -D1 (12.5 kDa), -G (14 kDa), and -D2 (15 kDa), respectively. Furthermore, we found the trypanosome SmB (T. brucei) and SmD3 (Trypanosoma cruzi) homologues through database searches, thus completing a set of seven canonical Sm proteins. Sequence comparisons of the trypanosome proteins revealed several deviations in highly conserved positions from the Sm consensus motif. We have identified a network of specific heterodimeric and -trimeric Sm protein interactions in vitro. These results are summarized in a model of the trypanosome Sm core, which argues for a strong conservation of the Sm particle structure. The conservation extends also to the functional level, because at least one trypanosome Sm protein, SmG, was able to specifically complement a corresponding mutation in yeast. PMID: 10900267 [PubMed - indexed for MEDLINE] 427: Nature 2000 Jul 6;406(6791):94-8 Forkhead-like transcription factors recruit Ndd1 to the chromatin of G2/M-specific promoters. Koranda M, Schleiffer A, Endler L, Ammerer G. Department of Biochemistry and Molecular Cell Biology, Ludwig Boltzmann Forschungsstelle, University of Vienna, Austria. Many cell-cycle-specific events are supported by stage-specific gene expression. In budding yeast, at least three different nuclear factors seem to cooperate in the periodic activation of G2/M-specific genes. Here we show, by using chromatin immunoprecipitation polymerase chain reaction assays, that a positive regulator, Ndd1, becomes associated with G2/M promoter regions in manner that depends on the stage in cell cycle. Its recruitment depends on a permanent protein-DNA complex consisting of the MADS box protein, Mcm1, and a recently identified partner Fkh2, a forkhead/winged helix related transcription factor. The lethality of Ndd1 depletion is suppressed by fkh2 null mutations, which indicates that Fkh2 may also have a negative regulatory role in the transcription of G2/M-induced RNAs. We conclude that Ndd1-Fkh2 interactions may be the transcriptionally important process targeted by Cdk activity. PMID: 10894549 [PubMed - indexed for MEDLINE] 428: Mol Cell Biol 2000 Aug;20(15):5700-11 Histone-histone interactions and centromere function. Glowczewski L, Yang P, Kalashnikova T, Santisteban MS, Smith MM. Department of Microbiology and Cancer Center, University of Virginia, Charlottesville, Virginia 22908, USA. Cse4p is a structural component of the core centromere of Saccharomyces cerevisiae and is a member of the conserved CENP-A family of specialized histone H3 variants. The histone H4 allele hhf1-20 confers defects in core centromere chromatin structure and mitotic chromosome transmission. We have proposed that Cse4p and histone H4 interact through their respective histone fold domains to assemble a nucleosome-like structure at centromeric DNA. To test this model, we targeted random mutations to the Cse4p histone fold domain and isolated three temperature-sensitive cse4 alleles in an unbiased genetic screen. Two of the cse4 alleles contain mutations at the Cse4p-H4 interface. One of these requires two widely separated mutations demonstrating long-range cooperative interactions in the structure. The third cse4 allele is mutated at its helix 2-helix 3 interface, a region required for homotypic H3 fold dimerization. Overexpression of wild-type Cse4p and histone H4 confer reciprocal allele-specific suppression of cse4 and hhf1 mutations, providing strong evidence for Cse4p-H4 protein interaction. Overexpression of histone H3 is dosage lethal in cse4 mutants, suggesting that histone H3 competes with Cse4p for histone H4 binding. However, the relative resistance of the Cse4p-H4 pathway to H3 interference argues that centromere chromatin assembly must be highly regulated. PMID: 10891506 [PubMed - indexed for MEDLINE] 429: Biochemistry 2000 Jul 11;39(27):7886-94 Investigations of the active site of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase using fluorescent labeled dolichyl-phosphate derivatives. Xing J, Forsee WT, Lamani E, Maltsev SD, Danilov LL, Shibaev VN, Schutzbach JS, Cheung HC, Jedrzejas MJ. Department of Biochemistry and Molecular Genetics and Department of Microbiology, The University of Alabama at Birmingham, 933 19th Street South, Birmingham, Alabama 35295-2041, USA. Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the biosynthesis of N-linked oligosaccharides as well as for O-linked oligosaccharides on yeast glycoproteins, and for the synthesis of the glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins. It is synthesized by Dol-P-Man synthase which is the only glycosyltransferase in the dolichol pathway that has been expressed as an active protein, solubilized and purified in large enough quantities for structural investigations. Earlier studies showed that the enzyme is closely associated with membranes of endoplasmic reticulum with unique lipid requirements for its maximal activity. This potential target of antibiotic therapy is now being investigated at the molecular level to establish information about the structure of the enzyme as well as determine the nature and properties of the enzyme-phospholipid interactions. In this paper, we have determined the activities of the fluorescent labeled dolichyl-phosphate derivatives as well as the intramolecular distances between amino acid residues near the active site and/or the fluorophores of the substrate derivatives using fluorescence energy resonance transfer. These results also show that the conserved consensus sequence is not required by Dol-P-Man synthase neither for the recognition of Dol-P nor for the catalytic activity. PMID: 10891068 [PubMed - indexed for MEDLINE] 430: Methods Enzymol 2000;318:374-84 Translational repression assay procedure: a method to study RNA-protein interactions in yeast. Paraskeva E, Hentze MW. Zentrum fur Molekulare Biologie, Universitat Heidelberg, Germany. PMID: 10890000 [PubMed - indexed for MEDLINE] 431: J Biochem Biophys Methods 2000 Jul 10;44(1-2):95-107 Fast, isotope-free methods for the assay of thiamine-binding proteins and for the determination of their affinities to thiamine-related compounds. Mickowska B, Dulinski R, Kozik A. Department of Biochemistry, The Jan Zurzycki Institute of Molecular Biology, Jagiellonian University, Al. Mickiewicza 3, 31-120, Krakow, Poland. A fast, isotope-free method for the determination of parameters for the interactions of proteins with thiamine and related compounds was developed. The free and bound forms of a ligand (thiamine or a fluorogenic analogue) were separated by ultrafiltration using commercially available centrifugal protein microconcentrators (Nanosep, Pall Filtron). The free thiamine concentration in the filtrate was analysed by (i) a pre-column derivatisation of thiamine to thiochrome with the use of alkaline potassium hexacyanoferrate(III) followed by reverse-phase HPLC (isocratic, analytical ODS column, 10 mM potassium phosphate, pH 7.8, 5% tetrahydrofuran) with fluorometric detection (excitation at 365 nm, emission at 430 nm), or (ii) an ion-pair reverse-phase HPLC (isocratic, ODS column, 0.08% trifluoroacetic acid-0.08% sodium octanesulfonate-25% tetrahydrofuran) with post-column derivatisation and fluorometric detection. The 'saturation-binding' version (single ligand added in increasing doses to the protein samples) of this method allowed the determination of low micromolar concentrations of thiamine-binding proteins and of the dissociation constants of their complexes with thiamine or fluorogenic thiamine analogues in the range of 0.3-10 microM. Using the other, 'competitive displacement' version (constant amount of thiamine plus increasing doses of a competing ligand), dissociation constants at least one order of magnitude higher could successfully be determined. PMID: 10889280 [PubMed - indexed for MEDLINE] 432: Mol Biol Cell 2000 Jul;11(7):2335-47 The Skn7 response regulator of Saccharomyces cerevisiae interacts with Hsf1 in vivo and is required for the induction of heat shock genes by oxidative stress. Raitt DC, Johnson AL, Erkine AM, Makino K, Morgan B, Gross DS, Johnston LH. Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom. desmond_raitt@dfci.harvard.edu The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein-protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress. PMID: 10888672 [PubMed - indexed for MEDLINE] 433: J Inorg Biochem 2000 May 30;80(1-2):161-8 Oxo-vanadium as a spin probe for the investigation of the metal coordination environment of imidazole glycerol phosphate dehydratase. Petersen J, Hawkes TR, Lowe DJ. Nitrogen Fixation Laboratory, John Innes Centre, Norwich, UK. Imidazole glycerol phosphate dehydratase (IGPD) catalyses the dehydration of imidazole glycerol phosphate to imidazole acetol phosphate, an important late step in the biosynthesis of histidine. IGPD, isolated as a low molecular weight and inactive apo-form, assembles with specific divalent metal cations to form a catalytically active high molecular weight metalloenzyme. Oxo-vanadium ions also assemble the protein into, apparently, the same high molecular weight form but, uniquely, yield a protein without catalytic activity. The VO2+ derivative of IGPD has been investigated by electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopy. The spin Hamiltonian parameters indicate the presence of multiple 14N nuclei in the inner coordination sphere of VO2+ which is corroborated by ENDOR and ESEEM spectra showing resonances attributable to interactions with 14N nuclei. The isotropic superhyperfine coupling component of about 7 MHz determined by ENDOR is consistent with a nitrogen of coordinated histidine imidazole(s). The ESEEM Fourier-transform spectra further support the notion that the VO2+ substituted enzyme contains inner-sphere nitrogen ligands. The isotropic and anisotropic 14N superhyperfine coupling components are similar to those reported for other equatorially coordinated enzymatic histidine imidazole systems. ESEEM resonances from axial 14N ligands are discussed. Publication Types: Review Review, Tutorial PMID: 10885480 [PubMed - indexed for MEDLINE] 434: Mol Cell 2000 May;5(5):865-76 Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mossessova E, Lima CD. Biochemistry Department, Weill Medical College of Cornell University, New York, New York 10021, USA. Modification of cellular proteins by the ubiquitin-like protein SUMO is essential for nuclear processes and cell cycle progression in yeast. The Ulp1 protease catalyzes two essential functions in the SUMO pathway: (1) processing of full-length SUMO to its mature form and (2) deconjugation of SUMO from targeted proteins. Selective reduction of the proteolytic reaction produced a covalent thiohemiacetal transition state complex between a Ulp1 C-terminal fragment and its cellular substrate Smt3, the yeast SUMO homolog. The Ulp1-Smt3 crystal structure and functional testing of elements within the conserved interface elucidate determinants of SUMO recognition, processing, and deconjugation. Genetic analysis guided by the structure further reveals a regulatory element N-terminal to the proteolytic domain that is required for cell growth in yeast. PMID: 10882122 [PubMed - indexed for MEDLINE] 435: Eur J Biochem 2000 Jul;267(14):4566-76 Reconstitution of ethanolic fermentation in permeabilized spheroplasts of wild-type and trehalose-6-phosphate synthase mutants of the yeast Saccharomyces cerevisiae. Noubhani A, Bunoust O, Rigoulet M, Thevelein JM. Laboratorium voor Moleculaire Celbiologie, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Flanders, Belgium. In the yeast Saccharomyces cerevisiae, TPS1-encoded trehalose-6-phosphate synthase (TPS) exerts an essential control on the influx of glucose into glycolysis, presumably by restricting hexokinase activity. Deletion of TPS1 results in severe hyperaccumulation of sugar phosphates and near absence of ethanol formation. To investigate whether trehalose 6-phosphate (Tre6P) is the sole mediator of hexokinase inhibition, we have reconstituted ethanolic fermentation from glucose in permeabilized spheroplasts of the wild-type, tps1Delta and tps2Delta (Tre6P phosphatase) strains. For the tps1Delta strain, ethanol production was significantly lower and was associated with hyperaccumulation of Glu6P and Fru6P. A tps2Delta strain shows reduced accumulation of Glu6P and Fru6P both in intact cells and in permeabilized spheroplasts. These results are not consistent with Tre6P being the sole mediator of hexokinase inhibition. Reconstitution of ethanolic fermentation in permeabilized spheroplasts with glycolytic intermediates indicates additional target site(s) for the Tps1 control. Addition of Tre6P partially shifts the ethanol production rate and the metabolite pattern in permeabilized tps1Delta spheroplasts to those of the wild-type strain, but only with glucose as substrate. This is observed at a very high ratio of glucose to Tre6P. Inhibition of hexokinase activity by Tre6P is less efficiently counteracted by glucose in permeabilized spheroplasts compared to cell extracts, and this effect is largely abolished by deletion of TPS2 but not TPS1. In permeabilized spheroplasts, hexokinase activity is significantly lower in a tps2Delta strain compared to a wild-type strain and this difference is strongly reduced by additional deletion of TPS1. These results indicate that Tps1-mediated protein-protein interactions are important for control of glucose influx into yeast glycolysis, that Tre6P inhibition of hexokinase might not be competitive with respect to glucose in vivo and that also Tps2 appears to play a role in the control of hexokinase activity. PMID: 10880982 [PubMed - indexed for MEDLINE] 436: Genetics 2000 Jul;155(3):1069-81 MPH1, a yeast gene encoding a DEAH protein, plays a role in protection of the genome from spontaneous and chemically induced damage. Scheller J, Schurer A, Rudolph C, Hettwer S, Kramer W. Abteilung Molekulare Genetik und Praparative Molekularbiologie, Institut fur Mikrobiologie und Genetik, Georg-August-Universitat Gottingen, 37077 Gottingen, Germany. We have characterized the MPH1 gene from Saccharomyces cerevisiae. mph1 mutants display a spontaneous mutator phenotype. Homologs were found in archaea and in the EST libraries of Drosophila, mouse, and man. Mph1 carries the signature motifs of the DEAH family of helicases. Selected motifs were shown to be necessary for MPH1 function by introducing missense mutations. Possible indirect effects on translation and splicing were excluded by demonstrating nuclear localization of the protein and splicing proficiency of the mutant. A mutation spectrum did not show any conspicuous deviations from wild type except for an underrepresentation of frameshift mutations. The mutator phenotype was dependent on REV3 and RAD6. The mutant was sensitive to MMS, EMS, 4-NQO, and camptothecin, but not to UV light and X rays. Epistasis analyses were carried out with representative mutants from various repair pathways (msh6, mag1, apn1, rad14, rad52, rad6, mms2, and rev3). No epistatic interactions were found, either for the spontaneous mutator phenotype or for MMS, EMS, and 4-NQO sensitivity. mph1 slightly increased the UV sensitivity of mms2, rad6, and rad14 mutants, but no effect on X-ray sensitivity was observed. These data suggest that MPH1 is not part of a hitherto known repair pathway. Possible functions are discussed. PMID: 10880470 [PubMed - indexed for MEDLINE] 437: Genetics 2000 Jul;155(3):1033-44 Isolation and characterization of HRT1 using a genetic screen for mutants unable to degrade Gic2p in saccharomyces cerevisiae. Blondel M, Galan JM, Peter M. Swiss Institute for Experimental Cancer Research (ISREC), 1066 Epalinges/VD, Switzerland. Skp1p-cullin-F-box (SCF) protein complexes are ubiquitin ligases required for degradation of many regulatory proteins involved in cell cycle progression, morphogenesis, and signal transduction. Using a genetic screen, we have isolated a novel allele of the HRT1/RBX1 gene in budding yeast (hrt1-C81Y). hrt1-C81Y mutant cells exhibited an aberrant morphology but were viable at all temperatures. The cells displayed multiple genetic interactions with mutations in known SCF components and were defective for the degradation of several SCF targets including Gic2p, Far1p, Sic1p, and Cln2p. In addition, they also failed to degrade the F-box proteins Grr1p, Cdc4p, and Met30p. Wild-type Hrt1p but not Hrt1p-C81Y was able to bind multiple F-box proteins in an F-box-dependent manner. Hrt1p-C81Y harbors a single mutation in its ring-finger domain, which is conserved in subunits of distinct E3 ligases. Finally, Hrt1p was localized in both nucleus and cytoplasm and despite a short half-life was expressed constitutively throughout the cell cycle. Taken together, these results suggest that Hrt1p is a core subunit of multiple SCF complexes. PMID: 10880467 [PubMed - indexed for MEDLINE] 438: EMBO J 2000 Jul 3;19(13):3215-22 The yeast prion [URE3] can be greatly induced by a functional mutated URE2 allele. Fernandez-Bellot E, Guillemet E, Cullin C. Centre de Genetique Moleculaire, Centre National de la Recherche Scientifique, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. The non-Mendelian element [URE3] of yeast is considered to be a prion form of the Ure2 protein. The [URE3] phenotype occurs at a frequency of 10(-5) in haploid yeast strains, is reversible, and its frequency is increased by overexpressing the URE2 gene. We created a new mutant of the Ure2 protein, called H2p, which results in a 1000-fold increase in the rate of [URE3] occurrence. To date, only the overexpression of various C-terminal truncated mutants of Ure2p gives rise to a comparable level. The h2 allele is, thus, the first characterized URE2 allele that induces prion formation when expressed at a low level. By shuffling mutated and wild-type domains of URE2, we also created the first mutant Ure2 protein that is functional and induces prion formation. We demonstrate that the domains of URE2 function synergistically in cis to induce [URE3] formation, which highlights the importance of intramolecular interactions in Ure2p folding. Additionally, we show using a green fluorescent protein (GFP) fusion protein that the h2 allele exhibits numerous filiform structures that are not generated by the wild-type protein. PMID: 10880435 [PubMed - indexed for MEDLINE] 439: J Biol Chem 2000 Sep 15;275(37):28816-25 Subunit interactions within the Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon ) complex. Demonstration of a dimeric pol epsilon. Dua R, Edwards S, Levy DL, Campbell JL. Braun Laboratories, California Institute of Technology, Pasadena, California 91125, USA. Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon) is essential for chromosomal replication. A major form of pol epsilon purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay. The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits. We propose that Pol2p.Dpb2p and Dpb3p.Dpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes. We present biochemical evidence to support the proposal that pol epsilon may be dimeric in vivo. Gel filtration of the Pol2p.Dpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2p.Dpb2p. Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p. This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication. PMID: 10878005 [PubMed - indexed for MEDLINE] 440: J Biol Chem 2000 Jul 7;275(27):20527-32 Influence of FAD structure on its binding and activity with the C406A mutant of recombinant human liver monoamine oxidase A. Nandigama RK, Edmondson DE. Departments of Biochemistry and Chemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA. The FAD binding site of human liver monoamine oxidase A (MAO A) has been investigated by mutagenesis of the amino acid site of covalent FAD attachment (Cys-406) to an alanyl residue. Expression of the C406A mutant in Saccharomyces cerevisiae results in the formation of an active enzyme, as found previously with the rat liver enzyme. The activity of this mutant enzyme is labile to solubilization, thus requiring all experiments to be done with membrane preparations. C406A MAO A was expressed in a rib 5(-) strain of S. cerevisiae in the presence of 16 different riboflavin analogues. Inactive apoC406A MAO A is formed by induction of the enzyme in the absence of riboflavin. FAD but not FMN or riboflavin restores catalytic activity with an apparent K(d) of 62 +/- 5 nm. The results from both in vivo and in vitro reconstitution experiments show increased activity levels (up to approximately 7-fold higher) with those analogues exhibiting higher oxidation-reduction potentials than normal flavin and decreased activity levels with analogues exhibiting lower potentials. Analogues with substituents on the pyrimidine ring bind to C406A MAO A more weakly than normal FAD, suggesting specific interactions with the N(3) and N(1) positions. Analogues with substituents in the 7 and 8 positions bind to C406A MAO A with affinities comparable with that of normal FAD. These results are discussed in regard to functional significance of 8alpha-covalent binding of flavins to proteins. PMID: 10877844 [PubMed - indexed for MEDLINE] 441: Curr Biol 2000 Jun 15;10(12):727-30 A myosin light chain mediates the localization of the budding yeast IQGAP-like protein during contractile ring formation. Shannon KB, Li R. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. Cytokinesis in animal cells is accomplished through constriction of an actomyosin ring [1] [2] [3], which must assemble at the correct time and place in order to ensure proper division of genetic material and organelles. Budding yeast is a useful model system for determining the biochemical pathway of contractile ring assembly. The budding yeast IQGAP-like protein, Cyk1/Iqg1p, has multiple roles in the assembly and contraction of the actomyosin ring [4] [5] [6]. Previously, the IQ motifs of Cyk1/Iqg1p were shown to be required for the localization of this protein at the bud neck [6]. We have investigated the binding partner of the IQ motifs, which are predicted to interact with calmodulin-like proteins. Mlc1p was originally identified as a light chain for a type V myosin, Myo2p; however, a cytokinesis defect associated with disruption of the MLC1 gene suggested that the essential function of Mlc1p may involve interactions with other proteins [7]. We show that Mlc1p binds the IQ motifs of Cyk1/Iqg1p and present evidence that this interaction recruits Cyk1/Iqg1p to the bud neck. Immunofluorescence staining shows that Mlc1p is localized to sites of polarized cell growth as well as the bud neck before and independently of Cyk1p. These results demonstrate that Mlc1p is important for the assembly of the actomyosin ring in budding yeast and that this function is mediated through interaction with Cyk1/Iqg1p. PMID: 10873803 [PubMed - indexed for MEDLINE] 442: Annu Rev Biochem 1999;68:649-86 MCM proteins in DNA replication. Tye BK. Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853-2703, USA. The MCM proteins are essential replication initiation factors originally identified as proteins required for minichromosome maintenance in Saccharomyces cerevisiae. The best known among them are a family of six structurally related proteins, MCM2-7, which are evolutionally conserved in all eukaryotes. The MCM2-7 proteins form a hexameric complex. This complex is a key component of the prereplication complex that assembles at replication origins during early G1 phase. New evidence suggests that the MCM2-7 proteins may be involved not only in the initiation but also in the elongation of DNA replication. Orchestration of the functional interactions between the MCM2-7 proteins and other components of the prereplication complex by cell cycle-dependent protein kinases results in initiation of DNA synthesis once every cell cycle. Publication Types: Review Review, Academic PMID: 10872463 [PubMed - indexed for MEDLINE] 443: Proc Natl Acad Sci U S A 2000 Jul 5;97(14):7916-20 A regulatory shortcut between the Snf1 protein kinase and RNA polymerase II holoenzyme. Kuchin S, Treich I, Carlson M. Department of Genetics and Development and Department of Microbiology, Columbia University, New York, NY 10032, USA. RNA polymerase II holoenzymes respond to activators and repressors that are regulated by signaling pathways. Here we present evidence for a "shortcut" mechanism in which the Snf1 protein kinase of the glucose signaling pathway directly regulates transcription by the yeast holoenzyme. In response to glucose limitation, the Snf1 kinase stimulates transcription by holoenzyme that has been artificially recruited to a reporter by a LexA fusion to a holoenzyme component. We show that Snf1 interacts physically with the Srb/mediator proteins of the holoenzyme in both two-hybrid and coimmunoprecipitation assays. We also show that a catalytically hyperactive Snf1, when bound to a promoter as a LexA fusion protein, activates transcription in a glucose-regulated manner; moreover, this activation depends on the integrity of the Srb/mediator complex. These results suggest that direct regulatory interactions between signal transduction pathways and RNA polymerase II holoenzyme provide a mechanism for transcriptional control in response to important signals. PMID: 10869433 [PubMed - indexed for MEDLINE] 444: Mol Cell Biol 2000 Jul;20(14):5321-9 The C terminus of the Saccharomyces cerevisiae alpha-factor receptor contributes to the formation of preactivation complexes with its cognate G protein. Dosil M, Schandel KA, Gupta E, Jenness DD, Konopka JB. Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York 11794-5222, USA. Binding of the alpha-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Galpha subunits in an alpha-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant Galpha subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the alpha-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for alpha-factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the alpha-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor-G-protein preactivation complexes. PMID: 10866688 [PubMed - indexed for MEDLINE] 445: J Biol Chem 2000 Sep 1;275(35):26925-34 The TATA-binding protein-associated factor yTafII19p functionally interacts with components of the global transcriptional regulator Ccr4-Not complex and physically interacts with the Not5 subunit. Lemaire M, Collart MA. Departement de Biochimie Medicale, Centre Medical Universitaire, 1 rue Michel Servet, 1211 Geneva 4, Switzerland. The Saccharomyces cerevisiae HIS3 gene is a model system to characterize transcription initiation from different types of core promoters. The NOT genes were identified by mutations that preferentially increased transcription of the HIS3 promoter lacking a canonical TATA sequence. They encode proteins associated in a complex that also contains the Caf1 and Ccr4 proteins. It has been suggested that the Ccr4-Not complex represses transcription by inhibiting factors more specifically required for promoters lacking a TATA sequence. A potential target is the yTaf(II)19 subunit of TFIID, which, when depleted, leads to a preferential decrease of HIS3 TATA-less transcription. We isolated conditional taf19 alleles that display synthetic growth phenotypes when combined with not4 or specific not5 alleles. Inactivation of yTaf(II)19p by shifting these mutants to the restrictive temperature led to a more rapid and striking decrease in transcription from promoters that do not contain a canonical TATA sequence. We demonstrated by the two-hybrid assay and directly in vitro that yTaf(II)19p and Not5p could interact. Finally, we found by the two-hybrid assay that yTaf(II)19p also interacted with many components of the Ccr4-Not complex. Taken together, our results provide evidence that interactions between Not5p and yTaf(II)19p may be involved in transcriptional regulation by the Ccr4-Not complex. PMID: 10864925 [PubMed - indexed for MEDLINE] 446: J Mol Biol 2000 Jun 30;300(1):11-6 A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase. Morgan CJ, Wilkins DK, Smith LJ, Kawata Y, Dobson CM. Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QT, UK. The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy. In the absence of denaturant, the hydrodynamic radius of 29.6(+/-0.25) A and the substantial chemical shift dispersion evident in the NMR spectrum are consistent with the highly structured dimeric native state of the protein. On the addition of 2. 2 M guanidine hydrochloride the effective hydrodynamic radius increases to 51.4(+/-0.43) A, consistent with that anticipated for the polypeptide chain in a highly unstructured random coil state. In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(+/-0.25) A, a value substantially decreased relative to that of the native dimeric state but very close to that anticipated for a monomeric species with native-like compaction (23. 5 A). The lack of chemical shift dispersion indicates, however, that few tertiary interactions persist within this species. Far UV CD and intrinsic fluorescence measurements show that this compact intermediate retains significant secondary structure and that on average the fluorophores are partially excluded from solvent. Such a species could be important in the formation of dimeric TIM from its unfolded state. Copyright 2000 Academic Press. PMID: 10864494 [PubMed - indexed for MEDLINE] 447: J Clin Invest 2000 Jun;105(12):1711-21 Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase. Hallows KR, Raghuram V, Kemp BE, Witters LA, Foskett JK. Renal-Electrolyte and Hypertension Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl(-) channel that regulates other epithelial transport proteins by uncharacterized mechanisms. We employed a yeast two-hybrid screen using the COOH-terminal 70 residues of CFTR to identify proteins that might be involved in such interactions. The alpha1 (catalytic) subunit of AMP-activated protein kinase (AMPK) was identified as a dominant and novel interacting protein. The interaction is mediated by residues 1420-1457 in CFTR and by the COOH-terminal regulatory domain of alpha1-AMPK. Mutations of two protein trafficking motifs within the 38-amino acid region in CFTR each disrupted the interaction. GST-fusion protein pull-down assays in vitro and in transfected cells confirmed the CFTR-alpha1-AMPK interaction and also identified alpha2-AMPK as an interactor with CFTR. AMPK is coexpressed in CFTR-expressing cell lines and shares an apical distribution with CFTR in rat nasal epithelium. AMPK phosphorylated full-length CFTR in vitro, and AMPK coexpression with CFTR in XENOPUS: oocytes inhibited cAMP-activated CFTR whole-cell Cl(-) conductance by approximately 35-50%. Because AMPK is a metabolic sensor in cells and responds to changes in cellular ATP, regulation of CFTR by AMPK may be important in inhibiting CFTR under conditions of metabolic stress, thereby linking transepithelial transport to cell metabolic state. PMID: 10862786 [PubMed - indexed for MEDLINE] 448: Yeast 2000 Jun 30;16(9):811-27 Mutational analysis of the karmellae-inducing signal in Hmg1p, a yeast HMG-CoA reductase isozyme. Profant DA, Roberts CJ, Wright RL. Department of Zoology, Box 351800, University of Washington, Seattle, WA 98195, USA. In response to elevated levels of HMG-CoA reductase, an integral endoplasmic reticulum (ER) membrane protein, cells assemble novel ER arrays. These membranes provide useful models for exploration of ER structure and function, as well as general features of membrane biogenesis and turnover. Yeast express two functional HMG-CoA reductase isozymes, Hmg1p and Hmg2p, each of which induces morphologically different ER arrays. Hmg1p induces stacks of paired nuclear-associated membranes called karmellae. In contrast, Hmg2p induces peripheral ER membrane arrays and short nuclear-associated membrane stacks. In spite of their ability to induce different cellular responses, both Hmg1p and Hmg2p have similar structures, including a polytopic membrane domain containing eight predicted transmembrane helices. By examining a series of recombinant HMG-CoA reductase proteins, our laboratory previously demonstrated that the last ER-lumenal loop (Loop G) of the Hmg1p membrane domain contains a signal needed for proper karmellae assembly. Our goal was to examine the primary sequence requirements within Loop G that were critical for proper function of this signal. To this end, we randomly mutagenized the Loop G sequence, expressed the mutagenized Hmg1p in yeast, and screened for inability to generate karmellae at wild-type levels. Out of approximately 4000 strains with Loop G mutations, we isolated 57 that were unable to induce wild-type levels of karmellae assembly. Twenty-nine of these mutants contained one or more point mutations in the Loop G sequence, including nine single point mutants, four of which had severe defects in karmellae assembly. Comparison of these mutations to single point mutations that did not affect karmellae assembly did not reveal obvious patterns of sequence requirements. For example, both conservative and non-conservative changes were present in both groups and changes that altered the total charge of the Loop G region were observed in both groups. Our hypothesis is that Loop G serves as a karmellae-inducing signal by mediating protein-protein or protein-lipid interactions and that amino acids revealed by this analysis may be important for maintaining the proper secondary structure needed for these interactions. Copyright 2000 John Wiley & Sons, Ltd. PMID: 10861905 [PubMed - indexed for MEDLINE] 449: EMBO J 2000 Jun 15;19(12):3016-27 Structure of the C-terminal domain of Tup1, a corepressor of transcription in yeast. Sprague ER, Redd MJ, Johnson AD, Wolberger C. Department of Biophysics and Biophysical Chemistry and Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. The Tup1-Ssn6 corepressor complex regulates the expression of several sets of genes, including genes that specify mating type in the yeast Saccharomyces cerevisiae. Repression of mating-type genes occurs when Tup1-Ssn6 is brought to the DNA by the Matalpha2 DNA-binding protein and assembled upstream of a- and haploid-specific genes. We have determined the 2.3 A X-ray crystal structure of the C-terminal domain of Tup1 (accesion No. 1ERJ), a 43 kDa fragment that contains seven copies of the WD40 sequence motif and binds to the Matalpha2 protein. Moreover, this portion of the protein can partially substitute for full-length Tup1 in bringing about transcriptional repression. The structure reveals a seven-bladed beta propeller with an N-terminal subdomain that is anchored to the side of the propeller and extends the beta sheet of one of the blades. Point mutations in Tup1 that specifically affect the Tup1-Matalpha2 interaction cluster on one surface of the propeller. We identified regions of Tup1 that are conserved among the fungal Tup1 homologs and may be important in protein-protein interactions with additional components of the Tup1-mediated repression pathways. PMID: 10856245 [PubMed - indexed for MEDLINE] 450: Chromosoma 2000;109(1-2):86-93 Meiotic recombination in RAD54 mutants of Saccharomyces cerevisiae. Schmuckli-Maurer J, Heyer WD. Institute of General Microbiology, University of Bern, Switzerland. The Rad54 protein is an important component of the recombinational DNA repair pathway in vegetative Saccharomyces cerevisiae cells. Unlike those in other members of the RAD52 group, the meiotic defect in rad54 is rather mild, reducing spore viability only to 26%-65%. A consistently greater requirement for Rad54p during meiosis was observed in hybrid strains, suggesting that Rad54p has a certain role in interhomolog interactions. Such a role is probably minor as no recombination defects were found in the surviving gametes in three genetic intervals on chromosome V. Also, the spore viability pattern in tetrads did not reflect an increase in nondisjunction at meiosis I indicative of a meiotic recombination defect. We suggest that the meiotic defect of rad54 cells lies in the failure to repair meiosis-specific double-strand breaks outside the context of the highly differentiated pathway leading to interhomolog joint molecules and meiotic crossovers that ensure accurate segregation at meiosis I. PMID: 10855498 [PubMed - indexed for MEDLINE] 451: Gene 2000 May 30;250(1-2):1-14 The continued evolution of two-hybrid screening approaches in yeast: how to outwit different preys with different baits. Fashena SJ, Serebriiskii I, Golemis EA. Division of Basic Science, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. The original two-hybrid system, an experimental approach designed to detect protein interactions, exploited the modular nature of many transcription factors. It has provided the intellectual and technical seed for the evolution of an array of innovative approaches, the application of which broadens the scope of experimentally feasible questions to include the interaction of proteins with diverse binding partners. The available array of modified and alternative approaches facilitates the analysis of complex cellular machinery and signaling networks that rely on multiple protein interactions. Such advances have facilitated the functional analysis of proteins on the genome level, a feat considered untenable a decade ago. Publication Types: Review Review, Tutorial PMID: 10854774 [PubMed - indexed for MEDLINE] 452: Curr Opin Microbiol 2000 Jun;3(3):303-8 Systematic and large-scale two-hybrid screens. Uetz P, Hughes RE. Department of Genetics, University of Washington, Box 357360, Seattle, WA 98195-7360, USA. uetz@u.washington.edu The increasing rate at which complete genome sequences become available necessitates rapid and robust methods for investigating the functions of their encoded proteins. Efforts have been made to study protein function by systematically screening large sets of proteins using the two-hybrid method. Analyses of the complete proteomes of baceriophage T7, the mammalian viruses hepatitis C and vaccinia, as well as of several protein complexes including RNA splicing proteins and RNA polymerase III from yeast, have been undertaken. Saccharomyces cerevisiae has been studied extensively by two-hybrid methods, with more than 2500 protein-protein interactions described. Systematic studies on metazoan proteomes are, however, still in their infancy. Publication Types: Review Review, Tutorial PMID: 10851163 [PubMed - indexed for MEDLINE] 453: Plant Cell Physiol 2000 Apr;41(4):523-33 Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors. Athwal GS, Lombardo CR, Huber JL, Masters SC, Fu H, Huber SC. US Department of Agriculture, Department of Horticultural Science, North Carolina State University, Raleigh 27695, USA. The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity. PMID: 10845467 [PubMed - indexed for MEDLINE] 454: Mol Cells 2000 Apr 30;10(2):232-5 Random changes of amino acid residues with expected frequency by saturated point mutagenesis. Kim SJ, Park H, Kim JK, Lee JY, Ahn K, Choe M, Choi YJ, Kim J. Graduate School of Biotechnology, Korea University, Seoul. The yeast transcriptional activator protein, Gcn4p from Saccharomyces cerevisiae binds to the specific sequence in the promoters of many amino acid biosynthetic genes for general control. A new random saturation mutagenesis method was developed to isolate Gcn4p derivatives with only one or two mutations in the DNA binding domain without using radioactive isotope. This will be used to identify the amino acids of Gcn4p involved in protein-protein interactions. Saturation mutagenesis in the DNA binding domain of Gcn4p was performed using spiked degenerate oligonucleotides containing randomized codon bases designed specifically for only one or two base changes in the mutagenized area. These oligonucleotides were synthesized to have two flanking restriction enzyme sites for cloning to the appropriate vector. The 3' ends were mutually primed after hybridization via the palindromic sequences of the restriction enzyme sites. These molecules were then converted to double stranded DNA upon treatment with DNA polymerase. Here, a library collection of 100,680 in an altered Gcn4p pool was generated by cloning a mixed-base oligonucleotide in the place of the sequence coding for the DNA binding domains. The quality of the library was examined by DNA sequencing and found to be in good agreement with the expected statistical values. Calculated mutation frequency was 66% of mutant nucleotide rate and actual sequencing data revealed 68% mutant nucleotide rates from the sequenced library. Thus, among 21 mutants, 16 had one point mutations and 5 had two point mutations. This approach appears to be an effective and general tool for creating proteins with one or two amino acid change(s) in their molecules. PMID: 10850667 [PubMed - indexed for MEDLINE] 455: Mol Cell Biol 2000 Jul;20(13):4838-48 Bypass of a meiotic checkpoint by overproduction of meiotic chromosomal proteins. Bailis JM, Smith AV, Roeder GS. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA. The Saccharomyces cerevisiae zip1 mutant, which exhibits defects in synaptonemal complex formation and meiotic recombination, triggers a checkpoint that causes cells to arrest at the pachytene stage of meiotic prophase. Overproduction of either the meiotic chromosomal protein Red1 or the meiotic kinase Mek1 bypasses this checkpoint, allowing zip1 cells to sporulate. Red1 or Mek1 overproduction also promotes sporulation of other mutants (zip2, dmc1, hop2) that undergo checkpoint-mediated arrest at pachytene. In addition, Red1 overproduction antagonizes interhomolog interactions in the zip1 mutant, substantially decreasing double-strand break formation, meiotic recombination, and homologous chromosome pairing. Mek1 overproduction, in contrast, suppresses checkpoint-induced arrest without significantly decreasing meiotic recombination. Cooverproduction of Red1 and Mek1 fails to bypass the checkpoint; moreover, overproduction of the meiotic chromosomal protein Hop1 blocks the Red1 and Mek1 overproduction phenotypes. These results suggest that meiotic chromosomal proteins function in the signaling of meiotic prophase defects and that the correct stoichiometry of Red1, Mek1, and Hop1 is needed to achieve checkpoint-mediated cell cycle arrest at pachytene. PMID: 10848609 [PubMed - indexed for MEDLINE] 456: Mol Cell Biol 2000 Jul;20(13):4806-13 Identification of amino acid residues in the Caenorhabditis elegans POU protein UNC-86 that mediate UNC-86-MEC-3-DNA ternary complex formation. Rockelein I, Rohrig S, Donhauser R, Eimer S, Baumeister R. Genzentrum, Ludwig-Maximilians-Universitat, D-81377 Munich, Germany. The POU homeodomain protein UNC-86 and the LIM homeodomain protein MEC-3 are essential for the differentiation of the six mechanoreceptor neurons in the nematode Caenorhabditis elegans. Previous studies have indicated that UNC-86 and MEC-3 bind cooperatively to at least three sites in the mec-3 promoter and synergistically activate transcription. However, the molecular details of the interactions of UNC-86 with MEC-3 and DNA have not been investigated so far. Here we used a yeast system to identify the functional domains in UNC-86 required for transcriptional activation and to characterize the interaction of UNC-86 with MEC-3 in vivo. Our results suggest that transcriptional activation is mediated by the amino terminus of UNC-86, whereas amino acids in the POU domain mediate DNA binding and interaction with MEC-3. By random mutagenesis, we identified mutations that only affect the DNA binding properties of UNC-86, as well as mutations that prevent coactivation by MEC-3. We demonstrated that both the POU-specific domain and the homeodomain of UNC-86, as well as DNA bases adjacent to the proposed UNC-86 binding site, are involved in the formation of a transcriptionally active complex with MEC-3. These data suggest that some residues involved in the contact of UNC-86 with MEC-3 also contribute to the interaction of the functionally nonrelated POU protein Oct-1 with Oca-B, whereas other positions have different roles. PMID: 10848606 [PubMed - indexed for MEDLINE] 457: Mol Endocrinol 2000 Jun;14(6):889-99 Characterization of transactivational property and coactivator mediation of rat mineralocorticoid receptor activation function-1 (AF-1). Fuse H, Kitagawa H, Kato S. Pharmacological Research Department, Teikoku Hormone Manufacturing Company, Ltd., Tokyo, Japan. The autonomous activation function-2 (AF-2) in the mineralocorticoid receptor (MR) E/F domain is known to play a major role in the ligand-induced transactivation function of MR; however, it remained unclear about the transactivation function of its A/B domain. We therefore tried to characterize the MR A/B domain as the AF-1 and further studied the actions of known coactivators for AF-2 in the E/F ligand-binding domain in the function of the MR A/B domain. Deletion analyses of rat and human MRs revealed that the A/B domains harbor a transactivation function acting as AF-1. The MR mutant (E959Q) with a point mutation in helix 12, which causes a complete loss of MR AF-2 activity, still retained ligand-induced transactivation function, indicating a significant role for AF-1 in the full activity of the ligand-induced MR function. Among the coactivators tested to potentiate the MR AF-2, TIF2 and p300 potentiated the MR AF-1 through two different core regions [amino acids (a.a.) 1-169, a.a. 451-603] and exhibited functional interactions with the MR A/B domain in the cultured cells. However, such interactions were undetectable in a yeast and in an in vitro glutathione-S-transferase pull-down assay, indicating that the functional interaction of TIF2 and p300 with the MR A/B domain to support the MR AF-1 activity require some unknown nuclear factor(s) or a proper modification of the A/B domain in the cells. PMID: 10847590 [PubMed - indexed for MEDLINE] 458: J Cell Biochem 2000 May;78(2):179-85 Recruitment of chromatin remodeling machines. Peterson CL, Logie C. Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. craig.peterson@umassmed.edu The assembly of eukaryotic DNA into folded nucleosomal arrays has drastic consequences for many nuclear processes that require access to the DNA sequence, including RNA transcription, DNA replication, recombination, and repair. Two types of highly conserved chromatin remodeling enzymes have been implicated as regulators of the repressive nature of chromatin structure: ATP-dependent remodeling complexes and nuclear histone acetyltransferases (HATs). Recent studies indicate that both types of enzymes can be recruited to chromosomal loci through either physical interactions with transcriptional activators or via the global accessibility of chromatin during S phase of the cell cycle. Here we review these recent observations and discuss the implications for gene-specific regulation by chromatin remodeling machines. Copyright 2000 Wiley-Liss, Inc. Publication Types: Review Review, Tutorial PMID: 10842313 [PubMed - indexed for MEDLINE] 459: Bioessays 2000 Jun;22(6):503-6 Building a protein interaction map: research in the post-genome era. Chen Z, Han M. Howard Hughes Medical Institute, University of Colorado at Boulder, Boulder, Colorado. With the extensive amount of information generated by genome-wide sequencing, the entire set of gene products in an organism can now be predicted. The challenge of understanding the function of each gene in the genome has led to the development of many large-scale and high-throughput experimental techniques. Recently, two papers, Walhout et al.(1) and Uetz et al.,(2) have described studies that add a new functional dimension to research conducted on a genome-wide scale. These two groups have utilized the yeast two-hybrid system to identify interactions among the entire complement of proteins encoded by the Caenorhabditis elegans and the Saccharomyces cerevisiae genomes, respectively. Using a set of 29 genes that have been previously characterized, Walhout et al. demonstrated the feasibility and efficiency of this technique by building an interaction matrix among a large number of proteins. On an even larger scale, Uetz et al. conducted two-hybrid analyses using proteins that represent over 87% of the total gene products in yeast and identified interactions for about 15% of the total yeast proteins. BioEssays 22:503-506, 2000. Copyright 2000 John Wiley & Sons, Inc. Publication Types: Review Review, Tutorial PMID: 10842303 [PubMed - indexed for MEDLINE] 460: J Biol Chem 2000 Sep 22;275(38):29368-76 Rap1p-binding sites in the saccharomyces cerevisiae GPD1 promoter are involved in its response to NaCl. Eriksson P, Alipour H, Adler L, Blomberg A. Department of Cell and Molecular Biology-Microbiology, Lundberg Laboratory, Goteborg University, Medicinaregatan 9C, S-413 90 Goteborg, Sweden. Mechanisms involved in transcriptional regulation of the osmotically controlled GPD1 gene in Saccharomyces cerevisiae were investigated by promoter analysis. The GPD1 gene encodes NAD(+)-dependent glycerol-3-phosphate dehydrogenase, a key enzyme in the production of the compatible solute glycerol. By analysis of promoter deletions, we identified a region at nucleotides -478 to -324, in relation to start of translation, to be of great importance for both basal activity and osmotic induction of GPD1. Electrophoretic mobility shift and DNase I footprint analyses demonstrated protein binding to parts of this region that contain three consensus sequences for Rap1p (repressor activator protein 1)-binding sites. Actual binding of Rap1p to this region was confirmed by demonstrating enhanced electrophoretic mobility of the protein-DNA complex with extracts containing an N-terminally truncated version of Rap1p. The detected Rap1p-DNA interactions were not affected by changes in the osmolarity of the growth medium. Specific inactivation of the Rap1p-binding sites by a C-to-A point mutation in the core of the consensus showed that this factor is a major determinant of GPD1 expression since mutations in all three putative binding sites for Rap1p strongly hampered osmotic induction and drastically lowered basal activity. We also show that the Rap1p-binding sites appear functionally distinct; the most distal site (core of the consensus at position -386) exhibited the highest affinity for Rap1p and was strictly required for low salt induction (< or =0.6 m NaCl), but not for the response at higher salinities (> or =0.8 m NaCl). This indicates tha different molecular mechanisms might be operational for low and high salt responses of the GPD1 promoter. PMID: 10842169 [PubMed - indexed for MEDLINE] 461: Biochemistry 2000 Jun 13;39(23):6910-7 Prenyl-flavonoids as potent inhibitors of the Pdr5p multidrug ABC transporter from Saccharomyces cerevisiae. Conseil G, Decottignies A, Jault JM, Comte G, Barron D, Goffeau A, Di Pietro A. Laboratoire de Biochimie Structurale et Fonctionnelle, Institut de Biologie et Chimie des Protinverted question markeines, UPR 412 du Centre National de la Recherche Scientifique, Lyon, France. The Pdr5p multidrug ABC ("ATP-binding cassette) transporter was highly overexpressed in plasma membranes from a yeast strain exhibiting both pdr1-3 gain-of-function mutation in the transcription factor-encoding gene PDR1 and disruption of genes encoding other plasma membrane ABC transporters. Solubilized and purified Pdr5p displayed a tryptophan-characteristic intrinsic fluorescence, whose quenching was used to monitor interactions with substrates and effectors. The transporter exhibited a magnesium-dependent binding affinity for ATP and its fluorescent analogue 2'(3')-N-methylanthraniloyl-ATP, producing a marked fluorescence resonance-energy transfer. It also bound a series of known drug substrates and modulators. Interestingly, yeast Pdr5p interacted with flavonoids recently found to bind to cancer cell P-glycoprotein and to the protozoan parasite multidrug transporter. The extent of high-affinity binding of prenyl-flavonoids to purified Pdr5p was correlated to their efficiency to inhibit energy-dependent quenching of rhodamine 6G fluorescence catalyzed by Pdr5p-enriched plasma membranes. The hydrophobic flavonoid derivative 6-(3, 3-dimethylallyl)galangin was the most efficient, with a K(i) of 0.18 microM for competitive inhibition of the MgATP-dependent quenching of rhodamine 6G fluorescence. In contrast, inhibition of either ATP or UTP hydrolysis occurred at much higher concentrations and appeared to be noncompetitive. Prenyl-flavonoids therefore behave as potent inhibitors of drug binding to the yeast Pdr5p ABC transporter. PMID: 10841772 [PubMed - indexed for MEDLINE] 462: Proc Natl Acad Sci U S A 2000 Jun 6;97(12):6306-10 Crystal structure of RPB5, a universal eukaryotic RNA polymerase subunit and transcription factor interaction target. Todone F, Weinzierl RO, Brick P, Onesti S. Blackett Laboratory and Department of Biochemistry, Imperial College, Exhibition Road, London SW7 2AZ, United Kingdom. Eukaryotic nuclei contain three different types of RNA polymerases (RNAPs), each consisting of 12-18 different subunits. The evolutionarily highly conserved RNAP subunit RPB5 is shared by all three enzymes and therefore represents a key structural/functional component of all eukaryotic RNAPs. Here we present the crystal structure of the RPB5 subunit from Saccharomyces cerevisiae. The bipartite structure includes a eukaryote-specific N-terminal domain and a C-terminal domain resembling the archaeal RNAP subunit H. RPB5 has been implicated in direct protein-protein contacts with transcription factor IIB, one of the components of the RNAP(II) basal transcriptional machinery, and gene-specific activator proteins, such as the hepatitis B virus transactivator protein X. The experimentally mapped regions of RPB5 involved in these interactions correspond to distinct and surface-exposed alpha-helical structures. PMID: 10841537 [PubMed - indexed for MEDLINE] 463: Biochim Biophys Acta 2000 May 31;1458(2-3):443-56 Organisation of the yeast ATP synthase F(0):a study based on cysteine mutants, thiol modification and cross-linking reagents. Velours J, Paumard P, Soubannier V, Spannagel C, Vaillier J, Arselin G, Graves PV. Institut de Biochimie et Genetique Cellulaires du CNRS, 1 rue Camille Saint Saens, 33077, cedex, Bordeaux, France. john.velours@ibgc.u-bordeaux2.fr A topological study of the yeast ATP synthase membranous domain was undertaken by means of chemical modifications and cross-linking experiments on the wild-type complex and on mutated enzymes obtained by site-directed mutagenesis of genes encoding ATP synthase subunits. The modification by non-permeant maleimide reagents of the Cys-54 of mutated subunit 4 (subunit b), of the Cys-23 in the N-terminus of subunit 6 (subunit a) and of the Cys-91 in the C-terminus of mutated subunit f demonstrated their location in the mitochondrial intermembrane space. Near-neighbour relationships between subunits of the complex were demonstrated by means of homobifunctional and heterobifunctional reagents. Our data suggest interactions between the first transmembranous alpha-helix of subunit 6, the two hydrophobic segments of subunit 4 and the unique membrane-spanning segments of subunits i and f. The amino acid residue 174 of subunit 4 is close to both oscp and the beta-subunit, and the residue 209 is close to oscp. The dimerisation of subunit 4 in the membrane revealed that this component is located in the periphery of the enzyme and interacts with other ATP synthase complexes. Publication Types: Review Review, Tutorial PMID: 10838057 [PubMed - indexed for MEDLINE] 464: Biochim Biophys Acta 2000 May 31;1458(2-3):428-42 Insights into ATP synthase assembly and function through the molecular genetic manipulation of subunits of the yeast mitochondrial enzyme complex. Devenish RJ, Prescott M, Roucou X, Nagley P. Department of Biochemistry and Molecular Biology, Monash University, P.O. Box 13D, Vic. 3800, Australia. Development of an increasingly detailed understanding of the eucaryotic mitochondrial ATP synthase requires a detailed knowledge of the stoichiometry, structure and function of F(0) sector subunits in the contexts of the proton channel and the stator stalk. Still to be resolved are the precise locations and roles of other supernumerary subunits present in mitochondrial ATP synthase complexes, but not found in the bacterial or chloroplast enzymes. The highly developed system of molecular genetic manipulation available in the yeast Saccharomyces cerevisiae, a unicellular eucaryote, permits testing for gene function based on the effects of gene disruption or deletion. In addition, the genes encoding ATP synthase subunits can be manipulated to introduce specific amino acids at desired positions within a subunit, or to add epitope or affinity tags at the C-terminus, enabling questions of stoichiometry, structure and function to be addressed. Newly emerging technologies, such as fusions of subunits with GFP are being applied to probe the dynamic interactions within mitochondrial ATP synthase, between ATP synthase complexes, and between ATP synthase and other mitochondrial enzyme complexes. Publication Types: Review Review, Tutorial PMID: 10838056 [PubMed - indexed for MEDLINE] 465: Curr Biol 2000 Jun 1;10(11):675-8 Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function. Brady DM, Hardwick KG. Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, UK. The spindle checkpoint delays the metaphase to anaphase transition in response to defects in kinetochore-microtubule interactions in the mitotic apparatus (see [1] [2] [3] [4] for reviews). The Mad and Bub proteins were identified as key components of the spindle checkpoint through budding yeast genetics [5] [6] and are highly conserved [3]. Most of the spindle checkpoint proteins have been localised to kinetochores, yet almost nothing is known about the molecular events which take place there. Mad1p forms a tight complex with Mad2p [7], and has been shown to recruit Mad2p to kinetochores [8]. Similarly, Bub3p binds to Bub1p [9] and may target it to kinetochores [10]. Here, we show that budding yeast Mad1p has a regulated association with Bub1p and Bub3p during a normal cell cycle and that this complex is found at significantly higher levels once the spindle checkpoint is activated. We find that formation of this complex requires Mad2p and Mps1p but not Mad3p or Bub2p. In addition, we identify a conserved motif within Mad1p that is essential for Mad1p-Bub1p-Bub3p complex formation. Mutation of this motif abolishes checkpoint function, indicating that formation of the Mad1p-Bub1p-Bub3p complex is a crucial step in the spindle checkpoint mechanism. PMID: 10837255 [PubMed - indexed for MEDLINE] 466: EMBO J 2000 Jun 1;19(11):2515-24 Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane. Navarre C, Goffeau A. Unite de Biochimie Physiologique, Universite Catholique de Louvain, Croix du Sud 2-20, 1348 Louvain-la-Neuve, Belgium. Yeast plasma membranes contain a small 55 amino acid hydrophobic polypeptide, Pmp3p, which has high sequence similarity to a novel family of plant polypeptides that are overexpressed under high salt concentration or low temperature treatment. The PMP3 gene is not essential under normal growth conditions. However, its deletion increases the plasma membrane potential and confers sensitivity to cytotoxic cations, such as Na(+) and hygromycin B. Interestingly, the disruption of PMP3 exacerbates the NaCl sensitivity phenotype of a mutant strain lacking the Pmr2p/Enap Na(+)-ATPases and the Nha1p Na(+)/H(+) antiporter, and suppresses the potassium dependency of a strain lacking the K(+) transporters, Trk1p and Trk2p. All these phenotypes could be reversed by the addition of high Ca(2+) concentration to the medium. These genetic interactions indicate that the major effect of the PMP3 deletion is a hyperpolarization of the plasma membrane potential that probably promotes a non-specific influx of monovalent cations. Expression of plant RCI2A in yeast could substitute for the loss of Pmp3p, indicating a common role for Pmp3p and the plant homologue. PMID: 10835350 [PubMed - indexed for MEDLINE] 467: Mol Cell Biol 2000 Jun;20(12):4199-209 Two regulators of Ste12p inhibit pheromone-responsive transcription by separate mechanisms. Olson KA, Nelson C, Tai G, Hung W, Yong C, Astell C, Sadowski I. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada. The yeast Saccharomyces cerevisiae transcription factor Ste12p is responsible for activating genes in response to MAP kinase cascades controlling mating and filamentous growth. Ste12p is negatively regulated by two inhibitor proteins, Dig1p (also called Rst1p) and Dig2p (also called Rst2p). The expression of a C-terminal Ste12p fragment (residues 216 to 688) [Ste12p(216-688)] from a GAL promoter causes FUS1 induction in a strain expressing wild-type STE12, suggesting that this region can cause the activation of endogenous Ste12p. Residues 262 to 594 are sufficient to cause STE12-dependent FUS1 induction when overexpressed, and this region of Ste12p was found to bind Dig1p but not Dig2p in yeast extracts. In contrast, recombinant glutathione S-transferase-Dig2p binds to the Ste12p DNA-binding domain (DBD). Expression of DIG2, but not DIG1, from a GAL promoter inhibits transcriptional activation by an Ste12p DBD-VP16 fusion. Furthermore, disruption of dig1, but not dig2, causes elevated transcriptional activation by a LexA-Ste12p(216-688) fusion. Ste12p has multiple regions within the C terminus (flanking residue 474) that can promote multimerization in vitro, and we demonstrate that these interactions can contribute to the activation of endogenous Ste12p by overproduced C-terminal fragments. These results demonstrate that Dig1p and Dig2p do not function by redundant mechanisms but rather inhibit pheromone-responsive transcription through interactions with separate regions of Ste12p. PMID: 10825185 [PubMed - indexed for MEDLINE] 468: J Biol Chem 2000 Aug 11;275(32):24928-34 The yeast histone acetyltransferase A2 complex, but not free Gcn5p, binds stably to nucleosomal arrays. Sendra R, Tse C, Hansen JC. Departament de Bioquimica i Biologia Molecular, Universitat de Valencia, E-46100 Valencia, Spain. We have investigated the structural basis for the differential catalytic function of the yeast Gcn5p-containing histone acetyltransferase (HAT) A2 complex and free recombinant yeast Gcn5p (rGcn5p). HAT A2 is shown to be a unique complex that contains Gcn5p, Ada2p, and Ada3p, but not proteins specific to other related HAT A complexes, e.g. ADA, SAGA. Nevertheless, HAT A2 produces the same unique polyacetylation pattern of nucleosomal substrates reported previously for ADA and SAGA, demonstrating that proteins specific to the ADA and SAGA complexes do not influence the enzymatic activity of Gcn5p within the HAT A2 complex. To investigate the role of substrate interactions in the differential behavior of free and complexed Gcn5p, sucrose density gradient centrifugation was used to characterize the binding of HAT A2 and free rGcn5p to intact and trypsinized nucleosomal arrays, H3/H4 tetramer arrays, and nucleosome core particles. We find that HAT A2 forms stable complexes with all nucleosomal substrates tested. In distinct contrast, rGcn5p does not interact stably with nucleosomal arrays, despite being able to specifically monoacetylate the H3 N terminus of nucleosomal substrates. Our data suggest that the ability of the HAT A2 complex to bind stably to nucleosomal arrays is functionally related to both local and global acetylation by the complexed and free forms of Gcn5p. PMID: 10825174 [PubMed - indexed for MEDLINE] 469: J Biol Chem 2000 Aug 4;275(31):23500-8 Replication protein A physically interacts with the Bloom's syndrome protein and stimulates its helicase activity. Brosh RM Jr, Li JL, Kenny MK, Karow JK, Cooper MP, Kureekattil RP, Hickson ID, Bohr VA. Laboratory of Molecular Genetics, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA. Bloom's syndrome is a rare autosomal recessive disorder characterized by genomic instability and predisposition to cancer. BLM, the gene defective in Bloom's syndrome, encodes a 159-kDa protein possessing DNA-stimulated ATPase and ATP-dependent DNA helicase activities. We have examined mechanistic aspects of the catalytic functions of purified recombinant BLM protein. Through analyzing the effects of different lengths of DNA cofactor on ATPase activity, we provide evidence to suggest that BLM translocates along single-stranded DNA in a processive manner. The helicase reaction catalyzed by BLM protein was examined as a function of duplex DNA length. We show that BLM catalyzes unwinding of short DNA duplexes (/=259-bp). The presence of the human single-stranded DNA-binding protein (human replication protein A (hRPA)) stimulates the BLM unwinding reaction on the 259-bp partial duplex DNA substrate. Heterologous single-stranded DNA-binding proteins fail to stimulate similarly the helicase activity of BLM protein. This is the first demonstration of a functional interaction between BLM and another protein. Consistent with a functional interaction between hRPA and the BLM helicase, we demonstrate a direct physical interaction between the two proteins mediated by the 70-kDa subunit of RPA. The interactions between BLM and hRPA suggest that the two proteins function together in vivo to unwind DNA duplexes during replication, recombination, or repair. PMID: 10825162 [PubMed - indexed for MEDLINE] 470: Science 2000 May 19;288(5469):1242-4 Distinct classes of yeast promoters revealed by differential TAF recruitment. Li XY, Bhaumik SR, Green MR. Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. The transcription factor TFIID contains the TATA box binding protein (TBP) and multiple TBP-associated factors (TAFs). Here, the association of TFIID components with promoters that either are dependent on multiple TAFs (TAFdep) or have no apparent TAF requirement (TAFind) is analyzed in yeast. At TAFdep promoters, TAFs are present at levels comparable to that of TBP, whereas at TAFind promoters, TAFs are present at levels that approximate background. After inactivation of several general transcription factors, including TBP, TAFs are still recruited by activators to TAFdep promoters. The results reveal two classes of promoters: at TAFind promoters, TBP is recruited in the apparent absence of TAFs, whereas at TAFdep promoters, TAFs are co-recruited with TBP in a manner consistent with direct activator-TAF interactions. PMID: 10817999 [PubMed - indexed for MEDLINE] 471: J Cell Biol 2000 May 15;149(4):863-74 Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. Adames NR, Cooper JA. Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck. Both movements depend on interactions of cytoplasmic microtubules with the cortex. We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively. We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex. In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin. Free microtubules can also slide along the cortex of both bud and mother. Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired. Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired. PMID: 10811827 [PubMed - indexed for MEDLINE] 472: EMBO J 2000 May 15;19(10):2323-31 The chromo domain protein chd1p from budding yeast is an ATP-dependent chromatin-modifying factor. Tran HG, Steger DJ, Iyer VR, Johnson AD. Department of Biochemistry and Biophysics and Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, CA 94143, USA. CHD proteins are members of the chromo domain family, a class of proteins involved in transcription, DNA degradation and chromatin structure. In higher eukaryotes, there are two distinct subfamilies of CHD proteins: CHD1 and CHD3/4. Analyses carried out in vitro indicate that the CHD3/4 proteins may regulate transcription via alteration of chromatin structure. However, little is known about the role of CHD proteins in vivo, particularly the CHD1 subfamily. To understand better the cellular function of CHD proteins, we initiated a study on the Chd1p protein from budding yeast. Using genomic DNA arrays, we identified genes whose expression is affected by the absence of Chd1p. A synthetic-lethal screen uncovered genetic interactions between SWI/SNF genes and CHD1. Biochemical experiments using Chd1p purified from yeast showed that it reconfigures the structure of nucleosome core particles in a manner distinct from the SWI-SNF complex. Taken together, these results suggest that Chd1p functions as a nucleosome remodeling factor, and that Chd1p may share overlapping roles with the SWI-SNF complex to regulate transcription. PMID: 10811623 [PubMed - indexed for MEDLINE] 473: Biochem Pharmacol 2000 Jan 15;59(2):177-85 Agonistic and synergistic activity of tamoxifen in a yeast model system. Graumann K, Jungbauer A. Institute for Applied Microbiology, University of the Agricultural Sciences, Vienna, Austria. The background of agonist/antagonist behaviour of the non-steroidal antiestrogen tamoxifen is still not fully understood. Depending on cell type, its activities range from full agonistic to antagonistic in different tissues. We investigated the transactivational properties of tamoxifen in a basic yeast model system which reconstitutes ligand-dependent human estrogen receptor-alpha (hER alpha) gene activation. Tamoxifen exerted low agonist activity in this system compared to 17 beta-estradiol (E2). Efficiencies and potencies of several isomers were calculated by fitting experimental data with a logistic dose-response function. Cis-, trans- and cis-transtamoxifen and trans-4-hydroxytamoxifen (4-OHT) showed comparable efficiencies and potencies in yeast. When subeffective doses of trans-, cis-/trans-, or trans-4-OH tamoxifen were combined with increasing 17 beta-estradiol concentrations, even a synergistic increase in efficiencies could be observed. Interestingly, the cis-isomer did not show this synergistic effect. Mutation of the N-terminus of the estrogen receptor changed the transactivational behaviour of tamoxifen and abolished the synergistic action with 17 beta-estradiol. Except for 4-OHT, the potencies of the investigated isomers, defined as ligand concentrations with half-maximal response, highly correlated with the binding affinities to hER alpha. Therefore, cis-, trans-, and cis-/trans-tamoxifen could be regarded as full agonists in yeast, while 4-OHT was regarded as a partial antagonist in yeast. Furthermore, these results indicate that the functional difference between trans-tamoxifen and trans-4-OHT is not due to their different affinities for the receptor protein. PMID: 10810452 [PubMed - indexed for MEDLINE] 474: J Biol Chem 2000 May 19;275(20):15350-6 Intramolecular interactions between the juxtamembrane domain and phosphatase domains of receptor protein-tyrosine phosphatase RPTPmu. Regulation of catalytic activity. Feiken E, van Etten I, Gebbink MF, Moolenaar WH, Zondag GC. Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. RPTPmu is a receptor-like protein-tyrosine phosphatase (RPTP) whose ectodomain mediates homotypic cell-cell interactions. The intracellular part of RPTPmu contains a relatively long juxtamembrane domain (158 amino acids; aa) and two conserved phosphatase domains (C1 and C2). The membrane-proximal C1 domain is responsible for the catalytic activity of RPTPmu, whereas the membrane-distal C2 domain serves an unknown function. The regulation of RPTP activity remains poorly understood, although dimerization has been proposed as a general mechanism of inactivation. Using the yeast two-hybrid system, we find that the C1 domain binds to an N-terminal noncatalytic region in RPTPmu, termed JM (aa 803-955), consisting of a large part of the juxtamembrane domain (120 aa) and a small part of the C1 domain (33 aa). When co-expressed in COS cells, the JM polypeptide binds to both the C1 and the C2 domain. Strikingly, the isolated JM polypeptide fails to interact with either full-length RPTPmu or with truncated versions of RPTPmu that contain the JM region, consistent with the JM-C1 and JM-C2 interactions being intramolecular rather than intermolecular. Furthermore, we find that large part of the juxtamembrane domain (aa 814-922) is essential for C1 to be catalytically active. Our findings suggest a model in which RPTPmu activity is regulated by the juxtamembrane domain undergoing intramolecular interactions with both the C1 and C2 domain. PMID: 10809770 [PubMed - indexed for MEDLINE] 475: J Biol Chem 2000 May 19;275(20):14979-84 Peptides selected to bind the Gal80 repressor are potent transcriptional activation domains in yeast. Han Y, Kodadek T. Departments of Internal Medicine and Biochemistry, Center for Biomedical Inventions, Ryburn Center for Molecular Cardiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8573, USA. The activation domain of the yeast Gal4 protein binds specifically to the Gal80 repressor and is also thought to associate with one or more coactivators in the RNA polymerase II holoenzyme and chromatin remodeling machines. This is a specific example of a common situation in biochemistry where a single protein domain can interact with multiple partners. Are these different interactions related chemically? To probe this point, phage display was employed to isolate peptides from a library based solely on their ability to bind Gal80 protein in vitro. Peptide-Gal80 protein association is shown to be highly specific and of moderate affinity. The Gal80 protein-binding peptides compete with the native activation domain for the repressor, suggesting that they bind to the same site. It was then asked if these peptides could function as activation domains in yeast when tethered to a DNA binding domain. Indeed, this is the case. Furthermore, one of the Gal80-binding peptides binds directly to a domain of the Gal11 protein, a known coactivator. The fact that Gal80-binding peptides are functional activation domains argues that repressor binding and activation/coactivator binding are intimately related properties. This peptide library-based approach should be generally useful for probing the chemical relationship of different binding interactions or functions of a given native domain. PMID: 10809742 [PubMed - indexed for MEDLINE] 476: Mol Endocrinol 2000 May;14(5):718-32 GCN5 and ADA adaptor proteins regulate triiodothyronine/GRIP1 and SRC-1 coactivator-dependent gene activation by the human thyroid hormone receptor. Anafi M, Yang YF, Barlev NA, Govindan MV, Berger SL, Butt TR, Walfish PG. Samuel Lunenfeld Research Institute, University of Toronto Medical School, Mount Sinai Hospital, Ontario, Canada. We have used yeast genetics and in vitro protein-protein interaction experiments to explore the possibility that GCN5 (general control nonrepressed protein 5) and several other ADA (alteration/deficiency in activation) adaptor proteins of the multimeric SAGA complex can regulate T3/GRIP1 (glucocorticoid receptor interacting protein 1) and SRC-1 (steroid receptor coactivator-1) coactivator-dependent activation of transcription by the human T3 receptor beta1 (hTRbeta1). Here, we show that in vivo activation of a T3/GRIP1 or SRC-1 coactivator-dependent T3 hormone response element by hTRbeta1 is dependent upon the presence of yeast GCN5, ADA2, ADA1, or ADA3 adaptor proteins and that the histone acetyltransferase (HAT) domains and bromodomain (BrD) of yGCN5 must be intact for maximal activation of transcription. We also observed that hTRbeta1 can bind directly to yeast or human GCN5 as well as hADA2, and that the hGCN5(387-837) sequence could bind directly to either GRIP1 or SRC-1 coactivator. Importantly, the T3-dependent binding of hTRbeta1 to hGCN5(387-837) could be markedly increased by the presence of GRIP1 or SRC1. Mutagenesis of GRIP1 nuclear receptor (NR) Box II and III LXXLL motifs also substantially decreased both in vivo activation of transcription and in vitro T3-dependent binding of hTRbeta1 to hGCN5. Taken together, these experiments support a multistep model of transcriptional initiation wherein the binding of T3 to hTRbeta1 initiates the recruitment of p160 coactivators and GCN5 to form a trimeric transcriptional complex that activates target genes through interactions with ADA/SAGA adaptor proteins and nucleosomal histones. PMID: 10809234 [PubMed - indexed for MEDLINE] 477: Proc Natl Acad Sci U S A 2000 May 9;97(10):5267-72 Distinct roles of the NH2- and COOH-terminal domains of the protein inhibitor of activated signal transducer and activator of transcription (STAT) 1 (PIAS1) in cytokine-induced PIAS1-Stat1 interaction. Liao J, Fu Y, Shuai K. Division of Hematology-Oncology, Department of Medicine, University of California, Los Angeles, CA 90095, USA. STATs are activated by tyrosine phosphorylation on cytokine stimulation. A tyrosine-phosphorylated STAT forms a functional dimer through reciprocal Src homology 2 domain (SH2)-phosphotyrosyl peptide interactions. IFN treatment induces the association of PIAS1 and Stat1, which results in the inhibition of Stat1-mediated gene activation. The molecular basis of the cytokine-dependent PIAS1-Stat1 interaction has not been understood. We report here that a region near the COOH terminus of PIAS1 (amino acids 392-541) directly interacts with the NH(2)-terminal domain of Stat1 (amino acids 1-191). A mutant PIAS1 lacking the Stat1-interacting domain failed to inhibit Stat1-mediated gene activation. By using a modified yeast two-hybrid assay, we demonstrated that PIAS1 specifically interacts with the Stat1 dimer, but not tyrosine-phosphorylated or -unphosphorylated Stat1 monomer. In addition, whereas the NH(2)-terminal region of PIAS1 does not interact with Stat1, it serves as a modulatory domain by preventing the interaction of the COOH-terminal domain of PIAS1 with the Stat1 monomer. Thus, the cytokine-induced PIAS1-Stat1 interaction is mediated through the specific recognition of the dimeric form of Stat1 by PIAS1. PMID: 10805787 [PubMed - indexed for MEDLINE] 478: Mol Cell Biol 2000 Jun;20(11):3965-76 Identification of domains and residues within the epsilon subunit of eukaryotic translation initiation factor 2B (eIF2Bepsilon) required for guanine nucleotide exchange reveals a novel activation function promoted by eIF2B complex formation. Gomez E, Pavitt GD. Department of Anatomy and Physiology, Medical Sciences Institute, University of Dundee, Dundee, United Kingdom. Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for protein synthesis initiation factor 2 (eIF2). Composed of five subunits, it converts eIF2 from a GDP-bound form to the active eIF2-GTP complex. This is a regulatory step of translation initiation. In vitro, eIF2B catalytic function can be provided by the largest (epsilon) subunit alone (eIF2Bepsilon). This activity is stimulated by complex formation with the other eIF2B subunits. We have analyzed the roles of different regions of eIF2Bepsilon in catalysis, in eIF2B complex formation, and in binding to eIF2 by characterizing mutations in the Saccharomyces cerevisiae gene encoding eIF2Bepsilon (GCD6) that impair the essential function of eIF2B. Our analysis of nonsense mutations indicates that the C terminus of eIF2Bepsilon (residues 518 to 712) is required for both catalytic activity and interaction with eIF2. In addition, missense mutations within this region impair the catalytic activity of eIF2Bepsilon without affecting its ability to bind eIF2. Internal, in-frame deletions within the N-terminal half of eIF2Bepsilon disrupt eIF2B complex formation without affecting the nucleotide exchange activity of eIF2Bepsilon alone. Finally, missense mutations identified within this region do not affect the catalytic activity of eIF2Bepsilon alone or its interactions with the other eIF2B subunits or with eIF2. Instead, these missense mutations act indirectly by impairing the enhancement of the rate of nucleotide exchange that results from complex formation between eIF2Bepsilon and the other eIF2B subunits. This suggests that the N-terminal region of eIF2Bepsilon is an activation domain that responds to eIF2B complex formation. PMID: 10805739 [PubMed - indexed for MEDLINE] 479: Nat Struct Biol 2000 May;7(5):375-9 Structural analysis of WW domains and design of a WW prototype. Macias MJ, Gervais V, Civera C, Oschkinat H. Forschungsinstitut fur Molekulare Pharmakologie, Alfred-Kowalke-Str. 4, 10315 Berlin, Germany. macias@EMBL-Heidelberg.de Two new NMR structures of WW domains, the mouse formin binding protein and a putative 84.5 kDa protein from Saccharomyces cerevisiae, show that this domain, only 35 amino acids in length, defines the smallest monomeric triple-stranded antiparallel beta-sheet protein domain that is stable in the absence of disulfide bonds, tightly bound ions or ligands. The structural roles of conserved residues have been studied using site-directed mutagenesis of both wild type domains. Crucial interactions responsible for the stability of the WW structure have been identified. Based on a network of highly conserved long range interactions across the beta-sheet structure that supports the WW fold and on a systematic analysis of conserved residues in the WW family, we have designed a folded prototype WW sequence. PMID: 10802733 [PubMed - indexed for MEDLINE] 480: J Biol Chem 2000 Jul 21;275(29):22470-8 An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin. Korman VL, Hatch V, Dixon KY, Craig R, Lehman W, Tobacman LS. Departments of Biochemistry and Internal Medicine, University of Iowa, College of Medicine, Iowa City, Iowa 52242, USA. Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+) to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca(2+) showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex. PMID: 10801864 [PubMed - indexed for MEDLINE] 481: Curr Opin Cell Biol 2000 Jun;12(3):361-71 The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm. Ryan KJ, Wente SR. Department of Cell Biology and Physiology, Washington University School of Medicine, Box 8228, St Louis, MO 63110, USA. kryan@cellbio. wustl.edu Compositional analysis of nuclear pore complexes (NPCs) is nearing completion, and efforts are now focused on understanding how these protein machines work. Recent analysis of soluble transport factor interactions with NPC proteins reveals distinct and overlapping pathways for movement between the nucleus and cytoplasm. New fluorescence- and microscopy-based strategies have been used to monitor the pathway of NPC assembly and to reveal the dynamics of the NPC during transport. Publication Types: Review Review, Tutorial PMID: 10801463 [PubMed - indexed for MEDLINE] 482: Biochem Biophys Res Commun 2000 May 10;271(2):464-8 Purification and polymerization properties of two lethal yeast actin mutants. Frieden C, Du J, Schriefer L, Buzan J. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, 63110, USA. frieden@biochem.wustl.edu The budding yeast Saccharomyces cerevisiae contains a single actin gene and the gene product, actin, is essential for growth. Two mutants of yeast actin that do not support yeast growth were prepared from yeast by coexpressing the mutant and a 6-histidine-tagged wild-type actin followed by separation of the wild-type and mutant actin using Ni-NTA chromatography as described elsewhere [Buzan, J., Du, J., Karpova, T., and Frieden, C. (1999) Proc. Natl. Acad. Sci. USA 96, 2823-2827]. The mutations, in muscle actin numbering, were at positions 334 (Glu334Lys) and 168 (Gly168Arg) and were chosen based on phenotypic changes observed in the behavior of actin mutants of Caenorhabditis elegans. Glu334 is located on the surface of actin between subdomains 1 and 3 while Gly168 is located in a region near actin-actin contacts in the actin filament. The Glu334Lys mutant polymerized slightly faster than wild-type yeast actin, suggesting that loss of interactions with some actin binding protein, rather than loss of actin-actin contacts, was responsible for its inability to support yeast growth. The Gly168Arg mutant polymerized at a rate similar to wild-type but the extent was considerably less, kinetic characteristics suggesting a high critical concentration (ca. 4 microM) without a large change in the ability to form nuclei for the nucleation-elongation process. Copyright 2000 Academic Press. PMID: 10799320 [PubMed - indexed for MEDLINE] 483: Mol Biol Cell 2000 May;11(5):1753-64 The yeast heat shock transcription factor changes conformation in response to superoxide and temperature. Lee S, Carlson T, Christian N, Lea K, Kedzie J, Reilly JP, Bonner JJ. Departments of Biology and Chemistry, Indiana University, Bloomington, Indiana 47405-3700, USA. In vitro DNA-binding assays demonstrate that the heat shock transcription factor (HSF) from the yeast Saccharomyces cerevisiae can adopt an altered conformation when stressed. This conformation, reflected in a change in electrophoretic mobility, requires that two HSF trimers be bound to DNA. Single trimers do not show this change, which appears to represent an alteration in the cooperative interactions between trimers. HSF isolated from stressed cells displays a higher propensity to adopt this altered conformation. Purified HSF can be stimulated in vitro to undergo the conformational change by elevating the temperature or by exposing HSF to superoxide anion. Mutational analysis maps a region critical for this conformational change to the flexible loop between the minimal DNA-binding domain and the flexible linker that joins the DNA-binding domain to the trimerization domain. The significance of these findings is discussed in the context of the induction of the heat shock response by ischemic stroke, hypoxia, and recovery from anoxia, all known to stimulate the production of superoxide. PMID: 10793149 [PubMed - indexed for MEDLINE] 484: Cell Struct Funct 2000 Feb;25(1):11-20 Overexpression of PRA2, a Rab/Ypt-family small GTPase from Pea Pisum sativum, aggravates the growth defect of yeast ypt mutants. Matsuda N, Ueda T, Sasaki Y, Nakano A. Molecular Membrane Biology Laboratory, RIKEN, Wako, Saitama, Japan. A large number of Rab/Ypt-family small GTPases have been identified from higher plants. While some of them can complement yeast ypt mutants, the expression of Arabidopsis Ara4 protein aggravated the growth defect of a subset of ypt mutants, probably because of the titration of common regulator(s) of yeast Ypt proteins [Ueda, T. et al. (1996) Plant Cell, 8: 2079-20911. PRA2 from pea Pisum sativum encodes an interesting Rab GTPase whose expression is regulated by light [Yoshida, K. et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 6636-6640]. We examined whether PRA2 complements any of the yeast ypt mutants and found again that PRA2 does not complement but rather confers the growth defect to some of the ypt mutants. No growth defect was observed when PRA2 was expressed in the wild-type yeast cells. Unlike the case of Ara4, neither Arabidopsis nor yeast GDI remedied the growth defect by Pra2, indicating that the mechanism of the exacerbation is different. Mutational analysis of PRA2 suggests that the growth inhibition can be ascribed to unidentified factor(s) which prefers the GTP-bound form of Pra2. This yeast system will be useful for identifying such putative regulatory factor(s) from yeast and plants and analyzing their interactions with Pra2. PMID: 10791890 [PubMed - indexed for MEDLINE] 485: Genetics 2000 May;155(1):69-83 Genetic interactions between GLC7, PPZ1 and PPZ2 in saccharomyces cerevisiae. Venturi GM, Bloecher A, Williams-Hart T, Tatchell K. Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 71130, USA. GLC7 encodes an essential serine/threonine protein type I phosphatase in Saccharomyces cerevisiae. Three other phosphatases (Ppz1p, Ppz2p, and Sal6p) share >59% identity in their catalytic region with Glc7p. ppz1 ppz2 null mutants have no apparent growth defect on rich media. However, null alleles of PPZ1 and PPZ2, in combination with mutant alleles of GLC7, confer a range of growth defects varying from slow growth to lethality. These results indicate that Glc7p, Ppz1p, and Ppz2p may have overlapping functions. To determine if this overlap extends to interaction with targeting subunits, Glc7p-binding proteins were tested for interaction in the two-hybrid system with the functional catalytic domain of Ppz1p. Ppz1p interacts strongly with a number of Glc7p regulatory subunits, including Glc8p, a protein that shares homology with mammalian PP1 inhibitor I2. Genetic data suggest that Glc8p positively affects both Glc7p and Ppz1p functions. Together our data suggest that Ppz1p and Ppz2p may have overlapping functions with Glc7p and that all three phosphatases may act through common regulatory proteins. PMID: 10790385 [PubMed - indexed for MEDLINE] 486: RNA 2000 Apr;6(4):638-50 REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export. Stutz F, Bachi A, Doerks T, Braun IC, Seraphin B, Wilm M, Bork P, Izaurralde E. Institute of Microbiology, Lausanne, Switzerland. Vertebrate TAP and its yeast ortholog Mex67p are involved in the export of messenger RNAs from the nucleus. TAP has also been implicated in the export of simian type D viral RNAs bearing the constitutive transport element (CTE). Although TAP directly interacts with CTE-bearing RNAs, the mode of interaction of TAP/Mex67p with cellular mRNAs is different from that with the CTE RNA and is likely to be mediated by protein-protein interactions. Here we show that Mex67p directly interacts with Yra1p, an essential yeast hnRNP-like protein. This interaction is evolutionarily conserved as Yra1p also interacts with TAP. Conditional expression in yeast cells implicates Yra1 p in the export of cellular mRNAs. Database searches revealed that Yra1p belongs to an evolutionarily conserved family of hnRNP-like proteins having more than one member in Mus musculus, Xenopus laevis, Caenorhabditis elegans, and Schizosaccharomyces pombe and at least one member in several species including plants. The murine members of the family directly interact with TAP. Because members of this protein family are characterized by the presence of one RNP-motif RNA-binding domain and exhibit RNA-binding activity, we called these proteins REF-bps for RNA and export factor binding proteins. Thus, Yra1p and members of the REF family of hnRNP-like proteins may facilitate the interaction of TAP/Mex67p with cellular mRNAs. PMID: 10786854 [PubMed - indexed for MEDLINE] 487: Cell 2000 Apr 14;101(2):223-33 A kaiC-interacting sensory histidine kinase, SasA, necessary to sustain robust circadian oscillation in cyanobacteria. Iwasaki H, Williams SB, Kitayama Y, Ishiura M, Golden SS, Kondo T. Division of Biological Science, Graduate School of Science, Nagoya University, Japan. Both regulated expression of the clock genes kaiA, kaiB, and kaiC and interactions among the Kai proteins are proposed to be important for circadian function in the cyanobacterium Synechococcus sp. strain PCC 7942. We have identified the histidine kinase SasA as a KaiC-interacting protein. SasA contains a KaiB-like sensory domain, which appears sufficient for interaction with KaiC. Disruption of the sasA gene lowered kaiBC expression and dramatically reduced amplitude of the kai expression rhythms while shortening the period. Accordingly, sasA disruption attenuated circadian expression patterns of all tested genes, some of which became arrhythmic. Continuous sasA overexpression eliminated circadian rhythms, whereas temporal overexpression changed the phase of kaiBC expression rhythm. Thus, SasA is a close associate of the cyanobacterial clock that is necessary to sustain robust circadian rhythms. PMID: 10786837 [PubMed - indexed for MEDLINE] 488: Eur J Biochem 2000 May;267(9):2680-7 Uncoupling proteins 2 and 3 interact with members of the 14.3.3 family. Pierrat B, Ito M, Hinz W, Simonen M, Erdmann D, Chiesi M, Heim J. Novartis Pharma Inc., Basle, Switzerland. Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two-hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms theta, beta, zeta were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the gamma isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 theta could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria. PMID: 10785390 [PubMed - indexed for MEDLINE] 489: J Biol Chem 2000 Jul 7;275(27):20562-71 Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II. Chang A, Cheang S, Espanel X, Sudol M. Department of Biochemistry and Molecular Biology, New York University/Mount Sinai School of Medicine, New York, New York 10029, USA. RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation. PMID: 10781604 [PubMed - indexed for MEDLINE] 490: Mol Cell Biol 2000 May;20(10):3597-607 Phospholipase C is involved in kinetochore function in Saccharomyces cerevisiae. Lin H, Choi JH, Hasek J, DeLillo N, Lou W, Vancura A. Department of Biological Sciences, St. John's University, Jamaica, New York 11439, USA. The budding yeast PLC1 gene encodes a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C. Here, we present evidence that Plc1p associates with the kinetochore complex CBF3. This association is mediated through interactions with two established kinetochore proteins, Ndc10p and Cep3p. We show by chromatin immunoprecipitation experiments that Plc1p resides at centromeric loci in vivo. Deletion of PLC1, as well as plc1 mutations which abrogate the interaction of Plc1p with the CBF3 complex, results in a higher frequency of minichromosome loss, nocodazole sensitivity, and mitotic delay. Overexpression of Ndc10p suppresses the nocodazole sensitivity of plc1 mutants, implying that the association of Plc1p with CBF3 is important for optimal kinetochore function. Chromatin extracts from plc1Delta cells exhibit reduced microtubule binding to minichromosomes. These results suggest that Plc1p associates with kinetochores and regulates some aspect of kinetochore function and demonstrate an intranuclear function of phospholipase C in eukaryotic cells. PMID: 10779349 [PubMed - indexed for MEDLINE] 491: Mol Cell Biol 2000 May;20(10):3538-49 Deletion of the PAT1 gene affects translation initiation and suppresses a PAB1 gene deletion in yeast. Wyers F, Minet M, Dufour ME, Vo LT, Lacroute F. Centre de Genetique Moleculaire, C.N.R.S., 91198 Gif sur Yvette, France. wyers@cgm.cnrs-gif.fr The yeast poly(A) binding protein Pab1p mediates the interactions between the 5' cap structure and the 3' poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. The PAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes. PMID: 10779343 [PubMed - indexed for MEDLINE] 492: J Biol Chem 2000 Apr 28;275(17):12926-33 Reversible transdominant inhibition of a metabolic pathway. In vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes in yeast. Velot C, Srere PA. Research Service of the Department of Veterans Affairs Medical Center, Dallas, Texas 75216, USA. poffenb1@airmail.net The enzymes of the Krebs tricarboxylic acid cycle in mitochondria are proposed to form a supramolecular complex, in which there is channeling of intermediates between enzyme active sites. While interactions have been demonstrated in vitro between most of the sequential tricarboxylic acid cycle enzymes, no direct evidence has been obtained in vivo for such interactions. We have isolated, in the Saccharomyces cerevisiae gene encoding the tricarboxylic acid cycle enzyme citrate synthase Cit1p, an "assembly mutation," i.e. a mutation that causes a tricarboxylic acid cycle deficiency without affecting the citrate synthase activity. We have shown that a 15-amino acid peptide from wild type Cit1p encompassing the mutation point inhibits the tricarboxylic acid cycle in a dominant manner, and that the inhibitory phenotype is overcome by a co-overexpression of Mdh1p, the mitochondrial malate dehydrogenase. These data provide the first direct in vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes, Cit1p and Mdh1p, and indicate that the characterization of assembly mutations by the reversible transdominant inhibition method may be a powerful way to study multienzyme complexes in their physiological context. PMID: 10777592 [PubMed - indexed for MEDLINE] 493: Nucleic Acids Res 2000 May 15;28(10):2060-8 DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair. Suter B, Wellinger RE, Thoma F. Institut fur Zellbiologie, ETH-Zurich, Honggerberg, CH-8093 Zurich, Switzerland. DNA damage formation and repair are tightly linked to protein-DNA interactions in chromatin. We have used minichromosomes in yeast as chromatin substrates in vivo to investigate how nucleotide excision repair (NER) and repair by DNA-photolyase (photoreactivation) remove pyrimidine dimers from an origin of replication ( ARS1 ). The ARS1 region is nuclease sensitive and flanked by nucleosomes on both sides. Photoreactivation was generally faster than NER at all sites. Site-specific heterogeneity of repair was observed for both pathways. This heterogeneity was different for NER and photoreactivation and it was altered in a minichromosome where ARS1 was transcribed. The results indicate distinct inter-actions of the repair systems with protein complexes bound in the ARS region (ORC, Abf1) and a predominant role of photolyase in CPD repair of an origin of replication. PMID: 10773073 [PubMed - indexed for MEDLINE] 494: Nucleic Acids Res 2000 May 15;28(10):2049-59 Domain specific interaction in the XRCC1-DNA polymerase beta complex. Marintchev A, Robertson A, Dimitriadis EK, Prasad R, Wilson SH, Mullen GP. Department of Biochemistry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032, USA. XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms complexes with DNA polymerase beta (beta-Pol), DNA ligase III and poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain-domain interaction in the XRCC1-beta-Pol complex has provided information on the specificity and mechanism of binding. The domain structure of XRCC1, determined using limited proteolysis, was found to include an N-terminal domain (NTD), a central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I-linker-BRCT-II C-terminal fragment and the linker-BRCT-II C-terminal fragment were relatively stable to proteolysis suggestive of a non-random conformation of the linker. A predicted inner domain was found not to be stable to proteolysis. Using cross-linking experiments, XRCC1 was found to bind intact beta-Pol and the beta-Pol 31 kDa domain. The XRCC1-NTD(1-183)(residues 1-183) was found to bind beta-Pol, the beta-Pol 31 kDa domain and the beta-Pol C-terminal palm-thumb (residues 140-335), and the interaction was further localized to XRCC1-NTD(1-157)(residues 1-157). The XRCC1-NTD(1-183)-beta-Pol 31 kDa domain complex was stable at high salt (1 M NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen, polypeptides expressed from two XRCC1 constructs, which included residues 36-355 and residues 1-159, were found to interact with beta-Pol, the beta-Pol 31 kDa domain, and the beta-Pol C-terminal thumb-only domain polypeptides expressed from the respective beta-Pol constructs. Neither the XRCC1-NTD(1-159), nor the XRCC1(36-355)polypeptide was found to interact with a beta-Pol thumbless polypeptide. A third XRCC1 polypeptide (residues 75-212) showed no interaction with beta-Pol. In quantitative gel filtration and analytical ultracentrifugation experiments, the XRCC1-NTD(1-183)was found to bind beta-Pol and its 31 kDa domain in a 1:1 complex with high affinity (K(d) of 0.4-2.4 microM). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD(1-159)and beta-Pol that is of an affinity comparable to other binding interactions involving beta-Pol. PMID: 10773072 [PubMed - indexed for MEDLINE] 495: Biochemistry 2000 Apr 25;39(16):4869-80 Participation of the amino-terminal domain in the self-association of the full-length yeast TATA binding protein. Daugherty MA, Brenowitz M, Fried MG. Department of Biochemistry, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA. The association of monomeric TATA binding protein with promoter DNA is an essential first step in many current models of eukaryotic transcription initiation. This step is followed by others in which additional transcription factors, and finally RNA polymerase, assemble at the promoter. Here we characterize the quaternary interactions of the Saccharomyces cerevisiae TATA-binding protein (yTBP), in the absence of other proteins or DNA. The data reveal a robust pattern in which yTBP monomers equilibrate with tetramers and octamers over a broad span of temperatures (4 degrees C or =1300 kDa that is sensitive both to RNase and NaCl. Using affinity-chromatography to isolate these complexes, we have identified two protein components other than Scp160p: poly(A) binding protein, Pab1p, and Bfr1p. The presence of Pab1p confirms these complexes to be mRNPs. The presence of Bfr1p is intriguing because the null phenotype for this gene is essentially the same as that reported for scp160 -null cells: increased cell size and aberrant DNA content. These results demonstrate that Scp160p associates with polyribosome-bound mRNP complexes in vivo, implicating a role for this protein in one or more levels of mRNA metabolism in yeast. PMID: 10710424 [PubMed - indexed for MEDLINE] 530: Bioinformatics 1999 Oct;15(10):776-84 Genes regulated cooperatively by one or more transcription factors and their identification in whole eukaryotic genomes. Wagner A. Department of Biology, University of New Mexico, Albuquerque, USA. wagnera@unm.edu MOTIVATION: The question addressed here is how cooperative interactions among transcription factors (TFs), a very frequent phenomenon in eukaryotic transcriptional regulation, can be used to identify genes that are regulated by one or more TFs with known DNA binding specificities. Cooperativity may be homotypic, involving binding of only one transcription factor to multiple sites in a gene's regulatory region. It may also be heterotypic, involving binding of more than one TF. Both types of cooperativity have in common that the binding sites for the respective TFs form tightly linked 'clusters', groups of binding sites often more closely associated than expected by chance alone. RESULTS: A statistical technique suitable for the identification of statistically significant homotypic or heterotypic TF binding site clusters in whole eukaryotic genomes is presented. It can be used to identify genes likely to be regulated by the TFs. Application of the technique is illustrated with two transcription factors involved in the cell cycle and mating control of the yeast Saccharomyces cerevisiae, indicating that the results obtained are biologically meaningful. This rapid and inexpensive computational method of generating hypotheses about gene regulation thus generates information that may be used to guide subsequent costly and laborious experimental approaches, and that may aid in the assignment of biological functions to putative open reading frames. PMID: 10705431 [PubMed - indexed for MEDLINE] 531: Proc Natl Acad Sci U S A 2000 Feb 15;97(4):1516-20 Studies on the role of the hydrophobic domain of Ost4p in interactions with other subunits of yeast oligosaccharyl transferase. Kim H, Park H, Montalvo L, Lennarz WJ. Department of Biochemistry, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, NY 11794-5215, USA. In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT), which catalyzes the transfer of dolichol-linked oligosaccharide chains to nascent polypeptides in the endoplasmic reticulum, consists of nine nonidentical membrane protein subunits. Genetic and biochemical evidence indicated these nine proteins exist in three subcomplexes. Three of the OT subunits (Ost4p, Ost3p, and Stt3p) have been proposed to exist in one subcomplex. To investigate the interaction of these three membrane proteins, initially we carried out a mutational analysis of Ost4p, which is an extraordinarily small membrane protein containing only 36 amino acid residues. This analysis indicated that when single amino acid residues in a region close to the luminal face of the putative transmembrane domain of Ost4p were changed into an ionizable amino acid such as Lys or Asp, growth at 37 degrees C and OT activity measured in vitro were impaired. In addition, using immunoprecipitation techniques and Western blot analysis, we found that with these mutations the interaction between Ost4p, Ost3p, and Stt3p was disrupted. Introduction of Lys or Asp residues at other positions in the putative transmembrane domain or at the N or C terminus of Ost4p had no effect on disrupting subunit interactions or impairing the activity of OT. These findings suggest that a localized region of the putative transmembrane domain of Ost4p mediates in stabilization of the interaction with the two other OT subunits (Ost3p and Stt3p) in a subcomplex in the endoplasmic reticulum membrane. PMID: 10677492 [PubMed - indexed for MEDLINE] 532: Proc Natl Acad Sci U S A 2000 Mar 14;97(6):2491-6 The interaction of nitric oxide (NO) with the yeast transcription factor Ace1: A model system for NO-protein thiol interactions with implications to metal metabolism. Shinyashiki M, Chiang KT, Switzer CH, Gralla EB, Valentine JS, Thiele DJ, Fukuto JM. Department of Pharmacology, University of California at Los Angeles Medical School, Center for the Health Sciences, Los Angeles, CA 90095-1735, USA. Nitric oxide (NO) was found to inhibit the copper-dependent induction of the yeast CUP1 gene. This effect is attributable to an inhibition of the copper-responsive CUP1 transcriptional activator Ace1. A mechanism is proposed whereby the metal binding thiols of Ace1 are chemically modified via NO- and O(2)-dependent chemistry, thereby diminishing the ability of Ace1 to bind and respond to copper. Moreover, it is proposed that demetallated Ace1 is proteolytically degraded in the cell, resulting in a prolonged inhibition of copper-dependent CUP1 induction. These findings indicate that NO may serve as a disrupter of yeast copper metabolism. More importantly, considering the similarity of Ace1 to other mammalian metal-binding proteins, this work lends support to the hypothesis that NO may regulate/disrupt metal homeostasis under both normal physiological and pathophysiological circumstances. PMID: 10694579 [PubMed - indexed for MEDLINE] 533: Genes Dev 2000 Feb 15;14(4):493-503 Progression of meiotic DNA replication is modulated by interchromosomal interaction proteins, negatively by Spo11p and positively by Rec8p. Cha RS, Weiner BM, Keeney S, Dekker J, Kleckner N. Department of Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 USA. Spo11p is a key mediator of interhomolog interactions during meiosis. Deletion of the SPO11 gene decreases the length of S phase by approximately 25%. Rec8p is a key coordinator of meiotic interhomolog and intersister interactions. Deletion of the REC8 gene increases S-phase length, by approximately 10% in wild-type and approximately 30% in a spo11Delta background. Thus, the progression of DNA replication is modulated by interchromosomal interaction proteins. The spo11-Y135F DSB (double strand break) catalysis-defective mutant is normal for S-phase modulation and DSB-independent homolog pairing but is defective for later events, formation of DSBs, and synaptonemal complexes. Thus, earlier and later functions of Spo11 are defined. We propose that meiotic S-phase progression is linked directly to development of specific chromosomal features required for meiotic interhomolog interactions and that this feedback process is built upon a more fundamental mechanism, common to all cell types, by which S-phase progression is coupled to development of nascent intersister connections and/or related aspects of chromosome morphogenesis. Roles for Rec8 and/or Spo11 in progression through other stages of meiosis are also revealed. PMID: 10691741 [PubMed - indexed for MEDLINE] 534: Proc Natl Acad Sci U S A 2000 Feb 29;97(5):2373-8 The movement protein NSm of tomato spotted wilt tospovirus (TSWV): RNA binding, interaction with the TSWV N protein, and identification of interacting plant proteins. Soellick T, Uhrig JF, Bucher GL, Kellmann JW, Schreier PH. Max-Planck-Institut fur Zuchtungsforschung, Carl-von-Linne-Weg 10, D-50829 Koln, Germany. The nonstructural NSm protein of tomato spotted wilt tospovirus (TSWV) represents a putative viral movement protein involved in cell-to-cell movement of nonenveloped ribonucleocapsid structures. To study the molecular basis of NSm function, we expressed the protein in Escherichia coli and investigated protein-protein and protein-RNA interactions of NSm protein in vitro. NSm specifically interacts with TSWV N protein and binds single-stranded RNA in a sequence-nonspecific manner. Using NSm as a bait in a yeast two-hybrid screen, we identified two homologous NSm-binding proteins of the DnaJ family from Nicotiana tabacum and Arabidopsis thaliana. PMID: 10688879 [PubMed - indexed for MEDLINE] 535: Biochemistry 2000 Feb 22;39(7):1716-24 Catalytic and DNA binding properties of the ogg1 protein of Saccharomyces cerevisiae: comparison between the wild type and the K241R and K241Q active-site mutant proteins. Guibourt N, Castaing B, Van Der Kemp PA, Boiteux S. Departement de Radiobiologie et Radiopathologie, UMR 217 CNRS-CEA "Radiobiologie Moleculaire et Cellulaire", Commissariat a l'Energie Atomique, DSV, BP6, 92265-Fontenay aux Roses, France. The Ogg1 protein of Saccharomyces cerevisiae belongs to a family of DNA glycosylases and apurinic/apyrimidinic site (AP) lyases, the signature of which is the alpha-helix-hairpin-alpha-helix-Gly/Pro-Asp (HhH-GPD) active site motif together with a conserved catalytic lysine residue, to which we refer as the HhH-GPD/K family. In the yeast Ogg1 protein, yOgg1, the HhH-GPD/K motif spans residues 225-260 and the conserved lysine is K241. In this study, we have purified the K241R and K241Q mutant proteins and compared their catalytic and DNA binding properties to that of the wild-type yOgg1. The results show that the K241R mutation greatly impairs both the DNA glycosylase and the AP lyase activities of yOgg1. Specificity constants for cleavage of a 34mer oligodeoxyribonucleotide containing a 7,8-dihydro-8-oxoguanine (8-OxoG) paired with a cytosine, [8-OxoG.C], are 56 x 10(-)(3) and 5 x 10(-)(3) min(-)(1) nM(-)(1) for the wild-type and the K241R protein, respectively. On the other hand, the K241Q mutation abolishes the DNA glycosylase and AP lyase activities of yOgg1. In contrast, the K241R and K241Q proteins have conserved wild-type DNA binding properties. K(dapp) values for binding of [8-OxoG.C] are 6.9, 7.4, and 4.8 nM for the wild-type, K241R, and K241Q proteins, respectively. The results also show that AP site analogues such as 1, 3-propanediol (Pr), tetrahydrofuran (F), or cyclopentanol (Cy) are not substrates but constitute good inhibitors of the wild-type yOgg1. Therefore, we have used a 59mer [Pr.C] duplex to further analyze the DNA binding properties of the wild-type, K241R, and K241Q proteins. Hydroxyl radical footprints of the wild-type yOgg1 show strong protection of six nucleotides centered around the Pr lesion in the damaged strand. On the complementary strand, only the cytosine placed opposite Pr was strongly protected. The same footprints were observed with the K241R and K241Q proteins, confirming their wild-type DNA binding properties. These results indicate that the K241Q mutant protein can be used to study interactions between yOgg1 and DNA containing metabolizable substrates such as 8-OxoG or an AP site. PMID: 10677220 [PubMed - indexed for MEDLINE] 536: Mol Cell Biol 2000 Mar;20(6):2209-17 Fourteen residues of the U1 snRNP-specific U1A protein are required for homodimerization, cooperative RNA binding, and inhibition of polyadenylation. Klein Gunnewiek JM, Hussein RI, van Aarssen Y, Palacios D, de Jong R, van Venrooij WJ, Gunderson SI. Department of Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands. It was previously shown that the human U1A protein, one of three U1 small nuclear ribonucleoprotein-specific proteins, autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA. The U1A autoregulatory complex requires two molecules of U1A protein to cooperatively bind a 50-nucleotide polyadenylation-inhibitory element (PIE) RNA located in the U1A 3' untranslated region. Based on both biochemical and nuclear magnetic resonance structural data, it was predicted that protein-protein interactions between the N-terminal regions (amino acids [aa] 1 to 115) of the two U1A proteins would form the basis for cooperative binding to PIE RNA and for inhibition of polyadenylation. In this study, we not only experimentally confirmed these predictions but discovered some unexpected features of how the U1A autoregulatory complex functions. We found that the U1A protein homodimerizes in the yeast two-hybrid system even when its ability to bind RNA is incapacitated. U1A dimerization requires two separate regions, both located in the N-terminal 115 residues. Using both coselection and gel mobility shift assays, U1A dimerization was also observed in vitro and found to depend on the same two regions that were found in vivo. Mutation of the second homodimerization region (aa 103 to 115) also resulted in loss of inhibition of polyadenylation and loss of cooperative binding of two U1A protein molecules to PIE RNA. This same mutation had no effect on the binding of one U1A protein molecule to PIE RNA. A peptide containing two copies of aa 103 to 115 is a potent inhibitor of polyadenylation. Based on these data, a model of the U1A autoregulatory complex is presented. PMID: 10688667 [PubMed - indexed for MEDLINE] 537: Nature 2000 Feb 10;403(6770):623-7 Comment in: Nature. 2000 Feb 10;403(6770):601-3. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P, Qureshi-Emili A, Li Y, Godwin B, Conover D, Kalbfleisch T, Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg JM. Department of Genetics, University of Washington, Seattle 98195-7360, USA. Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here. PMID: 10688190 [PubMed - indexed for MEDLINE] 538: Biopolymers 2000 Apr 5;53(4):293-307 Osmolyte-induced changes in protein conformational equilibria. Saunders AJ, Davis-Searles PR, Allen DL, Pielak GJ, Erie DA. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Examining solute-induced changes in protein conformational equilibria is a long-standing method for probing the role of water in maintaining protein stability. Interpreting the molecular details governing the solute-induced effects, however, remains controversial. We present experimental and theoretical data for osmolyte-induced changes in the stabilities of the A and N states of yeast iso-1-ferricytochrome c. Using polyol osmolytes of increasing size, we observe that osmolytes alone induce A-state formation from acid-denatured cytochrome c and N state formation from the thermally denatured protein. The stabilities of the A and N states increase linearly with osmolyte concentration. Interestingly, osmolytes stabilize the A state to a greater degree than the N state. To interpret the data, we divide the free energy for the reaction into contributions from nonspecific steric repulsions (excluded volume effects) and from binding interactions. We use scaled particle theory (SPT) to estimate the free energy contributions from steric repulsions, and we estimate the contributions from water-protein and osmolyte-protein binding interactions by comparing the SPT calculations to experimental data. We conclude that excluded volume effects are the primary stabilizing force, with changes in water-protein and solute-protein binding interactions making favorable contributions to stability of the A state and unfavorable contributions to the stability of the N state. The validity of our interpretation is strengthened by analysis of data on osmolyte-induced protein stabilization from the literature, and by comparison with other analyses of solute-induced changes in conformational equilibria. Copyright 2000 John Wiley & Sons, Inc. PMID: 10685050 [PubMed - indexed for MEDLINE] 539: Nucleic Acids Res 2000 Mar 15;28(6):1407-17 A new double-stranded RNA-binding protein that interacts with PKR. Coolidge CJ, Patton JG. Department of Molecular Biology, Box 1820, Station B, Vanderbilt University, Nashville, TN 37235, USA. We have identified a 74 kDa double-stranded (ds)RNA-binding protein that shares extensive homology with the mouse spermatid perinuclear RNA-binding (Spnr) protein. p74 contains two dsRNA-binding motifs (dsRBMs) that are essential for preferential binding to dsRNA. Previously, dsRNA-binding proteins were shown to undergo homo- and heterodimerization, raising the possibility that regulation of activity could be controlled by interactions between different family members. Homodimerization is required to activate the dsRNA-dependent protein kinase PKR, whereas hetero-dimerization between PKR and other dsRNA-binding proteins can inhibit kinase activity. We have found that p74 also interacts with PKR, both the wild-type enzyme and a catalytically defective mutant (K296R). While co-expression of p74 and wild-type PKR in the yeast Saccharomyces cerevisiae did not alter PKR activity, co-expression of p74 and the catalytically defective K296R mutant surprisingly resulted in abnormal morphology and cell death in transformants that maintained a high level of p74 expression. These transformants could be rescued by overexpression of the alpha-subunit of wild-type eukaryotic translation initiation factor 2 (eIF2alpha), one of the known substrates for PKR. We hypothesize that competing heterodimers between p74-K296R PKR and eIF2alpha-K296R PKR may control cell growth such that stabilization of the p74-K296R PKR heterodimer induces abnormal morphology and cell death. PMID: 10684936 [PubMed - indexed for MEDLINE] 540: Nucleic Acids Res 2000 Mar 15;28(6):1332-9 Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues. Saparbaev M, Mani JC, Laval J. Groupe 'Reparation des lesions Radio- et Chimio-Induites', UMR 8532 CNRS, Institut Gustave Roussy, 94805 Villejuif Cedex, France. In DNA, the deamination of dAMP generates 2'-deoxy-inosine 5'-monophosphate (dIMP). Hypoxanthine (HX) residues are mutagenic since they give rise to A.T-->G.C transition. They are excised, although with different efficiencies, by an activity of the 3-methyl-adenine (3-meAde)-DNA glycosylases from Escherichia coli (AlkA protein), human cells (ANPG protein), rat cells (APDG protein) and yeast (MAG protein). Comparison of the kinetic constants for the excision of HX residues by the four enzymes shows that the E.coli and yeast enzymes are quite inefficient, whereas for the ANPG and the APDG proteins they repair the HX residues with an efficiency comparable to that of alkylated bases, which are believed to be the primary substrates of these DNA glycosylases. Since the use of various substrates to monitor the activity of HX-DNA glycosylases has generated conflicting results, the efficacy of the four 3-meAde-DNA glycosylases of different origin was compared using three different substrates. Moreover, using oligo-nucleotides containing a single dIMP residue, we investigated a putative sequence specificity of the enzymes involving the bases next to the HX residue. We found up to 2-5-fold difference in the rates of HX excision between the various sequences of the oligonucleotides studied. When the dIMP residue was placed opposite to each of the four bases, a preferential recognition of dI:T over dI:dG, dI:dC and dI:dA mismatches was observed for both human (ANPG) and E.coli (AlkA) proteins. At variance, the yeast MAG protein removed more efficiently HX from a dI:dG over dI:dC, dI:T and dI:dA mismatches. PMID: 10684927 [PubMed - indexed for MEDLINE] 541: J Biol Chem 2000 Feb 25;275(8):5767-72 The assembly factor Atp11p binds to the beta-subunit of the mitochondrial F(1)-ATPase. Wang ZG, Ackerman SH. Department of Surgery, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. Atp11p is a protein of Saccharomyces cerevisiae required for the assembly of the F(1) component of the mitochondrial F(1)F(0)-ATP synthase. This study presents evidence that Atp11p binds selectively to the beta-subunit of F(1). Under conditions in which avidin-Sepharose beads specifically adsorbed biotinylated Atp11p from yeast mitochondrial extracts, the F(1) beta-subunit coprecipitated with the tagged Atp11p protein. Binding interactions between Atp11p and the entire beta-subunit of F(1) or fragments of the beta-subunit were also revealed by a yeast two-hybrid screen: Atp11p bound to a region of the nucleotide-binding domain of the beta-subunit located between Gly(114) and Leu(318). Certain elements of this sequence that would be accessible to Atp11p in the free beta-subunit make contact with adjacent alpha-subunits in the assembled enzyme. This observation suggests that the alpha-subunits may exchange for bound Atp11p during the process of F(1) assembly. PMID: 10681564 [PubMed - indexed for MEDLINE] 542: Proc Natl Acad Sci U S A 2000 Feb 29;97(5):2011-6 Anatomy of a proficient enzyme: the structure of orotidine 5'-monophosphate decarboxylase in the presence and absence of a potential transition state analog. Miller BG, Hassell AM, Wolfenden R, Milburn MV, Short SA. Department of Biochemistry, University of North Carolina, Chapel Hill, NC 27599, USA. Research Triangle Park, NC 27709, USA. Orotidine 5'-phosphate decarboxylase produces the largest rate enhancement that has been reported for any enzyme. The crystal structure of the recombinant Saccharomyces cerevisiae enzyme has been determined in the absence and presence of the proposed transition state analog 6-hydroxyuridine 5'-phosphate, at a resolution of 2.1 A and 2.4 A, respectively. Orotidine 5'-phosphate decarboxylase folds as a TIM-barrel with the ligand binding site near the open end of the barrel. The binding of 6-hydroxyuridine 5'-phosphate is accompanied by protein loop movements that envelop the ligand almost completely, forming numerous favorable interactions with the phosphoryl group, the ribofuranosyl group, and the pyrimidine ring. Lysine-93 appears to be anchored in such a way as to optimize electrostatic interactions with developing negative charge at C-6 of the pyrimidine ring, and to donate the proton that replaces the carboxylate group at C-6 of the product. In addition, H-bonds from the active site to O-2 and O-4 help to delocalize negative charge in the transition state. Interactions between the enzyme and the phosphoribosyl group anchor the pyrimidine within the active site, helping to explain the phosphoribosyl group's remarkably large contribution to catalysis despite its distance from the site of decarboxylation. PMID: 10681417 [PubMed - indexed for MEDLINE] 543: EMBO J 2000 Feb 15;19(4):683-90 The novel coactivator C1 (HCF) coordinates multiprotein enhancer formation and mediates transcription activation by GABP. Vogel JL, Kristie TM. Laboratory of Viral Diseases, National Institutes of Health, Building 4, Room 133, 4 Center Drive, Bethesda, MD 20892, USA. Transcription of the herpes simplex virus 1 (HSV-1) immediate early (IE) genes is determined by multiprotein enhancer complexes. The core enhancer assembly requires the interactions of the POU-homeodomain protein Oct-1, the viral transactivator alphaTIF and the cellular factor C1 (HCF). In this context, the C1 factor interacts with each protein to assemble the stable enhancer complex. In addition, the IE enhancer cores contain adjacent binding sites for other cellular transcription factors such as Sp1 and GA-binding protein (GABP). In this study, a direct interaction of the C1 factor with GABP is demonstrated, defining the C1 factor as the critical coordinator of the enhancer complex assembly. In addition, mutations that reduce the GABP transactivation potential also impair the C1-GABP interaction, indicating that the C1 factor functions as a novel coactivator of GABP-mediated transcription. The interaction and coordinated assembly of the enhancer proteins by the C1 factor may be critical for the regulation of the HSV lytic-latent cycle. PMID: 10675337 [PubMed - indexed for MEDLINE] 544: EMBO J 2000 Feb 15;19(4):581-8 Crystal structure of a class I alpha1,2-mannosidase involved in N-glycan processing and endoplasmic reticulum quality control. Vallee F, Lipari F, Yip P, Sleno B, Herscovics A, Howell PL. Structural Biology and Biochemistry, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, M5G 1X8, Ontario. Mannose trimming is not only essential for N-glycan maturation in mammalian cells but also triggers degradation of misfolded glycoproteins. The crystal structure of the class I alpha1, 2-mannosidase that trims Man(9)GlcNAc(2) to Man(8)GlcNAc(2 )isomer B in the endoplasmic reticulum of Saccharomyces cerevisiae reveals a novel (alphaalpha)(7)-barrel in which an N-glycan from one molecule extends into the barrel of an adjacent molecule, interacting with the essential acidic residues and calcium ion. The observed protein-carbohydrate interactions provide the first insight into the catalytic mechanism and specificity of this eukaryotic enzyme family and may be used to design inhibitors that prevent degradation of misfolded glycoproteins in genetic diseases. PMID: 10675327 [PubMed - indexed for MEDLINE] 545: Gene 2000 Jan 11;241(2):309-15 A highly representative two-hybrid genomic library for the yeast Yarrowia lipolytica. Kabani M, Boisrame A, Beckerich JM, Gaillardin C. Laboratoire de Genetique Moleculaire et Cellulaire, INRA-INA.PG-CNRS BP 01 78850, Thiverval-Grignon, France. kabani@platon.grignon.inra.fr Since its description by Fields and Song in 1989 (Nature 340, 245-246), the yeast two-hybrid system has been used extensively to study protein-protein interactions, becoming increasingly efficient with technological and methodological improvements. Here, we report the construction of a highly representative two-hybrid genomic library for the dimorphic yeast Yarrowia lipolytica based on the system described by James et al. (1996. Genetics 144, 1425-1436). The endoplasmic reticulum protein Slslp was then used as a bait in a functional test of the library. Indeed, we previously showed that the SLS1 gene product is involved in protein translocation across the endoplasmic reticulum membrane and interacts physically in a two-hybrid assay with Kar2p, an essential luminal member of the HSP70 family (Boisrame et al., 1998. J. Biol. Chem. 273, 30 903-30 908). We developed a mating strategy similar to that used for the Saccharomyces cerevisiae FRYL library (Fromont-Racine et al., 1997. Nat. Genet. 16, 277-282). No other partner than Kar2p was identified in this screen. As an interesting result, Kar2p interacts with Slslp through its ATPase domain, supporting our hypothesis that Slslp is a cofactor of the chaperone protein, modulating its activity during the HSP70 cycle. Our results indicate that we have constructed a new and powerful tool for the study of Yarrowia lipolytica, which we believe is a good alternative model to investigate such complex biological processes as secretion pathways. PMID: 10675043 [PubMed - indexed for MEDLINE] 546: Methods 2000 Feb;20(2):219-31 Identification of connexin-interacting proteins: application of the yeast two-hybrid screen. Jin C, Lau AF, Martyn KD. Molecular Carcinogenesis, Cancer Research Center of Hawaii, University of Hawaii at Manoa, 1236 Lauhala Street, Room 304, Honolulu, Hawaii 96813, USA. Protein-protein interactions are recognized as one of the fundamental mechanisms for relaying the intra- and intercellular signals that are required for normal cellular activities affecting growth, development, and maintenance of homeostasis in tissues and organs. The yeast two-hybrid screen has become a valuable tool for identifying protein-protein interactions. The gap junction protein connexin 43 (Cx43) has been implicated in a number of biological processes including development and cellular growth control. To further advance our understanding of the ways in which Cx43 may influence these cellular activities, and to extend our knowledge of the regulation of Cx43 function and/or processing, we have employed the yeast two-hybrid screen technique to identify Cx43-interacting proteins. We present detailed methods for the yeast two-hybrid screen of a mouse embryonic cDNA library using the C terminus of Cx43 as "bait." We also describe additional methods to confirm the interactions between Cx43 and the identified proteins. These methods include in vitro binding assays, coimmunoprecipitation, and subcellular localization using immunofluorescence microscopy. Copyright 2000 Academic Press. PMID: 10671315 [PubMed - indexed for MEDLINE] 547: Biol Cell 1999 Dec;91(9):649-63 The Saccharomyces cerevisiae Cdc14 phosphatase is implicated in the structural organization of the nucleolus. de Almeida A, Raccurt I, Peyrol S, Charbonneau M. UMR CNRS/ENS no 5665, Ecole Normale Superieure, Lyon, France. Cdc14, a dual-specificity protein phosphatase, has been previously implicated in triggering exit from mitosis in the yeast Saccharomyces cerevisiae. Using immunofluorescence microscopy and immunogold labeling, we demonstrate that a functional HA-tagged version of the phosphatase Cdc14 localizes to the nucleolus. Moreover, Cdc14-HA co-localized with the nucleolar NOP2 and GAR1 proteins. By immunofluorescence, Cdc14-HA was found in the nucleolus during most of the mitotic cell cycle, except during anaphase-telophase when it redistributed along the mitotic spindle. While this work was in progress, the same pattern of Cdc14 localization was described by others (Visintin et al, Nature 398 (1999) 818). Constitutive overexpression of CDC14 was toxic and led to cell cycle arrest of cells, mainly in G1. This correlated with the appearance of abnormal nuclear structures. A genetic search for suppressors of the lethality associated with CDC14 overexpression identified YJL076W. Because overproduction of Yj1076w buffered the toxic effect of Cdc14 overproduction, this suggested that it might be a substrate of Cdc14. This has indeed been found to be the case by others who recently described Yj1076w/Netl as a nucleolar protein that physically associates with Cdc14 (Shou et al, Cell 97 (1999) 233). The present data confirm several recently uncovered aspects of the regulation of Cdc14 localization and activity and suggest that the level of expression of CDC14 influences the structural organization of the nucleolus. PMID: 10668096 [PubMed - indexed for MEDLINE] 548: J Virol 2000 Mar;74(5):2372-82 A chimeric protein containing the N terminus of the adeno-associated virus Rep protein recognizes its target site in an in vivo assay. Cathomen T, Collete D, Weitzman MD. Laboratory of Genetics, The Salk Institute for Biological Studies, San Diego, California 92186, USA. The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration. They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19. Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization. In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo. Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression. Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and in Saccharomyces cerevisiae from a reporter cassette integrated into the yeast genome. Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers. These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications. PMID: 10666268 [PubMed - indexed for MEDLINE] 549: Curr Biol 2000 Jan 27;10(2):111-4 Two paralogs involved in transcriptional silencing that antagonistically control yeast life span. Roy N, Runge KW. Department of Molecular Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA. In the yeast Saccharomyces cerevisiae, one determinant of aging or life span is the accumulation of extrachromosomal copies of rDNA circles in old mother cells [1]. The production of rDNA circles depends upon intrachromosomal recombination within the rDNA tandem array, a process regulated by the protein Sir2 (Sir2p). Together with Sir1p, Sir3p, Sir4p and Orc1p, Sir2p is also involved in transcriptional silencing of genes at the silent mating-type cassettes, in the rDNA array, and at telomeres. Using a 'triple silencer' strain that can monitor an increase or decrease in gene expression at these three loci, we found that deletion of the ZDS1 gene caused an increase in silencing in the rDNA and at a silent mating-type cassette at the expense of telomere silencing. The zds1 deletion also resulted in an increase in life span and a decrease in Sir3p phosphorylation. In contrast, deletion of its paralog ZDS2 caused a decrease in rDNA silencing, a decrease in life span and an increase in Sir3p phosphorylation. As Zds2p, but not Zds1p, had strong two-hybrid interactions with Orc1p and the four Sir proteins, Zds1p might indirectly control Sir3p through a Sir3p kinase. PMID: 10662670 [PubMed - indexed for MEDLINE] 550: Virology 2000 Feb 15;267(2):185-98 Multiple interactions among proteins encoded by the mite-transmitted wheat streak mosaic tritimovirus. Choi IR, Stenger DC, French R. School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68583, USA. The genome organization of the mite-transmitted wheat streak mosaic virus (WSMV) appears to parallel that of members of the Potyviridae with monopartite genomes, but there are substantial amino acid dissimilarities with other potyviral polyproteins. To initiate studies on the functions of WSMV-encoded proteins, a protein interaction map was generated using a yeast two-hybrid system. Because the pathway of proteolytic maturation of the WSMV polyprotein has not been experimentally determined, random libraries of WSMV cDNA were made both in DNA-binding domain and activation domain plasmid vectors and introduced into yeast. Sequence analysis of multiple interacting pairs revealed that interactions largely occurred between domains within two groups of proteins. The first involved interactions among nuclear inclusion protein a, nuclear inclusion protein b, and coat protein (CP), and the second involved helper component-proteinase (HC-Pro) and cylindrical inclusion protein (CI). Further immunoblot and deletion mapping analyses of the interactions suggest that subdomains of CI, HC-Pro, and P1 interact with one another. The two-hybrid assay was then performed using full-length genes of CI, HC-Pro, P1, P3, and CP, but no heterologous interactions were detected. In vitro binding assay using glutathione-S-transferase fusion proteins and in vitro translation products, however, revealed mutual interactions among CI, HC-Pro, P1, and P3. The failure to detect interactions between full-length proteins by the two-hybrid assay might be due to adverse effects of expression of viral proteins in yeast cells. The capacity to participate in multiple homomeric and heteromeric molecular interactions is consistent with the pleiotropic nature of many potyviral gene mutants and suggests mechanisms for regulation of various viral processes via a network of viral protein complexes. Copyright 2000 Academic Press. PMID: 10662614 [PubMed - indexed for MEDLINE] 551: J Cell Biol 2000 Feb 7;148(3):441-52 Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast. Segal M, Clarke DJ, Maddox P, Salmon ED, Bloom K, Reed SI. Department of Molecular Biology, MB7, The Scripps Research Institute, La Jolla, California 92037, USA. The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Delta cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Delta cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly. PMID: 10662771 [PubMed - indexed for MEDLINE] 552: Mol Gen Genet 2000 Jan;262(6):1147-56 SLG1 plays a role during G1 in the decision to enter or exit the cell cycle. Ivanovska I, Rose MD. Department of Molecular Biology, Princeton University, NJ 08544-1014, USA. Saccharomyces cerevisiae cells decide to divide during G1. If nutrients are abundant, cells pass through START and coordinately undergo DNA replication, bud emergence, and spindle pole body duplication. Phenotypic analysis of the slg1delta mutant revealed that this mutation uncouples post-START events. At the nonpermissive temperature, slg1delta cells that have undergone bud emergence but not DNA replication or SPB duplication accumulate. Furthermore, while wild-type cells arrest in GO when starved, the slg1delta mutant fails to arrest at this point; instead, cells with small buds accumulate. The slg1delta mutation displayed genetic interactions with cdc34, which encodes a regulator of exit from G1. This is consistent with a role of SLG1 in G1 regulation. Epitope-tagged Slg1p cofractionated with the plasma membrane, suggesting that Slglp may function by integrating external cues and relaying them to the interior of the cell. We propose that SLG1 plays a regulatory role in bud emergence or stationary phase. PMID: 10660075 [PubMed - indexed for MEDLINE] 553: J Mol Biol 2000 Feb 11;296(1):7-17 Domain III of Saccharomyces cerevisiae 25 S ribosomal RNA: its role in binding of ribosomal protein L25 and 60 S subunit formation. van Beekvelt CA, Kooi EA, de Graaff-Vincent M, Riet J, Venema J, Raue HA. Department of Biochemistry and Molecular Biology, IMBW BioCentrum Amsterdam, Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. Domain III of Saccharomyces cerevisiae 25 S rRNA contains the recognition site for the primary rRNA-binding ribosomal protein L25, which belongs to the functionally conserved EL23/L25 family of ribosomal proteins. The EL23/L25 binding region is very complex, consisting of several irregular helices held together by long-distance secondary and tertiary interactions. Moreover, it contains the eukaryote-specific V9 (D7a) expansion segment. Functional characterisation of the structural elements of this site by a detailed in vitro and in vivo mutational analysis indicates the presence of two separate regions that are directly involved in L25 binding. In particular, mutation of either of two conserved nucleotides in the loop of helix 49 significantly reduces in vitro L25 binding, thus strongly supporting their role as attachment sites for the r-protein. Two other helices appear to be primarily required for the correct folding of the binding site. Mutations that abolish in vitro binding of L25 block accumulation of 25 S rRNA in vivo because they stall pre-rRNA processing at the level of its immediate precursor, the 27 S(B) pre-rRNA. Surprisingly, several mutations that do not significantly affect L25 binding in vitro cause the same lethal defect in 27 S(B) pre-rRNA processing. Deletion of the V9 expansion segment also leads to under-accumulation of mature 25 S rRNA and a twofold reduction in growth rate. We conclude that an intact domain III, including the V9 expansion segment, is essential for normal processing and assembly of 25 S rRNA. Copyright 2000 Academic Press. PMID: 10656814 [PubMed - indexed for MEDLINE] 554: J Mol Biol 2000 Jan 28;295(4):927-38 X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity. Albert A, Yenush L, Gil-Mascarell MR, Rodriguez PL, Patel S, Martinez-Ripoll M, Blundell TL, Serrano R. Grupo de Cristalografia Macromolecular y Biologia Estructural, Instituto de Quimica Fisica "Rocasolano", Consejo Superior de Investigaciones Cientificas, Serrano 119, Madrid, E-28006, Spain. xalbert@iqfr.csic.es The product of the yeast HAL2 gene (Hal2p) is an in vivo target of sodium and lithium toxicity and its overexpression improves salt tolerance in yeast and plants. Hal2p is a metabolic phosphatase which catalyses the hydrolysis of 3'-phosphoadenosine-5'-phosphate (PAP) to AMP. It is, the prototype of an evolutionarily conserved family of PAP phosphatases and the engineering of sodium insensitive enzymes of this group may contribute to the generation of salt-tolerant crops. We have solved the crystal structure of Hal2p in complex with magnesium, lithium and the two products of PAP hydrolysis, AMP and Pi, at 1.6 A resolution. A functional screening of random mutations of the HAL2 gene in growing yeast generated forms of the enzyme with reduced cation sensitivity. Analysis of these mutants defined a salt bridge (Glu238 ellipsis Arg152) and a hydrophobic bond (Va170 ellipsis Trp293) as important framework interactions determining cation sensitivity. Hal2p belongs to a larger superfamily of lithium-sensitive phosphatases which includes inositol monophosphatase. The hydrophobic interaction mutated in Hal2p is conserved in this superfamily and its disruption in human inositol monophosphatase also resulted in reduced cation sensitivity. Copyright 2000 Academic Press. PMID: 10656801 [PubMed - indexed for MEDLINE] 555: Nat Struct Biol 2000 Feb;7(2):113-7 The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix. Li M, Phylip LH, Lees WE, Winther JR, Dunn BM, Wlodawer A, Kay J, Gustchina A. Macromolecular Crystallography Laboratory, Program in Structural Biology, National Cancer Institute-FCRDC, Frederick, Maryland 21702, USA. Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae. Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule. Structures solved at 2.2 and 1.8 A, respectively, for complexes of proteinase A with full-length IA3 and with a truncated form consisting only of residues 2-34, reveal an unprecedented mode of inhibitor-enzyme interactions. Neither form of the free inhibitor has detectable intrinsic secondary structure in solution. However, upon contact with the enzyme, residues 2-32 become ordered and adopt a near-perfect alpha-helical conformation. Thus, the proteinase acts as a folding template, stabilizing the helical conformation in the inhibitor, which results in the potent and specific blockage of the proteolytic activity. PMID: 10655612 [PubMed - indexed for MEDLINE] 556: Proc Natl Acad Sci U S A 2000 Feb 1;97(3):1143-7 Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins. Ito T, Tashiro K, Muta S, Ozawa R, Chiba T, Nishizawa M, Yamamoto K, Kuhara S, Sakaki Y. Human Genome Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. tito@ims.u-tokyo.ac.jp Protein-protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell. As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae. We cloned all of the yeast ORFs individually as a DNA-binding domain fusion ("bait") in a MATa strain and as an activation domain fusion ("prey") in a MATalpha strain, and subsequently divided them into pools, each containing 96 clones. These bait and prey clone pools were systematically mated with each other, and the transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging. Our initial examination of approximately 4 x 10(6) different combinations, constituting approximately 10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport. The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein-protein interaction map. PMID: 10655498 [PubMed - indexed for MEDLINE] 557: Genetics 2000 Feb;154(2):557-71 A yeast heterogeneous nuclear ribonucleoprotein complex associated with RNA polymerase II. Conrad NK, Wilson SM, Steinmetz EJ, Patturajan M, Brow DA, Swanson MS, Corden JL. Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. Recent evidence suggests a role for the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) in pre-mRNA processing. The yeast NRD1 gene encodes an essential RNA-binding protein that shares homology with mammalian CTD-binding proteins and is thought to regulate mRNA abundance by binding to a specific cis-acting element. The present work demonstrates genetic and physical interactions among Nrd1p, the pol II CTD, Nab3p, and the CTD kinase CTDK-I. Previous studies have shown that Nrd1p associates with the CTD of pol II in yeast two-hybrid assays via its CTD-interaction domain (CID). We show that nrd1 temperature-sensitive alleles are synthetically lethal with truncation of the CTD to 9 or 10 repeats. Nab3p, a yeast hnRNP, is a high-copy suppressor of some nrd1 temperature-sensitive alleles, interacts with Nrd1p in a yeast two-hybrid assay, and coimmunoprecipitates with Nrd1p. Temperature-sensitive alleles of NAB3 are suppressed by deletion of CTK1, a kinase that has been shown to phosphorylate the CTD and increase elongation efficiency in vitro. This set of genetic and physical interactions suggests a role for yeast RNA-binding proteins in transcriptional regulation. PMID: 10655211 [PubMed - indexed for MEDLINE] 558: J Biol Chem 2000 Feb 4;275(5):3128-36 Analysis of the yeast arginine methyltransferase Hmt1p/Rmt1p and its in vivo function. Cofactor binding and substrate interactions. McBride AE, Weiss VH, Kim HK, Hogle JM, Silver PA. Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115, USA. Many eukaryotic RNA-binding proteins are modified by methylation of arginine residues. The yeast Saccharomyces cerevisiae contains one major arginine methyltransferase, Hmt1p/Rmt1p, which is not essential for normal cell growth. However, cells missing HMT1 and also bearing mutations in the mRNA-binding proteins Npl3p or Cbp80p can no longer survive, providing genetic backgrounds in which to study Hmt1p function. We now demonstrate that the catalytically active form of Hmt1p is required for its activity in vivo. Amino acid changes in the putative Hmt1p S-adenosyl-L-methionine-binding site were generated and shown to be unable to catalyze methylation of Npl3p in vitro and in vivo or to restore growth to strains that require HMT1. In addition these mutations affect nucleocytoplasmic transport of Npl3p. A cold-sensitive mutant of Hmt1p was generated and showed reduced methylation of Npl3p, but not of other substrates, at 14 degrees C. These results define new aspects of Hmt1 and reveal the importance of its activity in vivo. PMID: 10652296 [PubMed - indexed for MEDLINE] 559: Biochem Biophys Res Commun 2000 Feb 5;268(1):73-7 Investigation of Fanconi anemia protein interactions by yeast two-hybrid analysis. Huber PA, Medhurst AL, Youssoufian H, Mathew CG. Division of Medical Genetics, Guy's, King's and St. Thomas' School of Medicine, Guy's Hospital, 7th Floor, Guy's Tower, London, SE1 9RT, United Kingdom. pia.huber@kcl.ac.uk Fanconi anemia is a chromosomal breakage disorder with eight complementation groups (A-H), and three genes (FANCA, FANCC, and FANCG) have been identified. Initial investigations of the interaction between FANCA and FANCC, principally by co-immunoprecipitation, have proved controversial. We used the yeast two-hybrid assay to test for interactions of the FANCA, FANCC, and FANCG proteins. No activation of the reporter gene was observed in yeast co-expressing FANCA and FANCC as hybrid proteins, suggesting that FANCA does not directly interact with FANCC. However, a high level of activation was found when FANCA was co-expressed with FANCG, indicating strong, direct interaction between these proteins. Both FANCA and FANCG show weak but consistent interaction with themselves, suggesting that their function may involve dimerisation. The site of interaction of FANCG with FANCA was investigated by analysis of 12 mutant fragments of FANCG. Although both N- and C-terminal fragments did interact, binding to FANCA was drastically reduced, suggesting that more than one region of the FANCG protein is required for proper interaction with FANCA. Copyright 2000 Academic Press. PMID: 10652215 [PubMed - indexed for MEDLINE] 560: Eur J Biochem 2000 Feb;267(3):861-8 The structural basis of substrate activation in yeast pyruvate decarboxylase. A crystallographic and kinetic study. Lu G, Dobritzsch D, Baumann S, Schneider G, Konig S. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 A resolution. The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104-113 and 290-304) disordered. In the other subunit, these loop regions are well-ordered and shield the active site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 A away from residue C221. This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer-dimer interactions in the activated tetramer. No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule. Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder-order transition of two active-site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur. Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed. PMID: 10651824 [PubMed - indexed for MEDLINE] 561: Mol Cell Biol 2000 Feb;20(4):1361-9 Yeast meiosis-specific protein Hop1 binds to G4 DNA and promotes its formation. Muniyappa K, Anuradha S, Byers B. Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India. DNA molecules containing stretches of contiguous guanine residues can assume a stable configuration in which planar quartets of guanine residues joined by Hoogsteen pairing appear in a stacked array. This conformation, called G4 DNA, has been implicated in several aspects of chromosome behavior including immunoglobulin gene rearrangements, promoter activation, and telomere maintenance. Moreover, the ability of the yeast SEP1 gene product to cleave DNA in a G4-DNA-dependent fashion, as well as that of the SGS1 gene product to unwind G4 DNA, has suggested a crucial role for this structure in meiotic synapsis and recombination. Here, we demonstrate that the HOP1 gene product, which plays a crucial role in the formation of synaptonemal complex in Saccharomyces cerevisiae, binds robustly to G4 DNA. The apparent dissociation constant for interaction with G4 DNA is 2 x 10(-10), indicative of binding that is about 1,000-fold stronger than to normal duplex DNA. Oligonucleotides of appropriate sequence bound Hop1 protein maximally if the DNA was first subjected to conditions favoring the formation of G4 DNA. Furthermore, incubation of unfolded oligonucleotides with Hop1 led to their transformation into G4 DNA. Methylation interference experiments confirmed that modifications blocking G4 DNA formation inhibit Hop1 binding. In contrast, neither bacterial RecA proteins that preferentially interact with GT-rich DNA nor histone H1 bound strongly to G4 DNA or induced its formation. These findings implicate specific interactions of Hop1 protein with G4 DNA in the pathway to chromosomal synapsis and recombination in meiosis. PMID: 10648621 [PubMed - indexed for MEDLINE] 562: Mol Cell Biol 2000 Feb;20(4):1321-8 Regulatory interactions between the Reg1-Glc7 protein phosphatase and the Snf1 protein kinase. Sanz P, Alms GR, Haystead TA, Carlson M. Departments of Genetics and Development and Microbiology, Columbia University, New York, New York 10032, USA. Protein phosphatase 1, comprising the regulatory subunit Reg1 and the catalytic subunit Glc7, has a role in glucose repression in Saccharomyces cerevisiae. Previous studies showed that Reg1 regulates the Snf1 protein kinase in response to glucose. Here, we explore the functional relationships between Reg1, Glc7, and Snf1. We show that different sequences of Reg1 interact with Glc7 and Snf1. We use a mutant Reg1 altered in the Glc7-binding motif to demonstrate that Reg1 facilitates the return of the activated Snf1 kinase complex to the autoinhibited state by targeting Glc7 to the complex. Genetic evidence indicated that the catalytic activity of Snf1 negatively regulates its interaction with Reg1. We show that Reg1 is phosphorylated in response to glucose limitation and that this phosphorylation requires Snf1; moreover, Reg1 is dephosphorylated by Glc7 when glucose is added. Finally, we show that hexokinase PII (Hxk2) has a role in regulating the phosphorylation state of Reg1, which may account for the effect of Hxk2 on Snf1 function. These findings suggest that the phosphorylation of Reg1 by Snf1 is required for the release of Reg1-Glc7 from the kinase complex and also stimulates the activity of Glc7 in promoting closure of the complex. PMID: 10648618 [PubMed - indexed for MEDLINE] 563: J Cell Biol 2000 Jan 24;148(2):353-62 Comment in: J Cell Biol. 2000 Jan 24;148(2):219-21. A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex. Evangelista M, Klebl BM, Tong AH, Webb BA, Leeuw T, Leberer E, Whiteway M, Thomas DY, Boone C. Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada. Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly. PMID: 10648568 [PubMed - indexed for MEDLINE] 564: Structure Fold Des 1999 Dec 15;7(12):1557-66 The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins. Liu Z, Macias MJ, Bottomley MJ, Stier G, Linge JP, Nilges M, Bork P, Sattler M. European Molecular Biology Laboratory, Heidelberg, Germany. BACKGROUND: The HRDC (helicase and RNaseD C-terminal) domain is found at the C terminus of many RecQ helicases, including the human Werner and Bloom syndrome proteins. RecQ helicases have been shown to unwind DNA in an ATP-dependent manner. However, the specific functional roles of these proteins in DNA recombination and replication are not known. An HRDC domain exists in both of the human RecQ homologues that are implicated in human disease and may have an important role in their function. RESULTS: We have determined the three-dimensional structure of the HRDC domain in the Saccharomyces cerevisiae RecQ helicase Sgs1p by nuclear magnetic resonance (NMR) spectroscopy. The structure resembles auxiliary domains in bacterial DNA helicases and other proteins that interact with nucleic acids. We show that a positively charged region on the surface of the Sgs1p HRDC domain can interact with DNA. Structural similarities to bacterial DNA helicases suggest that the HRDC domain functions as an auxiliary domain in RecQ helicases. Homology models of the Werner and Bloom HRDC domains show different surface properties when compared with Sgs1p. CONCLUSIONS: The HRDC domain represents a structural scaffold that resembles auxiliary domains in proteins that are involved in nucleic acid metabolism. In Sgs1p, the HRDC domain could modulate the helicase function via auxiliary contacts to DNA. However, in the Werner and Bloom syndrome helicases the HRDC domain may have a role in their functional differences by mediating diverse molecular interactions. PMID: 10647186 [PubMed - indexed for MEDLINE] 565: J Biol Chem 2000 Jan 28;275(4):2627-35 PAR1 thrombin receptor-G protein interactions. Separation of binding and coupling determinants in the galpha subunit. Swift S, Sheridan PJ, Covic L, Kuliopulos A. Molecular Cardiology Research Institute, Division of Hematology, New England Medical Center, Boston, Massachusetts 02111, USA. Signal transfer between the protease-activated PAR1 thrombin receptor and membrane-associated heterotrimeric G proteins is mediated by protein-protein interactions. We constructed a yeast signaling system that resolves domain-specific functions of binding from coupling in the Galpha subunit. The endogenous yeast Galpha subunit, Gpa1, does not bind to PAR1 and served as a null structural template. N- and C-terminal portions of mammalian G(i2) and G(16) were substituted back into the Gpa1 template and gain-of-function assessed. The C-terminal third of G(16), but not of G(i2), provides sufficient interactions for coupling to occur with PAR1. The N-terminal two-thirds of G(i2) also contains sufficient determinants to bind and couple to PAR1 and overcome the otherwise negative or missing interactions supplied by the C-terminal third of Gpa1. Replacement of the N-terminal alpha-helix of G(i2), residues 1-34, with those of Gpa1 abolishes coupling but not binding to PAR1 or to betagamma subunits. These data support a model that the N-terminal alphaN helix of the Galpha subunit is physically interposed between PAR1 and the Gbeta subunit and directly assists in transferring the signal between agonist-activated receptor and G protein. PMID: 10644723 [PubMed - indexed for MEDLINE] 566: J Gen Virol 2000 Jan;81(Pt 1):209-18 Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent RNA polymerase, VP1. Tacken MG, Rottier PJ, Gielkens AL, Peeters BP. Institute for Animal Science and Health (ID-Lelystad), Department of Avian Virology, PO Box 65, NL-8200 AB Lelystad, The Netherlands. m.g.j.tacken@id.wag-ur.nl Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain. A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa. Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it interacted exclusively with the 95 kDa protein both in infected cells and in the culture supernatant. These results suggest that the VP1-VP3 complex is involved in replication and packaging of the IBDV genome. PMID: 10640560 [PubMed - indexed for MEDLINE] 567: Genes Dev 2000 Jan 1;14(1):97-107 ATP can be dispensable for prespliceosome formation in yeast. Perriman R, Ares M Jr. Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz, Santa Cruz, California 95064, USA. The first ATP-dependent step in pre-mRNA splicing involves the stable binding of U2 snRNP to form the prespliceosome. We show that a prespliceosome-like complex forms in the absence of ATP in yeast extracts lacking the U2 suppressor protein CUS2. These complexes display the same pre-mRNA and U snRNA requirements as authentic prespliceosomes and can be chased through the splicing pathway, indicating that they are a functional intermediate in the spliceosome assembly pathway. ATP-independent prespliceosome-like complexes are also observed in extracts containing a mutant U2 snRNA. Loss of CUS2 does not bypass the role of PRP5, an RNA helicase family member required for ATP-dependent prespliceosome formation. Genetic interactions between CUS2 and a heat-sensitive prp5 allele parallel those observed between CUS2 and U2, and suggest that CUS2 mediates functional interactions between U2 RNA and PRP5. We propose that CUS2 enforces ATP dependence during formation of the prespliceosome by brokering an interaction between PRP5 and the U2 snRNP that depends on correct U2 RNA structure. PMID: 10640279 [PubMed - indexed for MEDLINE] 568: Nucleic Acids Res 2000 Feb 1;28(3):809-17 Isolation and characterization of human orthologs of yeast CCR4-NOT complex subunits. Albert TK, Lemaire M, van Berkum NL, Gentz R, Collart MA, Timmers HT. Laboratory for Physiological Chemistry and Centre for Biomedical Genetics, Utrecht University, PO Box 80042, 3508 TA Utrecht, The Netherlands, The yeast CCR4-NOT protein complex is a global regulator of RNA polymerase II transcription. It is comprised of yeast NOT1 to NOT5, yeast CCR4 and additional proteins like yeast CAF1. Here we report the isolation of cDNAs encoding human NOT2, NOT3, NOT4 and a CAF1-like factor, CALIF. Analysis of their mRNA levels in different human tissues reveals a common ubiquitous expression pattern. A multitude of two-hybrid interactions among the human cDNAs suggest that their encoded proteins also form a complex in mammalian cells. Functional conservation of these proteins throughout evolution is supported by the observation that the isolated human NOT3 and NOT4 cDNAs can partially com-plement corresponding not mutations in yeast. Interestingly, human CALIF is highly homologous to, although clearly different from, a recently described human CAF1 protein. Conserved interactions of this factor with both NOT and CCR4 proteins and co-immunoprecipitation experiments suggest that CALIF is a bona fide component of the human CCR4-NOT complex. PMID: 10637334 [PubMed - indexed for MEDLINE] 569: Mol Biol Cell 2000 Jan;11(1):369-91 Kinetic analysis of a molecular model of the budding yeast cell cycle. Chen KC, Csikasz-Nagy A, Gyorffy B, Val J, Novak B, Tyson JJ. Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg Virginia 24061, USA. The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on budding yeast have dissected the roles of cyclin molecules (Cln1-3 and Clb1-6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation. These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation. Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities. Model in hand, we examine the molecular events controlling "Start" (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and "Finish" (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants. The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in budding yeast. PMID: 10637314 [PubMed - indexed for MEDLINE] 570: Mol Biol Cell 2000 Jan;11(1):339-54 Functions and functional domains of the GTPase Cdc42p. Kozminski KG, Chen AJ, Rodal AA, Drubin DG. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA. Cdc42p, a Rho family GTPase of the Ras superfamily, is a key regulator of cell polarity and morphogenesis in eukaryotes. Using 37 site-directed cdc42 mutants, we explored the functions and interactions of Cdc42p in the budding yeast Saccharomyces cerevisiae. Cytological and genetic analyses of these cdc42 mutants revealed novel and diverse phenotypes, showing that Cdc42p possesses at least two distinct essential functions and acts as a nodal point of cell polarity regulation in vivo. In addition, mapping the functional data for each cdc42 mutation onto a structural model of the protein revealed as functionally important a surface of Cdc42p that is distinct from the canonical protein-interacting domains (switch I, switch II, and the C terminus) identified previously in members of the Ras superfamily. This region overlaps with a region (alpha5-helix) recently predicted by structural models to be a specificity determinant for Cdc42p-protein interactions. PMID: 10637312 [PubMed - indexed for MEDLINE] 571: Mol Biol Cell 2000 Jan;11(1):277-86 The N terminus of the transmembrane protein BP180 interacts with the N-terminal domain of BP230, thereby mediating keratin cytoskeleton anchorage to the cell surface at the site of the hemidesmosome. Hopkinson SB, Jones JC. Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA. In epidermal cells, the keratin cytoskeleton interacts with the elements in the basement membrane via a multimolecular junction called the hemidesmosome. A major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid autoantigen (BP230/BPAG1), which connects directly to the keratin-containing intermediate filaments of the cytoskeleton via its C terminus. A second bullous pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component of the hemidesmosome. Using yeast two-hybrid technology and recombinant proteins, we show that an N-terminal fragment of BP230 can bind directly to an N-terminal fragment of BP180. We have also explored the consequences of expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in vitro. Unexpectedly, this fragment disrupts the distribution of BP180 in transfected cells but has no apparent impact on the organization of endogenous BP230 and alpha6beta4 integrin. We propose that the BP230 N terminus competes with endogenous BP230 protein for BP180 binding and inhibits incorporation of BP180 into the cell surface at the site of the hemidesmosome. These data provide new insight into those interactions of the molecules of the hemidesmosome that are necessary for its function in integrating epithelial and connective tissue types. PMID: 10637308 [PubMed - indexed for MEDLINE] 572: J Biol Chem 2000 Jan 21;275(3):2191-8 Lipid-dependent targeting of G proteins into rafts. Moffett S, Brown DA, Linder ME. Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. Domains rich in sphingolipids and cholesterol, or rafts, may organize signal transduction complexes at the plasma membrane. Raft lipids are believed to exist in a state similar to the liquid-ordered phase. It has been proposed that proteins with a high affinity for an ordered lipid environment will preferentially partition into rafts (Melkonian, K. A., Ostermeyer, A. G., Chen, J. Z., Roth, M. G., and Brown, D. A. (1999) J. Biol. Chem. 274, 3910-3917). We investigated the possibility that lipid-lipid interactions between lipid-modified proteins and raft lipids mediate targeting of proteins to these domains. G protein monomers or trimers were reconstituted in liposomes, engineered to mimic raft domains. Assay for partitioning of G proteins into rafts was based on Triton X-100 insolubility. Myristoylation and palmitoylation of Galpha(i) were necessary and sufficient for association with liposomes and partitioning into rafts. Strikingly, the amount of fatty-acylated Galpha(i) in rafts was significantly reduced when myristoylated Galpha(i) was thioacylated with cis-unsaturated fatty acids instead of saturated fatty acids such as palmitate. Prenylated betagamma subunits were excluded from rafts, whether reconstituted alone or with fatty-acylated alpha subunits. These results suggest that the structural difference between lipids that modify proteins is one basis for the selectivity of protein targeting to rafts. PMID: 10636925 [PubMed - indexed for MEDLINE] 573: J Biol Chem 2000 Jan 21;275(3):2130-6 The RNA interacting domain but not the protein interacting domain is highly conserved in ribosomal protein P0. Rodriguez-Gabriel MA, Remacha M, Ballesta JP. Centro de Biologia Molecular "Severo Ochoa," Universidad Autonoma de Madrid and Consejo Superior de Investigaciones Cientificas, Cantoblanco, 28049 Madrid. Protein P0 interacts with proteins P1alpha, P1beta, P2alpha, and P2beta, and forms the Saccharomyces cerevisiae ribosomal stalk. The capacity of RPP0 genes from Aspergillus fumigatus, Dictyostelium discoideum, Rattus norvegicus, Homo sapiens, and Leishmania infantum to complement the absence of the homologous gene has been tested. In S. cerevisiae W303dGP0, a strain containing standard amounts of the four P1/P2 protein types, all heterologous genes were functional except the one from L. infantum, some of them inducing an osmosensitive phenotype at 37 degrees C. The polymerizing activity and the elongation factor-dependent functions but not the peptide bond formation capacity is affected in the heterologous P0 containing ribosomes. The heterologous P0 proteins bind to the yeast ribosomes but the composition of the ribosomal stalk is altered. Only proteins P1alpha and P2beta are found in ribosomes carrying the A. fumigatus, R. norvegicus, and H. sapiens proteins. When the heterologous genes are expressed in a conditional null-P0 mutant whose ribosomes are totally deprived of P1/P2 proteins, none of the heterologous P0 proteins complemented the conditional phenotype. In contrast, chimeric P0 proteins made of different amino-terminal fragments from mammalian origin and the complementary carboxyl-terminal fragments from yeast allow W303dGP0 and D67dGP0 growth at restrictive conditions. These results indicate that while the P0 protein RNA-binding domain is functionally conserved in eukaryotes, the regions involved in protein-protein interactions with either the other stalk proteins or the elongation factors have notably evolved. PMID: 10636918 [PubMed - indexed for MEDLINE] 574: Mol Microbiol 2000 Jan;35(1):15-31 Recruitment of the yeast MADS-box proteins, ArgRI and Mcm1 by the pleiotropic factor ArgRIII is required for their stability. El Bakkoury M, Dubois E, Messenguy F. Institut de Recherches Microbiologiques J-M. Wiame, and Laboratoire de Microbiologie de l'Universit inverted question marke Libre de Bruxelles, Avenue E. Gryzon, 1, B-1070 Brussels, Belgium. Regulation of arginine metabolism requires the integrity of four regulatory proteins, ArgRI, ArgRII, ArgRIII and Mcm1. To characterize further the interactions between the different proteins, we used the two-hybrid system, which showed that ArgRI and Mcm1 interact together, and with ArgRII and ArgRIII, without an arginine requirement. To define the interacting domains of ArgRI and Mcm1 with ArgRIII, we fused portions of ArgRI and Mcm1 to the DNA-binding domain of Gal4 (GBD) and created mutations in GBD-ArgRI and GBD-Mcm1. The putative alpha helix present in the MADS-box domain of ArgRI and Mcm1 is their major region of interaction with ArgRIII. Interactions between the two MADS-box proteins and ArgRIII were confirmed using affinity chromatography. The requirement for ArgRIII in the control of arginine metabolism can be bypassed in vitro as well as in vivo by overproducing ArgRI or Mcm1, which indicates that ArgRIII is not present in the protein complex formed with the 'arginine boxes'. We show that the impairment of arginine regulation in an argRIII deletant strain is a result of a lack of stability of ArgRI and Mcm1. A mutation in ArgRI, impairing its interaction with ArgRIII, leads to an unstable ArgRI protein in a wild-type strain. ArgRIII integrity is crucial for Mcm1 function, as shown by the marked decreased expression of five genes controlled by Mcm1. However, ArgRIII is likely to recruit other proteins in the yeast cell, as overexpression of Mcm1 does not compensate some physiological defects observed in an argRIII deletant strain. PMID: 10632874 [PubMed - indexed for MEDLINE] 575: Nucleic Acids Res 2001 Jan 1;29(1):75-9 YPD, PombePD and WormPD: model organism volumes of the BioKnowledge library, an integrated resource for protein information. Costanzo MC, Crawford ME, Hirschman JE, Kranz JE, Olsen P, Robertson LS, Skrzypek MS, Braun BR, Hopkins KL, Kondu P, Lengieza C, Lew-Smith JE, Tillberg M, Garrels JI. Proteome, Inc., 100 Cummings Center, Suite 435M, Beverly, MA 01915, USA. mcc@proteome.com The BioKnowledge Library is a relational database and web site (http://www.proteome.com) composed of protein-specific information collected from the scientific literature. Each Protein Report on the web site summarizes and displays published information about a single protein, including its biochemical function, role in the cell and in the whole organism, localization, mutant phenotype and genetic interactions, regulation, domains and motifs, interactions with other proteins and other relevant data. This report describes four species-specific volumes of the BioKnowledge Library, concerned with the model organisms Saccharomyces cerevisiae (YPD), Schizosaccharomyces pombe (PombePD) and Caenorhabditis elegans (WormPD), and with the fungal pathogen Candida albicans (CalPD). Protein Reports of each species are unified in format, easily searchable and extensively cross-referenced between species. The relevance of these comprehensively curated resources to analysis of proteins in other species is discussed, and is illustrated by a survey of model organism proteins that have similarity to human proteins involved in disease. PMID: 11125054 [PubMed - indexed for MEDLINE] 576: Genetics 2000 Jan;154(1):83-97 Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network. Bensen ES, Costaguta G, Payne GS. Department of Biological Chemistry, School of Medicine, University of California, Los Angeles, California 90095, USA. Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane. To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae. Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone alpha-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment. By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN. One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences. The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1. Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53, but not the related INP51 and INP52 genes, resulted in alpha-factor maturation defects and exacerbated alpha-factor maturation defects when combined with chc1-521. Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN. PMID: 10628971 [PubMed - indexed for MEDLINE] 577: Genetics 2000 Jan;154(1):61-71 Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression. Ben-Yehuda S, Russell CS, Dix I, Beggs JD, Kupiec M. Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel. Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction. PMID: 10628969 [PubMed - indexed for MEDLINE] 578: Anal Biochem 2000 Jan 15;277(2):247-53 Yeast two-hybrid assay for examining human immunodeficiency virus protease heterodimer formation with dominant-negative inhibitors and multidrug-resistant variants. Todd S, Laboissiere MC, Craik CS. Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA. The yeast two-hybrid assay was used to study the dimerization of engineered and naturally occurring variants of human immunodeficiency virus (HIV) protease (PR) monomers. Defective monomers that were previously shown to exhibit a dominant-negative (D-N) effect in cultured mammalian cells were tested for their ability to interact in the two-hybrid assay. Similarly, monomers with dimer-interface substitutions and monomers harboring in vivo selected mutations that confer multidrug resistance (mdr) in an AIDS patient were tested for interaction in yeast. Dimer formation between wt monomers with catalytic aspartates was not detected in yeast, whereas the dimerization of PR monomers harboring the acid active site substitution D25N was readily demonstrated. The use of inactive monomers harboring the D25N substitution as a genetic background for studying additional HIV PR mutations allowed for the probing of interactions between monomers with mdr-associated mutations with those based on the HIV-1 HXB2R sequence. The HTLVIII/HIV-1 HXB2R clone has been the basis for a large number of HIV-related plasmids, primers, antibodies, and other specific reagents throughout the HIV research community. The results of our assay suggest that HXB2R-based D-N PR inhibitors associate with variant monomers based on the recently obtained nucleotide sequence from an AIDS patient with a multidrug-resistant virus. These results further encourage the use of D-N PR inhibitors as antiviral agents which may complement existing small-molecule combination therapies. Copyright 2000 Academic Press. PMID: 10625514 [PubMed - indexed for MEDLINE] 579: J Mol Biol 2000 Jan 21;295(3):393-409 Role of an alpha-helical bulge in the yeast heat shock transcription factor. Hardy JA, Walsh ST, Nelson HC. Department of Molecular Biology, University of California, Berkeley, CA, 94720-3206, USA. The heat shock transcription factor (HSF) is the master transcriptional regulator of the heat shock response. The identity of a majority of the genes controlled by HSF and the circumstances under which HSF becomes induced are known, but the details of the mechanism by which HSF is able to sense and respond to heat remains an enigma. For example, it is unclear whether HSF senses the heat shock directly or requires ancillary interactions from a heat-induced signaling pathway. We present the analysis of a series of mutations in an alpha-helical bulge in the DNA-binding domain of HSF. Deletion of residues in this bulged region increases the overall activity of the protein. Yeast containing the deletion mutant HSF are able to survive growth temperatures that are lethal to yeast containing wild-type HSF, and they are also constitutively thermotolerant. The increase in activity can be measured as an increase in both constitutive and induced transcriptional activity. The mutant proteins bind DNA more tightly than the wild-type protein does, but this is unlikely to account fully for the increase in transcriptional activity as yeast HSF is constitutively bound to its binding site in vivo. The stability of the mutant proteins to thermal denaturation is lower than wild-type, though their native-state structures are still well-folded. Therefore, the mutants may be structurally analogous to the heat-induced state of HSF, and suggest that the DNA-binding domain of HSF may be capable of sensing heat shock directly. Copyright 2000 Academic Press. PMID: 10623534 [PubMed - indexed for MEDLINE] 580: Biotechnol Bioeng 2000 Feb 5;67(3):300-11 Performance modeling and simulation of biochemical process sequences with interacting unit operations. Groep ME, Gregory ME, Kershenbaum LS, Bogle ID. Centre for Process Systems Engineering, Imperial College, London SW7 2BY. Many biochemical processes consist of a sequence of operations for which optimal operating conditions (setpoints) have to be determined. If such optimization is performed for each operation separately with respect to objectives defined for each operation individually, overall process performance is likely to be suboptimal. Interactions between unit operations have to be considered, and a unique objective has to be defined for the whole process. This paper shows how a suitable optimization problem can be formulated and solved to obtain the best overall set of operating conditions for a process. A typical enzyme production process has been chosen as an example. In order to arrive at a demonstrative model for the entire sequence of unit operations, it is shown how interaction effects may be accommodated in the models. Optimal operating conditions are then determined subject to a global process objective and are shown to be different from those resulting from optimization of each separate operation. As this strategy may result in an economic benefit, it merits further research into interaction modeling and performance optimization. Copyright 2000 John Wiley & Sons, Inc. PMID: 10620260 [PubMed - indexed for MEDLINE] 581: EMBO J 2000 Jan 4;19(1):37-47 The WD-repeat protein pfs2p bridges two essential factors within the yeast pre-mRNA 3'-end-processing complex. Ohnacker M, Barabino SM, Preker PJ, Keller W. Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. In the yeast Saccharomyces cerevisiae, pre-mRNA 3'-end processing requires six factors: cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor II (CF II), polyadenylation factor I (PF I), poly(A) polymerase (Pap1p) and poly(A)-binding protein I (Pab1p). We report the characterization of Pfs2p, a WD-repeat protein previously identified in a multiprotein complex carrying PF I-Pap1p activity. The 3'-end-processing defects of pfs2 mutant strains and the results of immunodepletion and immunoinactivation experiments indicate an essential function for Pfs2p in cleavage and polyadenylation. With a one-step affinity purification method that exploits protein A-tagged Pfs2p, we showed that this protein is part of a CF II-PF I complex. Pull-down experiments with GST fusion proteins revealed direct interactions of Pfs2p with subunits of CF II-PF I and CF IA. These results show that Pfs2p plays an essential role in 3'-end formation by bridging different processing factors and thereby promoting the assembly of the processing complex. PMID: 10619842 [PubMed - indexed for MEDLINE] 582: Carbohydr Res 1999 Oct 15;321(3-4):143-56 Synthesis and glycosidase inhibitory activity of 5-thioglucopyranosylamines. Molecular modeling of complexes with glucoamylase. Randell KD, Frandsen TP, Stoffer B, Johnson MA, Svensson B, Pinto BM. Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada. The synthesis of a series of 5-thio-D-glucopyranosylarylamines by reaction of 5-thio-D-glucopyranose pentaacetate with the corresponding arylamine and mercuric chloride catalyst is reported. The products were obtained as anomeric mixtures of the tetraacetates which can be separated and crystallized. The tetraacetates were deprotected to give alpha/beta mixtures of the parent compounds which were evaluated as inhibitors of the hydrolysis of maltose by glucoamylase G2 (GA). A transferred NOE NMR experiment with an alpha/beta mixture of 7 in the presence of GA showed that only the alpha isomer is bound by the enzyme. The Ki values, calculated on the basis of specific binding of the alpha isomers, are 0.47 mM for p-methoxy-N-phenyl-5-thio-D-glucopyranosylamine (7), 0.78 mM for N-phenyl-5-thio-D-glucopyranosylamine (8), 0.27 mM for p-nitro-N-phenyl-5-thio-D-glucopyranosylamine (9) and 0.87 mM for p-trifluoromethyl-N-phenyl-5-thio-D-glucopyranosylamine (10), and the K(m) values for the substrates maltose and p-nitrophenyl alpha-D-glucopyranoside are 1.2 and 3.7 mM, respectively. Methyl 4-amino-4-deoxy-4-N-(5'-thio-alpha-D-glucopyranosyl)-alpha-D-glucopyrano side (11) is a competitive inhibitor of GA wild-type (Ki 4 microM) and the active site mutant Trp120-->Phe GA (Ki 0.12 mM). Compounds 7, 8, and 11 are also competitive inhibitors of alpha-glucosidase from brewer's yeast, with Ki values of 1.05 mM, > 10 mM, and 0.5 mM, respectively. Molecular modeling of the inhibitors in the catalytic site of GA was used to probe the ligand-enzyme complementary interactions and to offer insight into the differences in inhibitory potencies of the ligands. PMID: 10614065 [PubMed - indexed for MEDLINE] 583: J Cell Biol 1999 Dec 27;147(7):1493-502 Adenine nucleotide translocase-1, a component of the permeability transition pore, can dominantly induce apoptosis. Bauer MK, Schubert A, Rocks O, Grimm S. Max-Planck-Institute for Biochemistry, 82152 Martinsried, Germany. Here, we describe the isolation of adenine nucleotide translocase-1 (ANT-1) in a screen for dominant, apoptosis-inducing genes. ANT-1 is a component of the mitochondrial permeability transition complex, a protein aggregate connecting the inner with the outer mitochondrial membrane that has recently been implicated in apoptosis. ANT-1 expression led to all features of apoptosis, such as phenotypic alterations, collapse of the mitochondrial membrane potential, cytochrome c release, caspase activation, and DNA degradation. Both point mutations that impair ANT-1 in its known activity to transport ADP and ATP as well as the NH(2)-terminal half of the protein could still induce apoptosis. Interestingly, ANT-2, a highly homologous protein could not lead to cell death, demonstrating the specificity of the signal for apoptosis induction. In contrast to Bax, a proapoptotic Bcl-2 gene, ANT-1 was unable to elicit a form of cell death in yeast. This and the observed repression of apoptosis by the ANT-1-interacting protein cyclophilin D suggest that the suicidal effect of ANT-1 is mediated by specific protein-protein interactions within the permeability transition pore. PMID: 10613907 [PubMed - indexed for MEDLINE] 584: Annu Rev Cell Dev Biol 1999;15:63-80 Cooperation between microtubule- and actin-based motor proteins. Brown SS. Department of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor 48109, USA. susanbb@umich.edu Organelle transport has been proposed to proceed in two steps: long-range transport along microtubules and local delivery via actin filaments. This model is supported by recent studies of pigment transport in several cell types and transport in neurons, and in several cases, class V myosin has been implicated as the actin-based motor. Mutations in mice (dilute) and yeast (myo2) have also implicated this class of myosin in organelle transport, and genetic interactions in yeast have indicated that a kinesin-related protein (Smy1p) plays a supporting role. This link between members of two different motor superfamilies has now taken a surprising turn: There is evidence for a physical interaction between class V myosins and kinesin or Smy1p in both mice and yeast. Publication Types: Review Review, Tutorial PMID: 10611957 [PubMed - indexed for MEDLINE] 585: Biochimie 1999 Dec;81(12):1079-87 Display of Ras on filamentous phage through cysteine replacement. Wind T, Kjaer S, Clark BF. University of Aarhus, Department of Molecular and Structural Biology, Denmark. Phage display technology has been used in a variety of contexts to understand and manipulate biomolecular interactions between proteins and other biomolecules. In this paper we describe the establishment of a phage display system for elucidation of the interactions between the GTPase Ras and its panel of effectors. It is shown how technical problems associated with phage display of a protein with unpaired cysteines, likely to be caused by the oxidizing environment of the bacterial periplasm into which the protein is directed, can be overcome by cysteine replacement based on functional and structural studies. First, the catalytic domain (residues 1-166) of mammalian H-Ras (Ras) was observed to be displayed on phage in an incorrect conformation not detectable by antibodies recognizing conformational epitopes on Ras. Although truncation of the phage coat protein used as fusion partner (g3p) resulted in minor improvements in the display, Ras was tailored for phage display by cysteine replacement. By replacing the three cysteines at positions 51, 80 and 118 of Ras with the corresponding residues in Saccharomyces cerevisiae RAS1, the resulting fusion-phage is recognized by the conformation-dependent anti-Ras antibodies. Furthermore, display of cysteine-free Ras is demonstrated by GTP-analogue dependent binding to the Ras-binding domain of the Ras-effector Raf1. These data pave the way for analysis of Ras-effector interactions using phage display technology yet demonstrate that phage display of proteins with normally reduced cysteines should be approached with caution. PMID: 10607402 [PubMed - indexed for MEDLINE] 586: RNA 1999 Dec;5(12):1615-31 Splicing factor SF1 from Drosophila and Caenorhabditis: presence of an N-terminal RS domain and requirement for viability. Mazroui R, Puoti A, Kramer A. Departement de Biologie Cellulaire, Universite de Geneve, Switzerland. Splicing factor SF1 contributes to the recognition of the 3' splice site by interacting with U2AF65 and binding to the intron branch site during the formation of the early splicing complex E. These interactions and the essential functional domains of SF1 are highly conserved in Saccharomyces cerevisiae. We have isolated cDNAs encoding SF1 from Drosophila (Dm) and Caenorhabditis (Ce). The encoded proteins share the U2AF65 interaction domain, a hnRNP K homology domain, and one or two zinc knuckles required for RNA binding as well as Pro-rich C-terminal sequences with their yeast and mammalian counterparts. In contrast to SF1 in other species, DmSF1 and CeSF1 are characterized by an N-terminal region enriched in Ser, Arg, Lys, and Asp residues with homology to the RS domains of several splicing proteins. These domains mediate protein-protein or protein-RNA interactions, suggesting an additional role for DmSF1 and CeSF1 in pre-mRNA splicing. Human (Hs), fly, and worm SF1 interact equally well with HsU2AF65 or the Drosophila homolog DmU2AF50. Moreover, DmSF1 lacking its N terminus is functional in prespliceosome formation in a HeLa splicing system, emphasizing the conserved nature of interactions at an early step in spliceosome assembly. The Ce-SF1 gene is located in a polycistronic transcription unit downstream of the genes encoding U2AF35 (uaf-2) and a cyclophilin (cyp-13), implying the coordinate transcriptional regulation of these genes. Injection of double-stranded RNA into C. elegans results in embryonic lethality; thus, the SF1 gene is essential not only in yeast but also in at least one metazoan. PMID: 10606272 [PubMed - indexed for MEDLINE] 587: RNA 1999 Dec;5(12):1526-34 Crystallographic structure of the amino terminal domain of yeast initiation factor 4A, a representative DEAD-box RNA helicase. Johnson ER, McKay DB. Department of Structural Biology, Stanford University School of Medicine, California 94305-5400, USA. The eukaryotic translation initiation factor 4A (elF4A) is a representative of the DEAD-box RNA helicase protein family. We have solved the crystallographic structure of the amino-terminal domain (residues 1-223) of yeast elF4A. The domain is built around a core scaffold, a parallel alpha-beta motif with five beta strands, that is found in other RNA and DNA helicases, as well as in the RecA protein. The amino acid sequence motifs that are conserved within the helicase family are localized to the beta strand-->alpha helix junctions within the core. The core of the amino terminal domain of elF4A is amplified with additional structural elements that differ from those of other helicases. The phosphate binding loop (the Walker A motif) is in an unusual closed conformation. The crystallographic structure reveals specific interactions between amino acid residues of the phosphate binding loop, the DEAD motif, and the SAT motif, whose alteration is known to impair coupling between the ATPase cycle and the RNA unwinding activity of elF4A. PMID: 10606264 [PubMed - indexed for MEDLINE] 588: EMBO J 1999 Dec 15;18(24):7041-55 SIR repression of a yeast heat shock gene: UAS and TATA footprints persist within heterochromatin. Sekinger EA, Gross DS. Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, LA 71130-3932, USA. Previous work has suggested that products of the Saccharomyces cerevisiae Silent Information Regulator (SIR) genes form a complex with histones, nucleated by cis-acting silencers or telomeres, which represses transcription in a position-dependent but sequence-independent fashion. While it is generally thought that this Sir complex works through the establishment of heterochromatin, it is unclear how this structure blocks transcription while remaining fully permissive to other genetic processes such as recombination or integration. Here we examine the molecular determinants underlying the silencing of HSP82, a transcriptionally potent, stress-inducible gene. We find that HSP82 is efficiently silenced in a SIR-dependent fashion, but only when HMRE mating-type silencers are configured both 5' and 3' of the gene. Accompanying dominant repression are novel wrapped chromatin structures within both core and upstream promoter regions. Strikingly, DNase I footprints mapping to the binding sites for heat shock factor (HSF) and TATA-binding protein (TBP) are strengthened and broadened, while groove-specific interactions, as detected by dimethyl sulfate, are diminished. Our data are consistent with a model for SIR repression whereby transcriptional activators gain access to their cognate sites but are rendered unproductive by a co-existing heterochromatic complex. PMID: 10601026 [PubMed - indexed for MEDLINE] 589: J Mol Biol 1999 Dec 17;294(5):1311-25 Crystal structure of the histone acetyltransferase Hpa2: A tetrameric member of the Gcn5-related N-acetyltransferase superfamily. Angus-Hill ML, Dutnall RN, Tafrov ST, Sternglanz R, Ramakrishnan V. Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84132, USA. We report the crystal structure of the yeast protein Hpa2 in complex with acetyl coenzyme A (AcCoA) at 2.4 A resolution and without cofactor at 2.9 A resolution. Hpa2 is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, a family of enzymes with diverse substrates including histones, other proteins, arylalkylamines and aminoglycosides. In vitro, Hpa2 is able to acetylate specific lysine residues of histones H3 and H4 with a preference for Lys14 of histone H3. Hpa2 forms a stable dimer in solution and forms a tetramer upon binding AcCoA. The crystal structure reveals that the Hpa2 tetramer is stabilized by base-pair interactions between the adenine moieties of the bound AcCoA molecules. These base-pairs represent a novel method of stabilizing an oligomeric protein structure. Comparison of the structure of Hpa2 with those of other GNAT superfamily members illustrates a remarkably conserved fold of the catalytic domain of the GNAT family even though members of this family share low levels of sequence homology. This comparison has allowed us to better define the borders of the four sequence motifs that characterize the GNAT family, including a motif that is not discernable in histone acetyltransferases by sequence comparison alone. We discuss implications of the Hpa2 structure for the catalytic mechanism of the GNAT enzymes and the opportunity for multiple histone tail modification created by the tetrameric Hpa2 structure. Copyright 1999 Academic Press. PMID: 10600387 [PubMed - indexed for MEDLINE] 590: Protein Sci 1999 Nov;8(11):2465-73 Structures of yeast vesicle trafficking proteins. Tishgarten T, Yin FF, Faucher KM, Dluhy RA, Grant TR, Fischer von Mollard G, Stevens TH, Lipscomb LA. Department of Biochemistry & Molecular Biology, University of Georgia, Athens 30602, USA. In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE, is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes. PMID: 10595551 [PubMed - indexed for MEDLINE] 591: Mol Cell Biol 2000 Jan;20(1):104-12 Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II. Rodriguez CR, Cho EJ, Keogh MC, Moore CL, Greenleaf AL, Buratowski S. Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115, USA. The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription. PMID: 10594013 [PubMed - indexed for MEDLINE] 592: Mol Cell Biol 2000 Jan;20(1):26-33 Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation. Shima F, Okada T, Kido M, Sen H, Tanaka Y, Tamada M, Hu CD, Yamawaki-Kataoka Y, Kariya K, Kataoka T. Department of Physiology II, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, Japan. Posttranslational modification, in particular farnesylation, of Ras is crucial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1). Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Ras, we postulated that the associated CAP might contribute to the formation of a Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary Ras-binding site, the leucine-rich repeat domain. Here, we observed a posttranslational modification-dependent association of Ras with a complex between CAP and CYR1 C-terminal region. When CAP mutants defective in Ras signaling but retaining the CYR1-binding activity were isolated by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by modified Ras and exhibited a severely impaired ability to bind Ras, providing a genetic evidence for the importance of the physical association with Ras at the second Ras-binding site. On the other hand, CYR1, complexed with the other CAP mutant, failed to be activated by Ras but exhibited a greatly enhanced binding to Ras. Conversely, a Ras mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 efficiently. Thus, the strength of interaction at the second Ras-binding site appears to be a critical determinant of CYR1 regulation by Ras: too-weak and too-strong interactions are both detrimental to CYR1 activation. These results, taken together with those obtained with mammalian Raf, suggest the importance of the second Ras-binding site in effector regulation. PMID: 10594005 [PubMed - indexed for MEDLINE] 593: Mol Cell Biol 2000 Jan;20(1):12-25 Pan1p, End3p, and S1a1p, three yeast proteins required for normal cortical actin cytoskeleton organization, associate with each other and play essential roles in cell wall morphogenesis. Tang HY, Xu J, Cai M. Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609, Singapore. The EH domain proteins Pan1p and End3p of budding yeast have been known to form a complex in vivo and play important roles in organization of the actin cytoskeleton and endocytosis. In this report, we describe new findings concerning the function of the Pan1p-End3p complex. First, we found that the Pan1p-End3p complex associates with Sla1p, another protein known to be required for the assembly of cortical actin structures. Sla1p interacts with the first long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and the C-terminal repeat region of End3p, undisturbed. Second, Pan1p, End3p, and Sla1p are also required for normal cell wall morphogenesis. Each of the Pan1-4, sla1Delta, and end3Delta mutants displays the abnormal cell wall morphology previously reported for the act1-1 mutant. These cell wall defects are also exhibited by wild-type cells overproducing the C-terminal region of Sla1p that is responsible for interactions with Pan1p and End3p. These results indicate that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may depend on the formation of a heterotrimeric complex. Interestingly, the cell wall abnormalities exhibited by these cells are independent of the actin cytoskeleton organization on the cell cortex, as they manifest despite the presence of apparently normal cortical actin cytoskeleton. Examination of several act1 mutants also supports this conclusion. These observations suggest that the Pan1p-End3p-Sla1p complex is required not only for normal actin cytoskeleton organization but also for normal cell wall morphogenesis in yeast. PMID: 10594004 [PubMed - indexed for MEDLINE] 594: Mol Cell Biol 2000 Jan;20(1):1-11 Scanning mutagenesis of Mcm1: residues required for DNA binding, DNA bending, and transcriptional activation by a MADS-box protein. Acton TB, Mead J, Steiner AM, Vershon AK. Waksman Institute of Microbiology, Department of Molecular Biology, Rutgers University, Piscataway, New Jersey 08854-8020, USA. MCM1 is an essential gene in the yeast Saccharomyces cerevisiae and is a member of the MADS-box family of transcriptional regulatory factors. To understand the nature of the protein-DNA interactions of this class of proteins, we have made a series of alanine substitutions in the DNA-binding domain of Mcm1 and examined the effects of these mutations in vivo and in vitro. Our results indicate which residues of Mcm1 are important for viability, transcriptional activation, and DNA binding and bending. Substitution of residues in Mcm1 which are highly conserved among the MADS-box proteins are lethal to the cell and abolish DNA binding in vitro. These positions have almost identical interactions with DNA in both the serum response factor-DNA and alpha2-Mcm1-DNA crystal structures, suggesting that these residues make up a conserved core of protein-DNA interactions responsible for docking MADS-box proteins to DNA. Substitution of residues which are not as well conserved among members of the MADS-box family play important roles in contributing to the specificity of DNA binding. These results suggest a general model of how MADS-box proteins recognize and bind DNA. We also provide evidence that the N-terminal extension of Mcm1 may have considerable conformational freedom, possibly to allow binding to different DNA sites. Finally, we have identified two mutants at positions which are critical for Mcm1-mediated DNA bending that have a slow-growth phenotype. This finding is consistent with our earlier results, indicating that DNA bending may have a role in Mcm1 function in the cell. PMID: 10594003 [PubMed - indexed for MEDLINE] 595: Yeast 1999 Dec;15(16):1761-8 New tools for protein linkage mapping and general two-hybrid screening. Durfee T, Draper O, Zupan J, Conklin DS, Zambryski PC. Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. durf@nature.berkeley.edu The two-hybrid system has proved to be a facile method for detecting and analyzing protein-protein interactions. An expanded application of this system, protein linkage mapping, provides a means of identifying interactions on a global scale and should prove a powerful tool in analyzing whole genomes as their sequences become available. To overcome some of the inherent difficulties in such a large-scale approach, we have constructed a set of new strains and vectors that will allow for more efficient screening. The strains contain a GAL1-URA3 reporter for positive and negative selection, as well as a UAS(G)-lacZ reporter. The strains are of opposite mating types, permitting libraries present in one strain to be easily screened against a second library in the companion strain. We also constructed a family of CEN-based vectors for expression of both Gal4 DNA-binding and activation domain fusions. These plasmids include a hemagglutinin epitope tag and different polylinkers to increase the ease of subcloning. CEN-based vectors are maintained at 1-2 copies per cell, limiting the number of individual cells containing multiple plasmids that can confuse further analyses, and ensuring that fusions are not expressed at toxic levels. Using these vectors, both homo- and heterodimeric interactions resulted in up to 10-fold higher reporter gene transcription than obtained with 2micro;-based plasmids, despite significantly lower protein levels. In addition to protein linkage mapping, these reagents should be generally useful in standard two-hybrid applications. Copyright 1999 John Wiley & Sons, Ltd. PMID: 10590464 [PubMed - indexed for MEDLINE] 596: Yeast 1999 Dec;15(16):1719-31 RGD1 genetically interacts with MID2 and SLG1, encoding two putative sensors for cell integrity signalling in Saccharomyces cerevisiae. de Bettignies G, Barthe C, Morel C, Peypouquet MF, Doignon F, Crouzet M. Laboratoire de Biologie Moleculaire et de Sequencage, UPR CNRS 9026, BP 64, 146 rue Leo Saignat, 33076 Bordeaux cedex, France. The RGD1 gene was identified during systematic genome sequencing of Saccharomyces cerevisiae. To further understand Rgd1p function, we set up a synthetic lethal screen for genes interacting with RGD1. Study of one lethal mutant made it possible to identify the SLG1 and MID2 genes. The gene SLG1/HCS77/WSC1 was mutated in the original synthetic lethal strain, whereas MID2/SMS1 acted as a monocopy suppressor. The SLG1 gene has been described to be an upstream component in the yeast PKC pathway and encodes a putative cell surface sensor for the activation of cell integrity signalling. First identified by viability loss of shmoos after pheromone exposure, and since found in different genetic screens, MID2 was recently reported as also encoding an upstream activator of the PKC pathway. The RGD1 gene showed genetic interactions with both sensors of cell integrity pathway. The rgd1 slg1 synthetic lethality was rescued by osmotic stabilization, as expected for mutants altered in cell wall integrity. The slight viability defect of rgd1 in minimal medium, which was exacerbated by mid2, was not osmoremediated. As for mutants altered in PKC pathway, the accumulation of small-budded dead cells in slg1, rgd1 and mid2 after heat shock was prevented by 1 M sorbitol. In addition, the rgd1 strain also displayed dead shmoos after pheromone treatment, like mid2. Taken together, the present results indicate close functional links between RGD1, MID2 and SLG1 and suggest that RGD1 and MID2 interact in a cell integrity signalling functionally linked to the PKC pathway. Copyright 1999 John Wiley & Sons, Ltd. PMID: 10590461 [PubMed - indexed for MEDLINE] 597: Mol Biol Cell 1999 Dec;10(12):4121-33 The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity. Adamo JE, Rossi G, Brennwald P. Department of Cell Biology, Cell Biology, and Genetics, Weill Medical College of Cornell University, New York, New York 10021, USA. Budding yeast grow asymmetrically by the polarized delivery of proteins and lipids to specific sites on the plasma membrane. This requires the coordinated polarization of the actin cytoskeleton and the secretory apparatus. We identified Rho3 on the basis of its genetic interactions with several late-acting secretory genes. Mutational analysis of the Rho3 effector domain reveals three distinct functions in cell polarity: regulation of actin polarity, transport of exocytic vesicles from the mother cell to the bud, and docking and fusion of vesicles with the plasma membrane. We provide evidence that the vesicle delivery function of Rho3 is mediated by the unconventional myosin Myo2 and that the docking and fusion function is mediated by the exocyst component Exo70. These data suggest that Rho3 acts as a key regulator of cell polarity and exocytosis, coordinating several distinct events for delivery of proteins to specific sites on the cell surface. PMID: 10588647 [PubMed - indexed for MEDLINE] 598: Biochem J 1999 Dec 15;344 Pt 3:633-42 Polyamine transport in bacteria and yeast. Igarashi K, Kashiwagi K. Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. iga16077@p.chiba-u.ac.jp The polyamine content of cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) were ABC transporters, each consisting of a periplasmic substrate-binding protein, two transmembrane proteins and a membrane-associated ATPase. The crystal structures of the substrate-binding proteins (PotD and PotF) have been solved. They consist of two domains with an alternating beta-alpha-beta topology, similar to other periplasmic binding proteins. The polyamine-binding site is in a cleft between the two domains, as determined by crystallography and site-directed mutagenesis. Polyamines are mainly recognized by aspartic acid and glutamic acid residues, which interact with the NH(2)- (or NH-) groups, and by tryptophan and tyrosine residues that have hydrophobic interactions with the methylene groups of polyamines. The precursor of one of the substrate binding proteins, PotD, negatively regulates transcription of the operon for the spermidine-preferential uptake system, thus providing another level of regulation of cellular polyamines. The third transport system, catalysed by PotE, mediates both uptake and excretion of putrescine. Uptake of putrescine is dependent on membrane potential, whereas excretion involves an exchange reaction between putrescine and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport protein (TPO1) was identified. The properties of this protein are similar to those of PotE, and TPO1 is located on the vacuolar membrane. Publication Types: Review Review, Tutorial PMID: 10585849 [PubMed - indexed for MEDLINE] 599: Anal Biochem 1999 Dec 1;276(1):18-26 Usefulness of statistic experimental designs in enzymology: example with recombinant hCYP3A4 and 1A2. Bournique B, Petry M, Gousset G. Rhone-Poulenc Rorer, Drug Metabolism and Pharmacokinetics, and Pharmaceutical Sciences, 13 Quai Jules Guesdes, Vitry s/Seine Cedex, 94403, France. bruno.bournique@rp-rorer.fr First, the effects of 10 incubation factors were screened altogether on nifedipine dehydrogenase (NIF) and methoxyresorufin O-deethylase (MROD) activities catalyzed by recombinant human CYP3A4 and 1A2, respectively. Using a statistic experimental design, only 36 assays were needed to be exhaustive. Eight factors influenced CYP3A4-mediated NIF activity: buffer type, pH, temperature, Mg/EDTA, cytochrome b5, NADPH-P450 reductase, NADH, and solvent. Two factors had no significant effect: total ionic concentration and catalase/reduced glutathione. Six factors influenced CYP1A2-mediated MROD rates: buffer type, pH, temperature, Mg/EDTA, NADH, and glycerol. Four factors had no significant effect: total ionic concentration, cytochrome b5, reductase, and NAD+. Secondly, the combined effects of ionic strength and Mg concentration on NIF/CYP3A4 were studied and easily modeled using another statistic experimental design. The effect of Mg was strong at a constant ionic strength of 100 mM and negligible at a constant ionic strength of 500 mM. Thirdly, the effects of influencing factors and their interactions on MROD/CYP1A2 were modeled after 40 assays using a third statistic experimental design. Later experiments confirmed the predictivity of the models and the efficiency of optimized conditions. This approach can be applied to other biochemistry areas. Copyright 1999 Academic Press. PMID: 10585740 [PubMed - indexed for MEDLINE] 600: Mol Gen Genet 1999 Oct;262(3):508-14 Mechanism of transcription termination: PTRF interacts with the largest subunit of RNA polymerase I and dissociates paused transcription complexes from yeast and mouse. Jansa P, Grummt I. Division of Molecular Biology of the Cell II, German Cancer Research Center, Heidelberg. Transcription termination by RNA polymerase I (Pol I) is a stepwise process. First the elongating RNA polymerase is forced to pause by DNA-bound transcription termination factor (TTF-I). Then the ternary transcription complex is dissociated by PTRF, a novel factor that promotes release of both nascent transcripts and Pol I from the template. In this study we have investigated the ability of PTRF to liberate transcripts from ternary transcription complexes isolated from yeast and mouse. Using immobilized, tailed templates that contain terminator sequences from Saccharomyces cerevisiae and mouse, respectively, we demonstrate that PTRF promotes release of terminated transcripts, irrespective of whether mouse Pol I has interacted with the murine termination factor TTF-I or its yeast homolog Reb1p. In contrast, mouse Pol I paused by the lac repressor remains bound to the template both in the presence and absence of PTRF. We demonstrate that PTRF interacts with the largest subunit of murine Pol I, with TTF-I and Reb1p, but not the lac repressor. The results imply that Pol I transcription termination in yeast and mouse is mediated by conserved interactions between Pol I, Reb1p/TTF-I and PTRF. PMID: 10589839 [PubMed - indexed for MEDLINE] 601: Mol Gen Genet 1999 Oct;262(3):473-80 Budding yeast Cdc6p induces re-replication in fission yeast by inhibition of SCF(Pop)-mediated proteolysis. Wolf DA, McKeon F, Jackson PK. Department of Cancer Cell Biology, Harvard School of Public Health, Boston, MA 02115, USA. dwolf@hsph.harvard.edu In fission yeast, overexpression of the replication initiator protein Cdc18p induces re-replication, a phenotype characterized by continuous DNA synthesis in the absence of cell division. In contrast, overexpression of Cdc6p, the budding yeast homolog of Cdc18p, does not cause re-replication in S. cerevisiae. However, we have found that Cdc6p has the ability to induce rereplication in fission yeast. Cdc6p cannot functionally replace Cdc18p, but instead interferes with the proteolysis of both Cdc18p and Rum1p, the inhibitor of the protein kinase Cdc2p. This activity of Cdc6p is entirely contained within a short N-terminal peptide, which forms a tight complex with Cdc2p and the F-box/WD-repeat protein Sud1p/Pop2p, a component of the SCF(Pop) ubiquitin ligase in fission yeast. These interactions are mediated by two distinct regions within the N-terminal region of Cdc6p and depend on the integrity of its Cdc2p phosphorylation sites. The data suggest that disruption of re-replication control by overexpression of Cdc6p in fission yeast is a consequence of sequestration of Cdc2p and Pop2p, two factors involved in the negative regulation of Rum1p, Cdc18p and potentially other replication proteins. PMID: 10589835 [PubMed - indexed for MEDLINE] 602: Cell 1999 Nov 24;99(5):533-43 Structure and mechanism of yeast RNA triphosphatase: an essential component of the mRNA capping apparatus. Lima CD, Wang LK, Shuman S. Biochemistry Department, Weill Medical College of Cornell University, New York, New York 10021, USA. RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in cap formation. The 2.05 A crystal structure of yeast RNA triphosphatase Cet1p reveals a novel active site fold whereby an eight-stranded beta barrel forms a topologically closed triphosphate tunnel. Interactions of a sulfate in the center of the tunnel with a divalent cation and basic amino acids projecting into the tunnel suggest a catalytic mechanism that is supported by mutational data. Discrete surface domains mediate Cet1p homodimerization and Cet1p binding to the guanylyltransferase component of the capping apparatus. The structure and mechanism of fungal RNA triphosphatases are completely different from those of mammalian mRNA capping enzymes. Hence, RNA triphosphatase presents an ideal target for structure-based antifungal drug discovery. PMID: 10589681 [PubMed - indexed for MEDLINE] 603: J Biol Chem 1999 Dec 10;274(50):35583-90 Self-association of the alpha subunit of phosphorylase kinase as determined by two-hybrid screening. Ayers NA, Wilkinson DA, Fitzgerald TJ, Carlson GM. Division of Molecular Biology, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499, USA. The structural organization of the (alphabetagammadelta)(4) phosphorylase kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of alpha subunit interactions. By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the alpha subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the alpha subunit containing its C-terminal region. Domain mapping of binary interactions between alpha constructs revealed two regions involved in the self-association of the alpha subunit: residues 833-854, a previously unrecognized leucine zipper, and an unspecified region within residues 1015-1237. The cognate binding partner for the latter domain has been inferred to lie within the stretch from residues 864-1059. Indirect evidence from the literature suggests that the interacting domains contained within the latter two, overlapping regions may be further narrowed to the stretches from 1057 to 1237 and from 864 to 971. Cross-linking of the nonactivated holoenzyme with N-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced intramolecularly cross-linked alpha-alpha dimers, consistent with portions of two alpha subunits in the holoenyzme being in sufficient proximity to associate. This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase. Additionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed. PMID: 10585434 [PubMed - indexed for MEDLINE] 604: Nat Struct Biol 1999 Dec;6(12):1139-47 Comment in: Nat Struct Biol. 1999 Dec;6(12):1081-3. A novel loop-loop recognition motif in the yeast ribosomal protein L30 autoregulatory RNA complex. Mao H, White SA, Williamson JR. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. The yeast Saccharomyces cerevisiae ribosomal protein L30 negatively autoregulates its production by binding to a helix-loop-helix structure formed in its pre-mRNA and its mRNA. A three-dimensional solution structure of the L30 protein in complex with its regulatory RNA has been solved using NMR spectroscopy. In the complex, the helix-loop-helix RNA adopts a sharply bent conformation at the internal loop region. Unusual RNA features include a purine stack, a reverse Hoogsteen base pair (G11anti-G56syn) and highly distorted backbones. The L30 protein is folded in a three-layer alpha/beta/alpha sandwich topology, and three loops at one end of the sandwich make base-specific contacts with the RNA internal loop. The protein-RNA binding interface is divided into two clusters, including hydrophobic and aromatic stacking interactions centering around G56, and base-specific hydrogen-bonding contacts to A57, G58 and G10-U60 wobble base pair. Both the protein and the RNA exhibit a partially induced fit for binding, where loops in the protein and the internal loop in the RNA become more ordered upon complex formation. The specific interactions formed between loops on L30 and the internal loop on the mRNA constitute a novel loop-loop recognition motif where an intimate RNA-protein interface is formed between regions on both molecules that lack regular secondary structure. PMID: 10581556 [PubMed - indexed for MEDLINE] 605: Nat Struct Biol 1999 Dec;6(12):1081-3 Comment on: Nat Struct Biol. 1999 Dec;6(12):1139-47. If the loop fits... Frankel AD. The structure of the yeast L30 ribosomal protein bound to its autoregulatory RNA site has been determined by NMR spectroscopy. The intricate architecture of the RNA internal loop and the structure of the binding region of the protein both are stabilized in the complex, highlighting the importance of mutually-induced fit in RNA-protein interactions. Publication Types: Comment News PMID: 10581539 [PubMed - indexed for MEDLINE] 606: Appl Environ Microbiol 1999 Dec;65(12):5451-8 Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Thevissen K, Terras FR, Broekaert WF. F. A. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, B-3001 Heverlee-Leuven, Belgium. We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 microM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 microM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistant S. cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins. PMID: 10584003 [PubMed - indexed for MEDLINE] 607: EMBO J 1999 Dec 1;18(23):6832-44 The E2-E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain. Xie Y, Varshavsky A. Division of Biology, 147-75, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA. We dissected physical and functional interactions between the ubiquitin-conjugating (E2) enzyme Ubc2p and Ubr1p, the E3 component of the N-end rule pathway in Saccharomyces cerevisiae. The binding of the 20 kDa Ubc2p by the 225 kDa Ubr1p is shown to be mediated largely by the basic residue-rich (BRR) region of Ubr1p. However, mutations of the BRR domain that strongly decrease the interaction between Ubr1p and Ubc2p do not prevent the degradation of N-end rule substrates. In contrast, this degradation is completely dependent on the RING-H2 finger of Ubr1p adjacent to the BRR domain. Specifically, the first cysteine of RING-H2 is required for the ubiquitylation activity of the Ubr1p-Ubc2p complex, although this cysteine plays no detectable role in either the binding of N-end rule substrates by Ubr1p or the physical affinity between Ubr1p and Ubc2p. These results defined the topography of the Ubc2p-Ubr1p interaction and revealed the essential function of the RING-H2 finger, a domain that is present in many otherwise dissimilar E3 proteins of the ubiquitin system. PMID: 10581257 [PubMed - indexed for MEDLINE] 608: EMBO J 1999 Dec 1;18(23):6672-81 Gal83 mediates the interaction of the Snf1 kinase complex with the transcription activator Sip4. Vincent O, Carlson M. Departments of Genetics and Development, Columbia University, 701 West 168th Street, New York, NY 10032, USA. The Snf1/AMPK protein kinase family is widely conserved in eukaryotes. In Saccharomyces cerevisiae, the Snf1 kinase is an essential element of the glucose response pathway and has diverse regulatory roles. The Snf1 complex contains one of the related proteins Sip1, Sip2 and Gal83, which are also conserved in higher eukaryotes. Previous studies showed that the Sip1/Sip2/Gal83 component plays a structural role in the complex. We present evidence that this component also mediates the interaction of the Snf1 kinase complex with specific targets. We show that Gal83 mediates the association of the kinase with Sip4, a Snf1-regulated transcription activator of gluconeogenic genes. Gal83 interacts with Sip4 in two-hybrid assays in vivo, and bacterially expressed proteins bind in vitro. Moreover, Gal83 is required for the two-hybrid interaction of Sip4 with the Snf1 kinase. Gal83 also facilitates the rapid Snf1-dependent phosphorylation and activation of Sip4 in response to glucose limitation, indicating that Gal83 mediates the functional interaction of Snf1 with Sip4. Evidence indicates that Sip1 and Sip2 do not interact with Sip4. We propose that members of the Sip1/Sip2/Gal83 family confer specificity to the kinase complex in its interactions with target proteins. PMID: 10581241 [PubMed - indexed for MEDLINE] 609: EMBO J 1999 Dec 1;18(23):6662-71 A new regulatory domain on the TATA-binding protein. Cang Y, Auble DT, Prelich G. Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. Recognition of the TATA box by the TATA-binding protein (TBP) is a highly regulated step in RNA polymerase II-dependent transcription. Several proteins have been proposed to regulate TBP activity, yet the TBP domains responsive to all these regulators have not been defined. Here we describe a new class of TBP mutants that increase transcription from core promoters in vivo. The majority of these mutations alter amino acids that cluster tightly on the TBP surface, defining a new TBP regulatory domain. The mutant TBP proteins are defective for binding the transcriptional regulator yNC2, are resistant to inhibition by yNC2 in vitro and exhibit allele-specific genetic interactions with yNC2. These results provide strong biochemical and genetic evidence that TBP is directly repressed in vivo, and define a new TBP domain important for transcriptional regulation. PMID: 10581240 [PubMed - indexed for MEDLINE] 610: Biochem Biophys Res Commun 1999 Dec 9;266(1):135-40 Functional genomic analysis reveals the utility of the I/LWEQ module as a predictor of protein:actin interaction. McCann RO, Craig SW. Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland, 21205-2185, USA. The I/LWEQ module is a conserved sequence that we have identified as an actin-binding motif in the metazoan focal adhesion protein talin and the yeast protein Sla2p. Both of these proteins are associated with the actin cytoskeleton in cells. To better establish the value of the I/LWEQ module for prediction of actin-binding function, we have applied a functional genomics approach. Analysis of the 23 available I/LWEQ module sequences supports the division of I/LWEQ protein superfamily into four groups: (1) metazoan talin, (2) Dictyostelium discoideum talin homologs TalA/B, (3) metazoan Hip1p, and (4) yeast Sla2p. We show here that I/LWEQ modules from each major group bind to F-actin in vitro and that GFP-fusion proteins of the I/LWEQ modules of talin and Sla2p bind to F-actin in vivo. Therefore, the presence of an I/LWEQ module is strongly predictive of protein-actin interactions. The structural and functional conservation of the I/LWEQ module across the phylogenetic distance between cellular slime molds and mammals implies that the role of the I/LWEQ module is to connect diverse proteins involved in distinct cellular processes, including cell adhesion, cytoskeletal organization, and cell differentiation, to the actin cytoskeleton. Copyright 1999 Academic Press. PMID: 10581178 [PubMed - indexed for MEDLINE] 611: RNA 1999 Nov;5(11):1509-16 StreptoTag: a novel method for the isolation of RNA-binding proteins. Bachler M, Schroeder R, von Ahsen U. Institute of Microbiology & Genetics, University of Vienna, Austria. We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column containing streptomycin immobilized to Sepharose. The binding of the in vitro-assembled RNA-protein complex to streptomycin-Sepharose is mediated by the aptamer RNA and the specifically bound proteins are recovered from the affinity matrix by elution with the antibiotic. Employing two well-characterized RNA-protein interactions, we tested the performance of this new method. The spliceosomal U1A protein and the bacteriophage MS2 coat protein could be isolated via their appropriate RNA motif containing hybrid RNA from crude yeast extracts in high yield and purity after only one round of affinity purification. As the purification principle is independent of the extract source, this new affinity chromatography strategy that makes use of an in vitro-selected antibiotic-binding RNA as a tag, "StreptoTag," should be applicable to extracts from other organisms as well. Therefore, we propose StreptoTag to be a versatile tool for the isolation of unknown RNA-binding proteins. PMID: 10580480 [PubMed - indexed for MEDLINE] 612: RNA 1999 Nov;5(11):1470-81 Characterization of U6 snRNA-protein interactions. Vidal VP, Verdone L, Mayes AE, Beggs JD. Institute of Cell and Molecular Biology, University of Edinburgh, United Kingdom. Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome. Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA. The uridine-rich extreme 3' end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins. Interestingly, the Lsm proteins associate efficiently with the 3' half of U6, which contains the 3' stem-loop and uridine-rich 3' end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs. PMID: 10580475 [PubMed - indexed for MEDLINE] 613: Front Biosci 1999 Dec 1;4:D824-33 Transcription factors in DNA replication. Murakami Y, Ito Y. Department of Viral Oncology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. yota@virus.kyoto-u.ac.jp Accumulating evidence suggests the involvement of transcription factors in the regulation of DNA replication in eukaryotic cells. Almost all eukaryotic DNA viruses contain binding sites for transcription factors which function as auxiliary elements for DNA replication initiation at replication origins, and, indeed, the binding of transcription factors to these elements has been shown to stimulate DNA replication. Transcription factors also regulate some of the chromosome DNA replication origins of budding yeast, indicating that transcription factor involvement in DNA replication is not restricted to viruses. Consistent with this notion, recently determined replication origins of higher eukaryotes have been found occasionally to associate with transcription factor binding sites, although there is no direct evidence for the involvement of the factors that bind to these sequences in DNA replication. Analyses using viral and yeast systems have suggested that transcription factors stimulate the formation of the replication initiation complex by engaging in specific interactions with proteins of the initiation complex and/or by modulating the repressive chromatin structure around origins of replication. These mechanisms are analogous to those advanced to explain stimulation of transcription by transcription factors. The accumulated data suggests that transcription factors play a general role in the formation of functional complexes on chromosomes. Publication Types: Review Review, Tutorial PMID: 10577391 [PubMed - indexed for MEDLINE] 614: Biochimie 1999 Nov;81(11):1015-23 In vitro assembly of yeast 5S rRNA and a fusion protein containing ribosomal protein L5 and maltose binding protein. Pakhomova ON, Yeh LC, Monette J, Lee JC. Department of Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7760 USA. Binding of yeast ribosomal protein L5 with 5S rRNA has long been considered a promising model for studying molecular mechanisms of protein-RNA interactions. However, in vitro assembly of a ribonucleoprotein (RNP) complex from purified yeast ribosomal protein L5 (also known as L1, L1a, or YL3) and 5S rRNA proved to be difficult, thus limiting the utility of this model. In the present report, we present data on the successful in vitro assembly of a RNP complex using a fusion (MBP-L5) protein consisting of the yeast ribosomal protein L5 fused to the carboxyl terminus of the E. coli maltose-binding protein (MBP). We demonstrated that: 1) the MBP-L5 protein binds yeast 5S rRNA but not 5.8S rRNA in vitro; 2) the MBP protein itself does not bind yeast 5S rRNA; 3) formation of the RNP complex is proportional to the concentration of MBP-L5 protein and 5S rRNA; and 4) the MBP moiety of the fusion protein in the RNP complex can be removed with factor Xa. The electrophoretic mobility of the resultant RNP complex is indistinguishable from that of L5-5S rRNA complex isolated from the ribosome. Using this new experimental approach, we further showed that the RNA binding capability of a mutant L5 protein is decreased by 60% compared to the wild-type protein. Additionally, the mutant RNP complex migrates slower than the wild-type RNP complex suggesting that the mutant RNP complex has a less compact conformation. The finding provides a probable explanation for an earlier observation that the 60S ribosomal subunit containing the mutant protein is unstable. PMID: 10575356 [PubMed - indexed for MEDLINE] 615: Nature 1999 Nov 4;402(6757):83-6 Comment in: Nature. 1999 Nov 4;402(6757):23, 25-6. A combined algorithm for genome-wide prediction of protein function. Marcotte EM, Pellegrini M, Thompson MJ, Yeates TO, Eisenberg D. Molecular Biology Institute, UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, University of California, Los Angeles 90095, USA. The availability of over 20 fully sequenced genomes has driven the development of new methods to find protein function and interactions. Here we group proteins by correlated evolution, correlated messenger RNA expression patterns and patterns of domain fusion to determine functional relationships among the 6,217 proteins of the yeast Saccharomyces cerevisiae. Using these methods, we discover over 93,000 pairwise links between functionally related yeast proteins. Links between characterized and uncharacterized proteins allow a general function to be assigned to more than half of the 2,557 previously uncharacterized yeast proteins. Examples of functional links are given for a protein family of previously unknown function, a protein whose human homologues are implicated in colon cancer and the yeast prion Sup35. PMID: 10573421 [PubMed - indexed for MEDLINE] 616: Nucleic Acids Res 1999 Dec 15;27(24):4695-702 Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmc1 homolog, in Pyrococcus furiosus. Hayashi I, Morikawa K, Ishino Y. Department of Molecular Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan. Pyrococcus furiosus has an operon containing the DNA polymerase II (PolD) gene and three other genes. Using a two-hybrid screening to examine the interactions of the proteins encoded by the operon, we identified a specific interaction between the second subunit of PolD (DP1) and a Rad51/Dmc1 homologous protein (RadB). To ensure the specific interaction between these two proteins, each gene in the operon was expressed in Escherichia coli or insect cells separately and the products were purified. The in vitro analyses using the purified proteins also showed the interaction between DP1 and RadB. The deletion mutant analysis of DP1 revealed that a region important for binding with RadB is located in the central part of the sequence (amino acid residues 206-498). This region has an overlap to the C-terminal half (amino acids 334-613), which is highly conserved among euryarchaeal DP1s and is essential for the activity of PolD. Our results suggest that, although RadB does not noticeably affect the primer extension ability of PolD in vitro, PolD may utilize the RadB protein in DNA synthesis under certain conditions. PMID: 10572168 [PubMed - indexed for MEDLINE] 617: Proc Natl Acad Sci U S A 1999 Nov 23;96(24):13650-5 RNA-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein. Krol MA, Olson NH, Tate J, Johnson JE, Baker TS, Ahlquist P. Institute for Molecular Virology, University of Wisconsin, Madison, WI 53706, USA. Repeated, specific interactions between capsid protein (CP) subunits direct virus capsid assembly and exemplify regulated protein-protein interactions. The results presented here reveal a striking in vivo switch in CP assembly. Using cryoelectron microscopy, three-dimensional image reconstruction, and molecular modeling, we show that brome mosaic virus (BMV) CP can assemble in vivo two remarkably distinct capsids that selectively package BMV-derived RNAs in the absence of BMV RNA replication: a 180-subunit capsid indistinguishable from virions produced in natural infections and a previously unobserved BMV capsid type with 120 subunits arranged as 60 CP dimers. Each such dimer contains two CPs in distinct, nonequivalent environments, in contrast to the quasi-equivalent CP environments throughout the 180-subunit capsid. This 120-subunit capsid utilizes most of the CP interactions of the 180-subunit capsid plus nonequivalent CP-CP interactions. Thus, the CP of BMV, and perhaps other viruses, can encode CP-CP interactions that are not apparent from mature virions and may function in assembly or disassembly. Shared structural features suggest that the 120- and 180-subunit capsids share assembly steps and that a common pentamer of CP dimers may be an important assembly intermediate. The ability of a single CP to switch between distinct capsids by means of alternate interactions also implies reduced evolutionary barriers between different capsid structures. The in vivo switch between alternate BMV capsids is controlled by the RNA packaged: a natural BMV genomic RNA was packaged in 180-subunit capsids, whereas an engineered mRNA containing only the BMV CP gene was packaged in 120-subunit capsids. RNA features can thus direct the assembly of a ribonucleoprotein complex between alternate structural pathways. PMID: 10570127 [PubMed - indexed for MEDLINE] 618: Mol Pharmacol 1999 Dec;56(6):1105-15 Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity. Fiorani P, Amatruda JF, Silvestri A, Butler RH, Bjornsti MA, Benedetti P. Istituto di Biologia Cellulare, "Campus Adriano Buzzati-Traverso" Consiglio Nazionale delle Ricerche, Rome, Italy. DNA topoisomerase I (Top1p) relaxes supercoiled DNA by the formation of a covalent intermediate in which the active site tyrosine is transiently bound to the severed DNA strand. The antineoplastic agent camptothecin (Cpt) specifically targets Top1p and several mutations have been isolated that render the enzyme Cpt resistant. The mutated residues, although located in different regions of the enzyme, may constitute part of the Cpt binding site. To begin identifying the structural features of DNA Top1p important for Cpt-induced cytotoxicity, we developed a novel yeast genetic screen to isolate catalytically active, yet Cpt-resistant enzymes from a pool of human top1 mutants. Among the mutations isolated were substitutions of Ser or Val for Gly363, which like the Gly363 to Cys mutation previously reported by us, suppressed the Cpt sensitivity of Top1p. In contrast, each amino-acid substitution differed in its ability to suppress the lethal phenotype and catalytic activity of a human top1 mutant top1T718A that resembles Cpt by stabilizing the covalent intermediate. Biochemical analyses and molecular modeling support a model where interactions between two conserved domains, a central "lip" region containing residue Gly363 and the residues around the active site tyrosine (Tyr723), directly affect the formation of the Cpt-binding site and enzyme catalysis. PMID: 10570037 [PubMed - indexed for MEDLINE] 619: Biochemistry 1999 Nov 23;38(47):15580-6 DNA topoisomerases as targets for the anticancer drug TAS-103: DNA interactions and topoisomerase catalytic inhibition. Fortune JM, Velea L, Graves DE, Utsugi T, Yamada Y, Osheroff N. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA. TAS-103 is a novel anticancer drug that kills cells by increasing levels of DNA cleavage mediated by topoisomerase II. While most drugs that stimulate topoisomerase II-mediated DNA scission (i.e., topoisomerase II poisons) also inhibit the catalytic activity of the enzyme, they typically do so only at concentrations above the clinical range. TAS-103 is unusual in that it reportedly inhibits the catalytic activity of both topoisomerase I and II and does so at physiologically relevant concentrations [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. Without a topoisomerase activity to relieve accumulating torsional stress, the DNA tracking systems that promote the action of TAS-103 as a topoisomerase II poison would be undermined. Therefore, the effects of TAS-103 on the catalytic activity of topoisomerase I and II were characterized. DNA binding and unwinding assays indicate that the drug intercalates into DNA with an apparent dissociation constant of approximately 2.2 microM. Furthermore, DNA strand passage assays with mammalian topoisomerase I indicate that TAS-103 does not inhibit the catalytic activity of the type I enzyme. Rather, the previously reported inhibition of topoisomerase I-catalyzed DNA relaxation results from a drug-induced alteration in the apparent topology of the nucleic acid substrate. TAS-103 does inhibit the catalytic activity of human topoisomerase IIalpha, apparently by blocking the DNA religation reaction of the enzyme. The lack of inhibition of topoisomerase I catalytic activity by TAS-103 explains how the drug is able to function as a topoisomerase II poison in treated cells. PMID: 10569942 [PubMed - indexed for MEDLINE] 620: Biochemistry 1999 Nov 23;38(47):15573-9 DNA topoisomerases as targets for the anticancer drug TAS-103: primary cellular target and DNA cleavage enhancement. Byl JA, Fortune JM, Burden DA, Nitiss JL, Utsugi T, Yamada Y, Osheroff N. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA. TAS-103 is a novel antineoplastic agent that is active against in vivo tumor models [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. This drug is believed to be a dual topoisomerase I/II-targeted agent, because it enhances both topoisomerase I- and topoisomerase II-mediated DNA cleavage in treated cells. However, the relative importance of these two enzymes for the cytotoxic actions of TAS-103 is not known. Therefore, the primary cellular target of the drug and its mode of action were determined. TAS-103 stimulated DNA cleavage mediated by mammalian topoisomerase I and human topoisomerase IIalpha and beta in vitro. The drug was less active than camptothecin against the type I enzyme but was equipotent to etoposide against topoisomerase IIalpha. A yeast genetic system that allowed manipulation of topoisomerase activity and drug sensitivity was used to determine the contributions of topoisomerase I and II to drug cytotoxicity. Results indicate that topoisomerase II is the primary cellular target of TAS-103. In addition, TAS-103 binds to human topoisomerase IIalpha in the absence of DNA, suggesting that enzyme-drug interactions play a role in formation of the ternary topoisomerase II.drug.DNA complex. TAS-103 induced topoisomerase II-mediated DNA cleavage at sites similar to those observed in the presence of etoposide. Like etoposide, it enhanced cleavage primarily by inhibiting the religation reaction of the enzyme. Based on these findings, it is suggested that TAS-103 be classified as a topoisomerase II-targeted drug. PMID: 10569941 [PubMed - indexed for MEDLINE] 621: FEBS Lett 1999 Nov 19;461(3):253-7 Interactions between the full complement of human RNA polymerase II subunits. Schaller S, Grandemange S, Shpakovski GV, Golemis EA, Kedinger C, Vigneron M. Institut de Genetique et de Biologie Moleculaire et Cellulaire (CNRS/INSERM/ULP), BP 163, 67404, Illkirch, France. As an approach to elucidating the rules governing the assembly of human RNA polymerase II (hRPB), interactions between its subunits have been systematically analyzed. Eleven of the 12 expected hRPB subunits have previously been tested for reciprocal interactions (J. Biol. Chem. 272 (1997) 16815-16821). We now report the results obtained for the last subunit (hRPB4; Mol. Cell. Biol. 18 (1998) 1935-1945) and propose an essentially complete picture of the potential interactions occurring within hRPB. Finally, complementation experiments in yeast indicated that hRPB4 expression efficiently cured both heat and cold-sensitivity of RPB4-lacking strains, supporting the existence of conserved functional subunit interactions. PMID: 10567706 [PubMed - indexed for MEDLINE] 622: Mol Cell Biol 1999 Dec;19(12):8673-85 Analysis of TFIIA function In vivo: evidence for a role in TATA-binding protein recruitment and gene-specific activation. Liu Q, Gabriel SE, Roinick KL, Ward RD, Arndt KM. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA. Activation of transcription can occur by the facilitated recruitment of TFIID to promoters by gene-specific activators. To investigate the role of TFIIA in TFIID recruitment in vivo, we exploited a class of yeast TATA-binding protein (TBP) mutants that is activation and DNA binding defective. We found that co-overexpression of TOA1 and TOA2, the genes that encode yeast TFIIA, overcomes the activation defects caused by the TBP mutants. Using a genetic screen, we isolated a new class of TFIIA mutants and identified three regions on TFIIA that are likely to be involved in TBP recruitment or stabilization of the TBP-TATA complex in vivo. Amino acid replacements in only one of these regions enhance TFIIA-TBP-DNA complex formation in vitro, suggesting that the other regions are involved in regulatory interactions. To determine the relative importance of TFIIA in the regulation of different genes, we constructed yeast strains to conditionally deplete TFIIA levels prior to gene activation. While the activation of certain genes, such as INO1, was dramatically impaired by TFIIA depletion, activation of other genes, such as CUP1, was unaffected. These data suggest that TFIIA facilitates DNA binding by TBP in vivo, that TFIIA may be regulated by factors that target distinct regions of the protein, and that promoters vary significantly in the degree to which they require TFIIA for activation. PMID: 10567590 [PubMed - indexed for MEDLINE] 623: Mol Microbiol 1999 Nov;34(4):780-91 Interaction between the F plasmid TraA (F-pilin) and TraQ proteins. Harris RL, Sholl KA, Conrad MN, Dresser ME, Silverman PM. Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA. Elaboration of conjugative (F) pili by F+ strains of Escherichia coli requires the activities of over a dozen F-encoded DNA transfer (Tra) proteins. The organization and functions of these proteins are largely unknown. Using the yeast two-hybrid assay, we have begun to analyse binary interactions among the Tra proteins required for F-pilus formation. We focus here on interactions involving F-pilin, the only known F-pilus subunit. Using a library of F tra DNA fragments that contained all the F genes required for F pilus formation in a yeast GAL4 activation domain vector (pACTII), we transformed yeast containing a plasmid (pAS1CYH2traA) encoding a GAL4 DNA-binding domain-F-pilin fusion. Doubly transformed cells were screened for GAL4-dependent gene expression. This screen repeatedly identified only a single Tra protein, TraQ, previously identified as a likely F-pilin chaperone. The F-pilin-TraQ interaction appeared to be specific, as no transcriptional activation was detected in yeast transformants containing pACTIItraQ plasmids and the Salmonella typhi pED208 traA gene cloned in pAS1CYH2. Two traQ segments isolated in the screen against F-pilin were tested for complementation of a traQ null allele in E. coli. One, lacking the first 11 (of 94) TraQ amino acids, restored DNA donor activity, donor-specific bacteriophage sensitivity and membrane F-pilin accumulation to wild-type levels. The second, lacking the first 21 amino acids, was much less effective in these assays. Both TraQ polypeptides accumulated in E. coli as transmembrane proteins. The longer, biologically active segment was fused to the GAL4 DNA-binding domain gene of pAS1CYH2 and used to screen the tra fragment library. The only positives from this screen identified traA segments. The fusion sites between the traA and GAL4 segments identified the hydrophobic, C-terminal domain IV of F-pilin as sufficient for the interaction. As TraQ is the only Tra protein required for the accumulation of inner membrane F-pilin, the interaction probably reflects a specific, chaperone-like function for TraQ in E. coli. Attempts to isolate an F-pilin-TraQ complex from E. coli were unsuccessful, suggesting that the interaction between the two is normally transient, as expected from previous studies of the kinetics of TraA membrane insertion and processing to F-pilin. PMID: 10564517 [PubMed - indexed for MEDLINE] 624: Biochim Biophys Acta 1999 Nov 16;1435(1-2):147-52 A novel mammalian nuclear protein similar to Schizosaccharomyces pombe Prp1p/Zer1p and Saccharomyces cerevisiae Prp6p pre-mRNA splicing factors. Nishikimi A, Mukai J, Kioka N, Yamada M. Laboratory of Reproductive Physiology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan. We have cloned a novel 100-kDa mammalian protein, which was recognized by an anti-peptide antibody against an epitope-containing nuclear localization signal of NF-kappaB p65 subunit. Predicted amino acid sequence of the protein is similar to those of yeast splicing factors, Prp1p/Zer1p of Schizosaccharomyces pombe and Prp6p of Saccharomyces cerevisiae. Among these proteins, tetratrico peptide repeat (TPR) motif, which mediates protein-protein interactions, is conserved, whereas leucine zipper motif is found only in the 100-kDa protein. Indirect immunofluorescent staining showed that the 100-kDa protein localized in the nucleus in HeLa cells. PMID: 10561546 [PubMed - indexed for MEDLINE] 625: Mol Biol Cell 1999 Nov;10(11):3549-65 Shr3p mediates specific COPII coatomer-cargo interactions required for the packaging of amino acid permeases into ER-derived transport vesicles. Gilstring CF, Melin-Larsson M, Ljungdahl PO. Ludwig Institute for Cancer Research, S-171 77 Stockholm, Sweden. The SHR3 gene of Saccharomyces cerevisiae encodes an integral membrane component of the endoplasmic reticulum (ER) with four membrane-spanning segments and a hydrophilic, cytoplasmically oriented carboxyl-terminal domain. Mutations in SHR3 specifically impede the transport of all 18 members of the amino acid permease (aap) gene family away from the ER. Shr3p does not itself exit the ER. Aaps fully integrate into the ER membrane and fold properly independently of Shr3p. Shr3p physically associates with the general aap Gap1p but not Sec61p, Gal2p, or Pma1p in a complex that can be purified from N-dodecylmaltoside-solubilized membranes. Pulse-chase experiments indicate that the Shr3p-Gap1p association is transient, a reflection of the exit of Gap1p from the ER. The ER-derived vesicle COPII coatomer components Sec13p, Sec23p, Sec24p, and Sec31p but not Sar1p bind Shr3p via interactions with its carboxyl-terminal domain. The mutant shr3-23p, a nonfunctional membrane-associated protein, is unable to associate with aaps but retains the capacity to bind COPII components. The overexpression of either Shr3p or shr3-23p partially suppresses the temperature-sensitive sec12-1 allele. These results are consistent with a model in which Shr3p acts as a packaging chaperone that initiates ER-derived transport vesicle formation in the proximity of aaps by facilitating the membrane association and assembly of COPII coatomer components. PMID: 10564255 [PubMed - indexed for MEDLINE] 626: Genes Dev 1999 Nov 1;13(21):2811-27 A specific protein-protein interaction accounts for the in vivo substrate selectivity of Ptp3 towards the Fus3 MAP kinase. Zhan XL, Guan KL. Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA. The mitogen-activated protein kinases (MAPKs) play critical roles in many signal transduction processes. Several MAPKs have been found in Saccharomyces cerevisiae, including Fus3 in the mating pathway and Hog1 in the osmotic-stress response pathway. Cells lacking Fus3 or Hog1 activity are deficient in mating or adaptation to osmotic shock, respectively. However, constitutive activation of either Fus3 or Hog1 is lethal. Therefore, yeast cells have to tightly regulate both the activation and inactivation of Fus3 and Hog1 MAPKs, which are controlled mainly by phosphorylation and dephosphorylation. Previous studies have shown that Fus3 activity is negatively regulated by protein tyrosine phosphatase Ptp3. In contrast, the Hog1 MAPK is mainly dephosphorylated by Ptp2 even though the two phosphatases share a high degree of sequence similarity. To understand the mechanisms of MAPK regulation, we examined the molecular basis underlying the in vivo substrate specificity between phosphatases and MAPKs. We observed that the amino-terminal noncatalytic domain of Ptp3 directly interacts with Fus3 via CH2 (Cdc25 homology) domain conserved among yeast PTPases and mammalian MAP kinase phosphatases and is responsible for the in vivo substrate selectivity of the phosphatase. Interaction between Ptp3 and Fus3 is required for dephosphorylation and inactivation of Fus3 under physiological conditions. Mutations in either Ptp3 or Fus3 that abolish this interaction cause a dysregulation of the Fus3 MAPK. Our data demonstrate that the specificity of MAP kinase inactivation in vivo by phosphatases is determined by specific protein-protein interactions outside of the phosphatase catalytic domain. PMID: 10557209 [PubMed - indexed for MEDLINE] 627: J Mol Biol 1999 Nov 19;294(1):121-37 The dimerization/repression domain of RFX1 is related to a conserved region of its yeast homologues Crt1 and Sak1: a new function for an ancient motif. Katan-Khaykovich Y, Spiegel I, Shaul Y. Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, 76100, Israel. The RFX protein family includes members from yeast to humans, which function in various biological systems, and share a DNA-binding domain and a conserved C-terminal region. In the human transcription regulator RFX1, the conserved C terminus is an independent functional domain, which mediates dimerization and transcriptional repression. This dimerization domain has a unique ability to mediate the formation of two alternative homodimeric DNA-protein complexes, the upper of which has been linked to repression. Here, we localize the complex formation capacity to several different RFX1 C-terminal subregions, each of which can function independently to generate the upper complex and repress transcription, thus correlating complex formation with repression. To gain an evolutionary perspective, we have examined whether the different properties of the RFX1 C terminus exist in the two yeast RFX proteins, which are involved in signaling pathways. Replacement of the RFX1 C terminus with those of Sak1 and Crt1, its orthologues from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively, and analysis of fusions with the Gal4 DNA-binding domain, revealed that the ability to generate the two alternative complexes is conserved in the RFX family, from S. cerevisiae to man. While sharing this unique biochemical property, the three C termini differed from each other in their ability to mediate dimerization and transcriptional repression. In both functions, RFX1, Sak1, and Crt1 showed high capacity, moderate capacity, and no capacity, respectively. This comparative analysis of the RFX proteins, representing different evolutionary stages, suggests a gradual development of the conserved C terminus, from the appearance of the ancestral motif (Crt1), to the later acquisition of the dimerization/repression functions (Sak1), and finally to the enhancement of these functions to generate a domain mediating highly stable protein-protein interactions and potent transcriptional repression (RFX1). Copyright 1999 Academic Press. PMID: 10556033 [PubMed - indexed for MEDLINE] 628: Biochim Biophys Acta 1999 Nov 16;1422(3):235-54 Mechanisms of protein translocation into mitochondria. Voos W, Martin H, Krimmer T, Pfanner N. Institut fur Biochemie und Molekularbiologie, Universitat Freiburg, Hermann-Herder-Str. 7, D-79104, Freiburg, Germany. voos@ruf.uni-freiburg.de Mitochondrial biogenesis utilizes a complex proteinaceous machinery for the import of cytosolically synthesized preproteins. At least three large multisubunit protein complexes, one in the outer membrane and two in the inner membrane, have been identified. These translocase complexes cooperate with soluble proteins from the cytosol, the intermembrane space and the matrix. The translocation of presequence-containing preproteins through the outer membrane channel includes successive electrostatic interactions of the charged mitochondrial targeting sequence with a chain of import components. Translocation across the inner mitochondrial membrane utilizes the energy of the proton motive force of the inner membrane and the hydrolysis of ATP. The matrix chaperone system of the mitochondrial heat shock protein 70 forms an ATP-dependent import motor by interaction with the polypeptide chain in transit and components of the inner membrane translocase. The precursors of integral inner membrane proteins of the metabolite carrier family interact with newly identified import components of the intermembrane space and are inserted into the inner membrane by a second translocase complex. A comparison of the full set of import components between the yeast Sacccharomyces cerevisiae and the nematode Caenorhabditis elegans demonstrates an evolutionary conservation of most components of the mitochondrial import machinery with a possible greater divergence for the import pathway of the inner membrane carrier proteins. Publication Types: Review Review, Tutorial PMID: 10548718 [PubMed - indexed for MEDLINE] 629: J Cell Sci 1999 Nov;112 ( Pt 22):4123-34 Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation. Plemper RK, Bordallo J, Deak PM, Taxis C, Hitt R, Wolf DH. Institut fur Biochemie, Universitat Stuttgart, D-70569 Stuttgart, Germany. The endoplasmic reticulum contains a quality control system that subjects misfolded or unassembled secretory proteins to rapid degradation via the cytosolic ubiquitin proteasome system. This requires retrograde protein transport from the endoplasmic reticulum back to the cytosol. The Sec61 pore, the central component of the protein import channel into the endoplasmic reticulum, was identified as the core subunit of the retro-translocon as well. As import of mutated proteins into the endoplasmic reticulum lumen is successfully terminated, a new targeting mechanism must exist that mediates re-entering of misfolded proteins into the Sec61 pore from the lumenal side de novo. The previously identified proteins Der3p/Hrd1p and, as we show here, Hrd3p of the yeast Saccharomyces cerevisiae, are localised in the endoplasmic reticulum membrane and are essential for the degradation of several substrates of the endoplasmic reticulum degradation machinery. Based on genetic studies we demonstrate that they functionally interact with each other and with Sec61p, probably establishing the central part of the retro-translocon. In the absence of Hrd3p, the otherwise stable protein Der3p/Hrd1p becomes rapidly degraded. This depends on a functional ubiquitin proteasome system and the presence of substrate molecules of the endoplasmic reticulum degradation system. When overexpressed, Der3p/Hrd1p accelerates CPY* degradation in Delta(hrd3) cells. Our data suggest a recycling process of Der3p/Hrd1p through Hrd3p. The retro-translocon seems to be build up at least by the Sec61 pore, Der3p/Hrd1p and Hrd3p and mediates both retrograde transport and ubiquitination of substrate molecules. PMID: 10547371 [PubMed - indexed for MEDLINE] 630: Hum Mol Genet 1999 Nov;8(12):2263-73 Functional analysis of human FEN1 in Saccharomyces cerevisiae and its role in genome stability. Greene AL, Snipe JR, Gordenin DA, Resnick MA. Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA. The flap endonuclease, FEN1, is an evolutionarily conserved component of DNA replication from archaebacteria to humans. Based on in vitro results, it processes Okazaki fragments during replication and is involved in base excision repair. FEN1 removes the last primer ribonucleotide on the lagging strand and it cleaves a 5' flap that may result from strand displacement during replication or during base excision repair. Its biological importance has been revealed largely through studies in the yeast Saccharomyces cerevisiae where deletion of the homologous gene RAD27 results in genome instability and mutagen sensitivity. While the in vivo function of Rad27 has been well characterized through genetic and biochemical approaches, little is understood about the in vivo functions of human FEN1. Guided by our recent results with yeast RAD27, we explored the function of human FEN1 in yeast. We found that the human FEN1 protein complements a yeast rad27 null mutant for a variety of defects including mutagen sensitivity, genetic instability and the synthetic lethal interactions of a rad27 rad51 and a rad27 pol3-01 mutant. Furthermore, a mutant form of FEN1 lacking nuclease function exhibits dominant-negative effects on cell growth and genome instability similar to those seen with the homologous yeast rad27 mutation. This genetic impact is stronger when the human and yeast PCNA-binding domains are exchanged. These data indicate that the human FEN1 and yeast Rad27 proteins act on the same substrate in vivo. Our study defines a sensitive yeast system for the identification and characterization of mutations in FEN1. PMID: 10545607 [PubMed - indexed for MEDLINE] 631: EMBO J 1999 Nov 1;18(21):6095-105 Erratum in: EMBO J 2000 Jan 17;19(2):315 Cellular transcription factors recruit viral replication proteins to activate the Epstein-Barr virus origin of lytic DNA replication, oriLyt. Baumann M, Feederle R, Kremmer E, Hammerschmidt W. GSF-National Research Center for Environment and Health, Institute of Clinical Molecular Biology and Tumor Genetics, Department of Gene Vectors, Munchen, Germany. DNA replication of Epstein-Barr virus (EBV) during the productive phase of the life cycle of this herpesvirus depends on the cis-acting element oriLyt. It consists of two essential domains, the upstream and the downstream component. Whereas the upstream component contains several DNA-binding motifs for the viral activator protein BZLF1, the downstream component is known to be the binding site of several cellular proteins. We identified cellular transcription factors that bind synergistically to a functionally relevant subsequence of the downstream component, the TD element. Two of these transcription factors, ZBP-89 and Sp1, stimulate replication as shown by protein fusions with the GAL4 DNA-binding domain and a single GAL4 DNA-binding motif inserted into the TD element. In protein binding assays, we observed an interaction of Sp1 and ZBP-89 with the viral DNA polymerase and its processivity factor. Our data indicate that cellular transcriptional activators tether viral replication proteins to the lytic origin via direct protein-protein interactions to assemble the viral replication complex at oriLyt. PMID: 10545120 [PubMed - indexed for MEDLINE] 632: Toxicol Appl Pharmacol 1999 Nov 1;160(3):297-303 A human aryl hydrocarbon receptor signaling pathway constructed in yeast displays additive responses to ligand mixtures. Miller CA 3rd. Environmental Health Sciences Department, Tulane University School of Public Health and Tropical Medicine, Tulane-Xavier Center for Bioenvironmental Research, 1430 Tulane Avenue, New Orleans, Louisiana, 70112, USA. An optimized signal transduction pathway that reproduces the response of human aryl hydrocarbon (Ah) receptor to ligands has been established in Saccharomyces cerevisiae. Ligand treatment induced a 50-fold increase in beta-galactosidase activity from a reporter plasmid in yeast engineered to express human Ah receptor and Ah nuclear translocator (Arnt) proteins. The archetypal Ah receptor ligand, 2,3,7,8-tetrachlorodibenzo(p)dioxin, activated Ah receptor and induced lacZ reporter activity at concentrations of >/=0.3 nM. Mixtures of halogenated and nonhalogenated Ah receptor ligands produced additive signaling responses in this yeast bioassay. These results were consistent with the existence of a common binding site and mechanism of ligand-mediated Ah receptor activation. Although yeast have no natural counterpart to the Ah receptor pathway, expression of human Ah receptor and Arnt under the appropriate conditions provides a functional model system for studying Ah receptor activation and signal transduction. Copyright 1999 Academic Press. PMID: 10544064 [PubMed - indexed for MEDLINE] 633: Curr Biol 1999 Oct 7;9(19):1111-4 A retention mechanism for distribution of mitochondria during cell division in budding yeast. Yang HC, Palazzo A, Swayne TC, Pon LA. Department of Anatomy and Cell Biology Columbia University College of Physicians and Surgeons New York, New York, 10032, USA. Mitochondria are indispensable for normal eukaryotic cell function. As they cannot be synthesized de novo and are self-replicating, mitochondria must be transferred from mother to daughter cells. Studies in the budding yeast Saccharomyces cerevisiae indicate that mitochondria enter the bud immediately after bud emergence, interact with the actin cytoskeleton for linear, polarized movement of mitochondria from mother to bud, but are equally distributed among mother and daughter cells [1] [2] [3]. It is not clear how the mother cell maintains its own supply of mitochondria. Here, we found that mother cells retain mitochondria by immobilization of some mitochondria in the 'retention zone', the base of the mother cell distal to the bud. Retention requires the actin cytoskeleton as mitochondria colocalized with actin cables in the retention zone, and mutations that perturb actin dynamics or actin-mitochondrial interactions produced retention defects. Our results support the model that equal distribution of mitochondria during cell division is a consequence of two actin-dependent processes: movement of some mitochondria into the daughter bud and immobilization of others in the mother cell. PMID: 10531006 [PubMed - indexed for MEDLINE] 634: Appl Microbiol Biotechnol 1999 Sep;52(3):311-20 Yeast cells as tools for target-oriented screening. Munder T, Hinnen A. Hans-Knoll-Institut fur Naturstoff-Forschung e V. Jena, Germany. Information about biomolecular interaction networks is crucial for understanding cellular functions and the development of disease processes. Many diseases are known to be based on aberrations of DNA sequences encoding proteins with key functions in the cellular metabolism. Alterations in the respective proteins often lead to disturbances in biomolecular interactions caused by unbalanced stoichiometries, and thus result in alterations of molecule fluxes, cell architecture and signalling pathways. Drug discovery programmes have been designed to find promising chemical lead structures with the help of target-oriented bioassay systems. These are, in most cases, based upon the interaction of small molecules to specific macromolecular targets in vivo or in vitro, as exemplified by enzyme assays or small-ligand-based receptor systems. In addition, interactions between large biomolecules, such as proteins or nucleic acids, offer a huge arsenal of potential drug targets that can be addressed by small chemical compounds. This latter approach is gaining considerable attention because many potential target structures are becoming available through genomic research. Funnelling these new targets into high-throughput screening programs represents a major challenge for today's pharmaceutical research. An important outcome of the ongoing genome projects is the fact that the basic cellular structures, pathways and signalling principles show a high degree of conservation. Model organisms that are easily approachable by genetic, biochemical and physiological means can thus play an important role in the design of target-oriented screening systems. They offer the possibility to express individual proteins, nucleic acids or even more complex aggregates of biomolecules such as protein-interaction networks or transcription-initiation complexes, which can be addressed by small effector molecules in vivo. Combining these targets with biological signalling systems is an attractive way of creating robust cellular assay systems. Publication Types: Review Review, Tutorial PMID: 10531642 [PubMed - indexed for MEDLINE] 635: Infect Immun 1999 Nov;67(11):6040-7 Overexpression of the Candida albicans ALA1 gene in Saccharomyces cerevisiae results in aggregation following attachment of yeast cells to extracellular matrix proteins, adherence properties similar to those of Candida albicans. Gaur NK, Klotz SA, Henderson RL. Department of Research, Veterans Affairs Medical Center, Kansas City, Missouri 64128, USA. nkgaur@kuhub.cc.ukans.edu Candida albicans maintains a commensal relationship with human hosts, probably by adhering to mucosal tissue in a variety of physiological conditions. We show that adherence due to the C. albicans gene ALA1 when transformed into Saccharomyces cerevisiae, is comprised of two sequential steps. Initially, C. albicans rapidly attaches to extracellular matrix (ECM) protein-coated magnetic beads in small numbers (the attachment phase). This is followed by a relatively slower step in which cell-to-cell interactions predominate (the aggregation phase). Neither of these phases is observed in S. cerevisiae. However, expression of the C. albicans ALA1 gene from a low-copy vector causes S. cerevisiae transformants to attach to ECM-coated magnetic beads without appreciable aggregation. Expression of ALA1 from a high-copy vector results in both attachment and aggregation. Moreover, transcriptional fusion of ALA1 with the galactose-inducible promoters GALS, GALL, and GAL1, allowing for low, moderate, and high levels of inducible transcription, respectively, causes attachment and aggregation that correlates with the strength of the GAL promoter. The adherence of C. albicans and S. cerevisiae overexpressing ALA1 to a number of protein ligands occurs over a broad pH range, is resistant to shear forces generated by vortexing, and is unaffected by the presence of sugars, high salt levels, free ligands, or detergents. Adherence is, however, inhibited by agents that disrupt hydrogen bonds. The similarities in the adherence and aggregation properties of C. albicans and S. cerevisiae overexpressing ALA1 suggest a role in adherence and aggregation for ALA1 and ALA1-like genes in C. albicans. PMID: 10531265 [PubMed - indexed for MEDLINE] 636: Methods 1999 Oct;19(2):338-49 Genetic approaches to the study of protein-protein interactions. Appling DR. Department of Chemistry and Biochemistry, The Biochemical Institute, Austin, Texas 78712, USA. dappling@mail.utexas.edu This article describes genetic approaches to the study of heterologous protein-protein interactions, focusing on the yeast Saccharomyces cerevisiae as a useful eukaryotic model system. Several methods are described that can be used to search for new interactions, including extragenic suppression, multicopy suppression, synthetic lethality, and transdominant inhibition. Strategies for screening, genetic characterization, and clone identification are described, along with recent examples from the literature. In addition, genetic methods are discussed that can be used to further characterize a newly discovered protein-protein interaction. These include the creation of mutant libraries of a given protein by chemical mutagenesis or polymerase chain reaction, the production of dominant-negative mutants, and strategies for introducing these mutant alleles back into yeast for analysis. Although these genetic methods are quite powerful, they are often just a starting point for further biochemical or cell biological experiments. Copyright 1999 Academic Press. PMID: 10527736 [PubMed - indexed for MEDLINE] 637: Methods 1999 Oct;19(2):330-7 Applications of the yeast two-hybrid system. McAlister-Henn L, Gibson N, Panisko E. Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760, USA. henn@uthscsa.edu In recent years, the yeast two-hybrid system has become the method of choice for detection and analysis of protein-protein interactions in an in vivo context. This system, which capitalizes on the significant genetic history and ease of protocols for manipulation of Saccharomyces cerevisiae, is accessible to most laboratories and is applicable to the pursuit of a large variety of experimental goals. To date, the two-hybrid system has seen widespread application for identification of interaction partners by screening methods using a particular protein of interest as a "bait." Large-scale ventures are also in progress, for example, a cataloging of interactions among the cellular proteins in yeast. However, this method also has tremendous potential for more focused analyses of specific proteins and should become more routine as an alternative or adjunct approach for many structure-function investigations. Copyright 1999 Academic Press. PMID: 10527735 [PubMed - indexed for MEDLINE] 638: Mutat Res 1999 Sep 13;435(1):23-33 Erratum in: Mutat Res 2000 Mar 20;459(2):171-2 Protein complexes in nucleotide excision repair. Araujo SJ, Wood RD. Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, UK. The main pathway by which mammalian cells remove DNA damage caused by UV light and some other mutagens is nucleotide excision repair (NER). The best characterised components of the human NER process are those proteins defective in the inherited disorder xeroderma pigmentosum (XP). The proteins known to be involved in the first steps of the NER reaction (damage recognition and incision-excision) are heterotrimeric RPA, XPA, the 6 to 9 subunit TFIIH, XPC-hHR23B, XPG, and ERCC1-XPF. Many interactions between these proteins have been found in recent years using different methods both in mammalian cells and for the homologous proteins in yeast. There are virtually no quantitative measurements of the relative strengths of these interactions. Higher order associations between these proteins in solution and even the existence of a complete "repairosome" complex have been reported, which would have implications both for the mechanism of repair and for the interplay between NER and other cellular processes. Nevertheless, evidence for a completely pre-assembled functional repairosome in solution is inconclusive and the order of action of repair factors on damaged DNA is uncertain. Publication Types: Review Review, Tutorial PMID: 10526214 [PubMed - indexed for MEDLINE] 639: Mutat Res 1999 Sep 13;435(1):1-11 Genetic interactions between error-prone and error-free postreplication repair pathways in Saccharomyces cerevisiae. Xiao W, Chow BL, Fontanie T, Ma L, Bacchetti S, Hryciw T, Broomfield S. Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada. xiaow@sask.usask.ca Evidence obtained from recent studies supports the existence of an error-free postreplication repair (PRR) and a mutagenesis pathway within the Saccharomyces cerevisiae RAD6 DNA repair group. The MMS2 gene is the only known yeast gene involved in error-free PRR that, when mutated, significantly increases the spontaneous mutation rate. In this study, the mutational spectrum of the mms2 mutator was determined and compared to the wild type strain. In addition, mutagenenic effects and genetic interactions of the mms2 mutator and rev3 anti-mutator were examined with respect to forward mutations, frameshift reversions as well as amber and ochre suppressions. It was concluded from these results that the mms2 mutator phenotype is largely dependent on the functional REV3 gene. The synergistic effects of mms2 and rev3 mutations towards killing by a variety of DNA-damaging agents ruled out the possibility that MMS2 simply acts to suppress REV3 activity and favored the hypothesis that MMS2 and REV3 form two alternative subpathways within the RAD6 DNA repair pathway. Taken together, we propose that two pathways represented by MMS2 and REV3 deal with a similar range of endogenous and environmental DNA damage but with different biological consequences, namely, error-free repair and mutagenesis, respectively. PMID: 10526212 [PubMed - indexed for MEDLINE] 640: FEBS Lett 1999 Oct 15;459(3):458-62 The yeast Rgd1p is a GTPase activating protein of the Rho3 and rho4 proteins. Doignon F, Weinachter C, Roumanie O, Crouzet M. Laboratoire de Biologie Moleculaire et de Sequencage, UPR CNRS 9026, BP Box 64, 146 rue Leo Saignat, 33076, Bordeaux, France. The RGD1 gene, identified during sequencing of the Saccharomyces cerevisiae genome, encodes a protein with a Rho-GTPase activating protein (GAP) domain at the carboxy-terminal end. The Rgd1 protein showed two-hybrid interactions with the activated forms of Rho2p, Rho3p and Rho4p. Using in vitro assays, we demonstrated that Rgd1p stimulated the GTPase activity of both Rho3p and Rho4p; no stimulation was observed on Rho2p. In addition, the rho3Deltargd1Delta double mutant exhibited a dramatic growth defect compared to the single mutants, suggesting that Rgd1p has a GAP activity in vivo. The present study allowed the identification of the first GAP of Rho3p and Rho4p. PMID: 10526184 [PubMed - indexed for MEDLINE] 641: FEBS Lett 1999 Oct 15;459(3):427-32 Analysis by atomic force microscopy of Med8 binding to cis-acting regulatory elements of the SUC2 and HXK2 genes of saccharomyces cerevisiae. Moreno-Herrero F, Herrero P, Colchero J, Baro AM, Moreno F. Departamento de Bioquimica y Biologia Molecular, Instituto Universitario de Biotecnologia de Asturias, Universidad de Oviedo, 33006, Oviedo, Spain. Med8 protein is a regulator that specifically binds to upstream activating sequences (UASs) of SUC2 promoter, to downstream repressing sequences (DRSs) of the HXK2 gene and to the carboxy-terminal domain of the RNA polymerase II. Atomic force microscopy has allowed for direct visualization of Med8 interactions with a 305 bp fragment of SUC2 promoter and with a 676 bp fragment of HXK2 gene, containing respectively the UASs and DRSs regulatory regions. This approach has provided complementary information about the position and the structure of the DNA-protein complexes. Med8 binding to DNA results in total covering of one of the two existing 7 bp motives (consensus, (A/C)(A/G)GAAAT) in the studied DNA fragments. No preference for binding either of the two UASs of SUC2 promoter as well as for the two DRSs of HXK2 gene has been found. We also discuss whether this protein works as dimer or as a monomer. PMID: 10526178 [PubMed - indexed for MEDLINE] 642: Chromosoma 1999 Sep;108(5):278-90 Tel2p, a regulator of yeast telomeric length in vivo, binds to single-stranded telomeric DNA in vitro. Kota RS, Runge KW. The Lerner Research Institute, Cleveland Clinic Foundation, Department of Molecular Biology, NC 20, 9500 Euclid Avenue, Cleveland, OH 44195, USA. The telomeres of the yeast Saccharomyces cerevisiae consist of a duplex region of TG(1-3) repeats that acquire a single-stranded 3' extension of the TG(1-3) strand at the end of S-phase. The length of these repeats is kept within a defined range by regulators such as the TEL2-encoded protein (Tel2p). Here we show that Tel2p can specifically bind to single-stranded TG(1-3). Tel2p binding produced several shifted bands; however, only the slowest migrating band contained Tel2p. Methylation protection and interference experiments as well as gel shift experiments using inosine-containing probes indicated that the faster migrating bands resulted from Tel2p-mediated formation of DNA secondary structures held together by G-G interactions. Tel2p bound to single-stranded substrates that were at least 19 bases in length and contained 14 bases of TG(1-3), and also to double-stranded/single-stranded hybrid substrates with a 3' TG(1-3) overhang. Tel2p binding to a hybrid substrate with a 24 base single-stranded TG(1-3) extension also produced a band characteristic of G-G-mediated secondary structures. These data suggest that Tel2p could regulate telomeric length by binding to the 3' single-stranded TG(1-3) extension present at yeast telomeres. PMID: 10525964 [PubMed - indexed for MEDLINE] 643: Biotechniques 1999 Oct;27(4):790-6 Identification of ribonucleoprotein (RNP)-specific protein interactions using a yeast RNP interaction trap assay (RITA). Bouffard P, Briere F, Wellinger RJ, Boire G. Rheumatology Division, Faculty of Medicine, Universite de Sherbrooke, QC, Canada. We describe an adaptation of the yeast three-hybrid system that allows the reconstitution in vivo of tripartite (protein-RNA-protein) ribonucleoproteins (RNPs). To build and try this system that we called RNP interaction trap assay (RITA), we used as a model the autoantigenic Ro RNPs. hY RNAs bear distinct binding sites for Ro60 and La proteins, and Ro RNPs are thus physiologically tripartite (Ro60/hY RNA/La). Using recombinant La (rLa) and Ro60 (rRo60) proteins and recombinant hY RNAs (rhY) co-expressed in yeast, we found that RNPs made of rRo60/rhY/rLa were readily reassembled. Reconstitution of tripartite RNPs was critically dependent on the presence of an appropriate Ro60 binding site on the recombinant RNA. The RITA assay was further used to detect (rRo60/rhY RNP)-binding proteins from a HeLa cell cDNA library, allowing specific identification of La and of a novel Ro RNP-binding protein (RoBPI) in more than 70% of positive clones. RITA assay may complement already available two- and three-hybrid systems to characterize RNP-binding proteins by allowing the in vivo identification of interactions strictly dependent upon the simultaneous presence of a protein and of its cognate RNA. PMID: 10524322 [PubMed - indexed for MEDLINE] 644: Mol Cell Biol 1999 Nov;19(11):7661-71 Control of meiotic recombination and gene expression in yeast by a simple repetitive DNA sequence that excludes nucleosomes. Kirkpatrick DT, Wang YH, Dominska M, Griffith JD, Petes TD. Department of Biology University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA. Tandem repeats of the pentanucleotide 5'-CCGNN (where N indicates any base) were previously shown to exclude nucleosomes in vitro (Y. -H. Wang and J. D. Griffith, Proc. Natl. Acad. Sci. USA 93:8863-8867, 1996). To determine the in vivo effects of these sequences, we replaced the upstream regulatory sequences of the HIS4 gene of Saccharomyces cerevisiae with either 12 or 48 tandem copies of CCGNN. Both tracts activated HIS4 transcription. We found that (CCGNN)(12) tracts elevated meiotic recombination (hot spot activity), whereas the (CCGNN)(48) tract repressed recombination (cold spot activity). In addition, a "pure" tract of (CCGAT)(12) activated both transcription and meiotic recombination. We suggest that the cold spot activity of the (CCGNN)(48) tract is related to the phenomenon of the suppressive interactions of adjacent hot spots previously described in yeast (Q.-Q. Fan, F. Xu, and T. D. Petes, Mol. Cell. Biol. 15:1679-1688, 1995; Q.-Q. Fan, F. Xu, M. A. White, and T. D. Petes, Genetics 145:661-670, 1997; T.-C. Wu and M. Lichten, Genetics 140:55-66, 1995; L. Xu and N. Kleckner, EMBO J. 16:5115-5128, 1995). PMID: 10523654 [PubMed - indexed for MEDLINE] 645: J Biol Chem 1999 Oct 22;274(43):30393-401 An RNA binding motif in the Cbp2 protein required for protein-stimulated RNA catalysis. Tirupati HK, Shaw LC, Lewin AS. Department of Molecular Genetics, University of Florida College of Medicine, Gainesville, Florida 32605, USA. The fifth and terminal intron of yeast cytochrome b pre-mRNA (a group I intron) requires a protein encoded by the nuclear gene CBP2 for splicing. Because catalysis is intrinsic to the RNA, the protein is believed to promote formation of secondary and tertiary structure of the RNA, resulting in a catalytically competent intron. In vitro, this mitochondrial intron can be made to self-splice or undergo protein-facilitated splicing by varying the Mg(2+) and monovalent salt concentrations. This two-component system, therefore, provides a good model for understanding the role of proteins in RNA folding. A UV cross-linking experiment was initiated to identify RNA binding sites on Cbp2 and gain insights into Cbp2-intron interactions. A 12-amino acid region containing a presumptive contact site near the amino terminus was targeted for mutagenesis, and mutant proteins were characterized for RNA binding and stimulation of splicing. Mutations in this region resulted in partial or complete loss of function, demonstrating the importance of this determinant for stimulation of RNA splicing. Several of the mutations that severely reduced splicing did not significantly shift the overall binding isotherm of Cbp2 for the precursor RNA, suggesting that contacts critical for activity are not necessarily reflected in the dissociation constant. This analysis has identified a unique RNA binding motif of alternating basic and aromatic residues that is essential for protein facilitated splicing. PMID: 10521416 [PubMed - indexed for MEDLINE] 646: FEBS Lett 1999 Sep 10;458(1):11-6 Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors. Tanaka AS, Silva MM, Torquato RJ, Noguti MA, Sampaio CA, Fritz H, Auerswald EA. Departamento de Bioquimica, UNIFESP-EPM, Sao Paulo, Brazil. tanaka.bioq@epm.br The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V). The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions. PMID: 10518924 [PubMed - indexed for MEDLINE] 647: Proc Natl Acad Sci U S A 1999 Oct 12;96(21):11752-7 An internal targeting signal directing proteins into the mitochondrial intermembrane space. Diekert K, Kispal G, Guiard B, Lill R. Institut fur Zytobiologie und Zytopathologie der Philipps-Universitat Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany. Import of most nucleus-encoded preproteins into mitochondria is mediated by N-terminal presequences and requires a membrane potential and ATP hydrolysis. Little is known about the chemical nature and localization of other mitochondrial targeting signals or of the mechanisms by which they facilitate membrane passage. Mitochondrial heme lyases lack N-terminal targeting information. These proteins are localized in the intermembrane space and are essential for the covalent attachment of heme to c type cytochromes. For import of heme lyases, the translocase of the mitochondrial outer membrane complex is both necessary and sufficient. Here, we report the identification of the targeting signal of mitochondrial heme lyases in the third quarter of these proteins. The targeting sequence is highly conserved among all known heme lyases. Its chemical character is hydrophilic because of a large fraction of both positively and negatively charged amino acid residues. These features clearly distinguish this signal from classical presequences. When inserted into a cytosolic protein, the targeting sequence directs the fusion protein into the intermembrane space, even in the absence of a membrane potential or ATP hydrolysis. The heme lyase targeting sequence represents the first topogenic signal for energy-independent transport into the intermembrane space and harbors two types of information. It assures accurate recognition and translocation by the translocase of the mitochondrial outer membrane complex, and it is responsible for driving the import reaction by undergoing high-affinity interactions with components of the intermembrane space. PMID: 10518522 [PubMed - indexed for MEDLINE] 648: Mol Cell 1999 Sep;4(3):387-94 The FHA domain is a modular phosphopeptide recognition motif. Durocher D, Henckel J, Fersht AR, Jackson SP. Wellcome Trust and Cancer Research Campaign, Institute of Cancer and Developmental Biology, Cambridge, United Kingdom. FHA domains are conserved sequences of 65-100 amino acid residues found principally within eukaryotic nuclear proteins, but which also exist in certain prokaryotes. The FHA domain is thought to mediate protein-protein interactions, but its mode of action has yet to be elucidated. Here, we show that the two highly divergent FHA domains of Saccharomyces cerevisiae Rad53p, a protein kinase involved in cell cycle checkpoint control, possess phosphopeptide-binding specificity. We also demonstrate that other FHA domains bind peptides in a phospho-dependent manner. These findings indicate that the FHA domain is a phospho-specific protein-protein interaction motif and have important implications for mechanisms of intracellular signaling in both eukaryotes and prokaryotes. PMID: 10518219 [PubMed - indexed for MEDLINE] 649: Biotechnol Bioeng 1999 Dec 5;65(5):550-7 Visualizing integrated bioprocess designs through "windows of operation". Zhou YH, Titchener-Hooker NJ. The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. y.zhou@ucl.ac.uk This paper demonstrates a simple graphical approach for the design and analysis of a bioprocess flowsheet in which process interactions are significant. Results are presented showing how the feasible space for operation can be simulated and used both to address key design and operating decisions and to identify suitable trade-offs between operating variables, such as fermentation growth rate and disruption conditions, in order to achieve prespecified levels of process performance. Using verified models to describe the production and isolation of an intracellular protein alcohol dehydrogenase (ADH) in yeast as a test bed, a series of so-called "windows of operation" are developed at growth rates in the range of 0.06-0.28 h(-1) and for a range of overall process specifications. The effects of altering the process design performance specification as defined by the level of cell debris removal and the overall process productivity on the size and position of the feasible space were investigated to demonstrate the sensitivity of the flowsheet to changes in process objectives. Using the approach it has been possible to visualise the processing trade-offs required to increase performance in terms of the level of cell debris removal by 50% and the overall process productivity by 400% from a defined base level. The approach provides a convenient tool when designing integrated bioprocesses by enabling process options to be compared visually and can help in achieving better process designs and accelerating process development for the biological process industry. Copyright 1999 John Wiley & Sons, Inc. PMID: 10516581 [PubMed - indexed for MEDLINE] 650: J Biol Chem 1999 Oct 15;274(42):30052-8 Phospholipase C binds to the receptor-like GPR1 protein and controls pseudohyphal differentiation in Saccharomyces cerevisiae. Ansari K, Martin S, Farkasovsky M, Ehbrecht IM, Kuntzel H. Max-Planck-Institut fur Experimentelle Medizin, Hermann-Rein-Strasse 3, D-37075 Gottingen, Germany. The hormone receptor-like protein Gpr1p physically interacts with phosphatidylinositol-specific phospholipase C (Plc1p) and with the Galpha protein Gpa2p, as shown by two-hybrid assays and co-immune precipitation of epitope-tagged proteins. Plc1p binds to Gpr1p in either the presence or absence of Gpa2, whereas the Gpr1p/Gpa2p association depends on the presence of Plc1p. Genetic interactions between the null mutations plc1Delta, gpr1Delta, gpa2Delta, and ras2Delta suggest that Plc1p acts together with Gpr1p and Gpa2p in a growth control pathway operating in parallel to the Ras2p function. Diploid cells lacking Gpr1p, Plc1p, or Gpa2p fail to form pseudohyphae upon nitrogen depletion, and the filamentation defect of gpr1Delta and plc1Delta strains is rescued by activating a mitogen-activated protein kinase pathway via STE11-4 or by activating a cAMP pathway via overexpressed Tpk2p. Plc1p is also required for efficient expression of the FG(TyA)::lacZ reporter gene under nitrogen depletion. In conclusion, we have identified two physically interacting proteins, Gpr1p and Plc1p, as novel components of a nitrogen signaling pathway controlling the developmental switch from yeast-like to pseudohyphal growth. Our data suggest that phospholipase C modulates the interaction of the putative nutrient sensor Gpr1p with the Galpha protein Gpa2p as a downstream effector of filamentation control. PMID: 10514491 [PubMed - indexed for MEDLINE] 651: Mol Biol Cell 1999 Oct;10(10):3389-400 Association of the cell cycle transcription factor Mbp1 with the Skn7 response regulator in budding yeast. Bouquin N, Johnson AL, Morgan BA, Johnston LH. Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom. We previously isolated the SKN7 gene in a screen designed to isolate new components of the G1-S cell cycle transcription machinery in budding yeast. We have now found that Skn7 associates with Mbp1, the DNA-binding component of the G1-S transcription factor DSC1/MBF. SKN7 and MBP1 show several genetic interactions. Skn7 overexpression is lethal and is suppressed by a mutation in MBP1. Similarly, high overexpression of Mbp1 is lethal and can be suppressed by skn7 mutations. SKN7 is also required for MBP1 function in a mutant compromised for G1-specific transcription. Gel-retardation assays indicate that Skn7 is not an integral part of MBF. However, a physical interaction between Skn7 and Mbp1 was detected using two-hybrid assays and GST pulldowns. Thus, Skn7 and Mbp1 seem to form a transcription factor independent of MBF. Genetic data suggest that this new transcription factor could be involved in the bud-emergence process. PMID: 10512874 [PubMed - indexed for MEDLINE] 652: Biochemistry 1999 Sep 7;38(36):11634-42 An NMR analysis of ubiquitin recognition by yeast ubiquitin hydrolase: evidence for novel substrate recognition by a cysteine protease. Sakamoto T, Tanaka T, Ito Y, Rajesh S, Iwamoto-Sugai M, Kodera Y, Tsuchida N, Shibata T, Kohno T. Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194-8511, Japan. Yeast ubiquitin hydrolase 1 (YUH1), a cysteine protease that catalyzes the removal of ubiquitin C-terminal adducts, is important for the generation of monomeric ubiquitin. Heteronuclear NMR spectroscopy has been utilized to map the YUH1 binding surface on ubiquitin. When YUH1 was titrated into a sample of ubiquitin, approximately 50% of the (1)H-(15)N correlation peaks of ubiquitin were affected to some degree, as a result of binding to YUH1. It is noteworthy that the amide resonances of the basic residues (Arg42, Lys48, Arg72, and Lys74) were highly perturbed. These positively charged basic residues may be involved in direct interactions with the negatively charged acidic residues on YUH1. In addition to the electrostatic surface, the hydrophobic surfaces on ubiquitin (Leu8, Ile44, Phe45, Val70, Leu71, and Leu73) and YUH1 are also likely to contribute to the binding interaction. Furthermore, the amide resonances of Ile13, Leu43, Leu50, and Leu69, the side chains of which are not on the surface, were also highly perturbed, indicating substrate-induced changes in the environments of these residues as well. These large changes, observed from residues located throughout the five-stranded beta-sheet surface and the C-terminus, suggest that substrate recognition by YUH1 involves a wider area on ubiquitin. PMID: 10512618 [PubMed - indexed for MEDLINE] 653: Biospectroscopy 1999;5(5 Suppl):S42-52 Influence of protein environment on magnetic circular dichroism spectral properties of ferric and ferrous ligand complexes of yeast cytochrome c peroxidase. Pond AE, Sono M, Elenkova EA, Goodin DB, English AM, Dawson JH. Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208, USA. The addition of exogenous ligands to the ferric and ferrous states of yeast cytochrome c peroxidase (CCP) is investigated with magnetic circular dichroism (MCD) at 4 degrees C to determine the effect the protein environment may exercise on spectral properties. The MCD spectrum of each derivative is directly compared to those of analogous forms of horseradish peroxidase (HRP) and myoglobin (Mb), two well-characterized histidine-ligated heme proteins. The ferric azide adduct of CCP is a hexacoordinate, largely low-spin species with an MCD spectrum very similar to that of ferric azide HRP. This complex displays an MCD spectrum dissimilar from that of the Mb derivative, possibly because of the stabilizing interaction between the azide ligand and the distal arginine of CCP (Arg 48). For the ferric fluoride derivative all three proteins display varied MCD data, indicating that the differences in the distal pocket of each protein influences their respective MCD characteristics. The MCD data for the cyanoferric complexes are similar for all three proteins, demonstrating that a strong field ligand bound in the sixth axial position dominates the MCD characteristics of the derivative. Similarly, the ferric NO complexes of the three proteins show MCD spectra similar in feature position and shape, but vary somewhat in intensity. Reduction of CCP at neutral pH yields a typical pentacoordinate high-spin complex with an MCD spectrum similar to that of deoxyferrous HRP. Formation of the NO and cyanide complexes of ferrous CCP gives derivatives with MCD spectra similar to the analogous forms of HRP and Mb in both feature position and shape. Addition of CO to deoxyferrous CCP results in a ferrous-CO complex with MCD spectral similarity to that of ferrous-CO HRP but not Mb, indicating that interactions between the ligand and the distal residues affects the MCD characteristics. Examination of alkaline (pH 9.7) deoxyferrous CCP indicates that a pH dependent conformational change has occurred, leading to a coordination structure similar to that of ferrous cytochrome b5, a known bis-histidine complex. Exposure of this complex to CO further confirms that a conformational change has taken place in that the MCD spectral characteristics of the resulting complex are similar to those of ferrous-CO Mb but not ferrous-CO HRP. PMID: 10512537 [PubMed - indexed for MEDLINE] 654: Genetics 1999 Oct;153(2):643-52 Mutational analysis of yeast TFIIB. A functional relationship between Ssu72 and Sub1/Tsp1 defined by allele-specific interactions with TFIIB. Wu WH, Pinto I, Chen BS, Hampsey M. Department of Biochemistry, Louisiana State University Medical Center, Shreveport, Louisiana 71130, USA. TFIIB is an essential component of the RNA polymerase II core transcriptional machinery. Previous studies have defined TFIIB domains required for interaction with other transcription factors and for basal transcription in vitro. In the study reported here we investigated the TFIIB structural requirements for transcription initiation in vivo. A library of sua7 mutations encoding altered forms of yeast TFIIB was generated by error-prone polymerase chain reaction and screened for conditional growth defects. Twenty-two single amino acid replacements in TFIIB were defined and characterized. These replacements are distributed throughout the protein and occur primarily at phylogenetically conserved positions. Most replacements have little or no effect on the steady-state protein levels, implying that each affects TFIIB function rather than synthesis or stability. In contrast to the initial sua7 mutants, all replacements, with one exception, have no effect on start site selection, indicating that specific TFIIB structural defects affect transcriptional accuracy. This collection of sua7 alleles, including the initial sua7 alleles, was used to investigate the allele specificity of interactions between ssu72 and sub1, both of which were initially identified as either suppressors (SUB1 2mu) or enhancers (sub1Delta, ssu72-1) of sua7 mutations. We show that the interactions of ssu72-1 and sub1Delta with sua7 are allele specific; that the allele specificities of ssu72 and sub1 overlap; and that each of the sua7 alleles that interacts with ssu72 and sub1 affects the accuracy of transcription start site selection. These results demonstrate functional interactions among TFIIB, Ssu72, and Sub1 and suggest that these interactions play a role in the mechanism of start site selection by RNA polymerase II. PMID: 10511545 [PubMed - indexed for MEDLINE] 655: EMBO J 1999 Oct 1;18(19):5370-9 Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus. Egea-Cortines M, Saedler H, Sommer H. Max-Planck-Institut fur Zuchtungsforschung, Carl-von-Linne Weg 10, 50829 Koln, Germany. Marcos.Egea@etsia.upct.es In Antirrhinum, floral meristems are established by meristem identity genes. Floral meristems give rise to floral organs in whorls, with their identity established by combinatorial activities of organ identity genes. Double mutants of the floral meristem identity gene SQUAMOSA and organ identity genes DEFICIENS or GLOBOSA produce flowers in which whorled patterning is partially lost. In yeast, SQUA, DEF and GLO proteins form ternary complexes via their C-termini, which in gel-shift assays show increased DNA binding to CArG motifs compared with DEF/GLO heterodimers or SQUA/SQUA homodimers. Formation of ternary complexes by plant MADS-box factors increases the complexity of their regulatory functions and might be the molecular basis for establishment of whorled phyllotaxis and combinatorial interactions of floral organ identity genes. PMID: 10508169 [PubMed - indexed for MEDLINE] 656: J Biol Chem 1999 Oct 8;274(41):29211-9 Structure-function analysis of the protein-binding domains of Mac1p, a copper-dependent transcriptional activator of copper uptake in Saccharomyces cerevisiae. Serpe M, Joshi A, Kosman DJ. Department of Biochemistry, School of Medicine, State University of New York at Buffalo, Buffalo, New York 14214, USA. The Mac1 protein in Saccharomyces cerevisiae is essential for the expression of yeast high affinity copper uptake. A positive transcription factor, Mac1p binds via its N-terminal domain to GCTC elements in the promoters of CTR1 and FRE1, encoding a copper permease and metal reductase, respectively. Mac1p-dependent transcriptional activation is negatively regulated by copper. We have mapped the domains in Mac1p responsible for its nuclear localization and for the protein-protein interactions that underlie its transcriptional activity. Immunofluorescence studies indicate that Mac1p contains two nuclear localization signals, one each in the N- and C-terminal halves of the protein. Yeast one-hybrid analysis demonstrates that the copper-dependent transcriptional activity in Mac1p resides primarily in a cysteine-rich element encompassing residues 264-279. Two-hybrid analysis indicates that a copper-independent Mac1p-Mac1p interaction linked to DNA binding is due primarily to a predicted helix in the C-terminal region of the protein encompassing residues 388-406. Point mutations within this putative helix abrogate the Mac1-Mac1 interaction in vivo and formation of a ternary (Mac1p)(2).DNA complex in vitro. When produced in normal abundance, Mac1pI396D and Mac1pF400D helix mutants do not support transcriptional activation in vivo consistent with an essential Mac1p dimerization in transcriptional activation. Lastly, the one- and two-hybrid data indicate that an intramolecular interaction between the DNA-binding and transactivation domains negatively modulates Mac1p activity. PMID: 10506178 [PubMed - indexed for MEDLINE] 657: Mol Biotechnol 1999 Jun;11(3):213-20 Reconstitution of fibroblast growth factor receptor interactions in the yeast two hybrid system. Aloni-Grinstein R, Seddon A, Yayon A. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. Fibroblast growth factors (FGF) activate their receptors through the formation of trimolecular complexes, composed of a ligand, a receptor, and a heparan sulfate oligosaccharide, all of which are members of particularly large families capable of multiple interactions in a combinatorial fashion. Understanding this large network of interactions not only presents a great challenge, but is practically beyond the capacity of most classical techniques routinely used to study ligand receptor interactions. We have used the yeast two hybrid system to study protein-protein interactions in the FGF family. Both ligand and receptor ectodomains are properly folded and functional in the yeast. Basic FGF (bFGF) expressed in the yeast dimerizes spontaneously. This self-assembly occurs at low affinity, which can be greatly enhanced by the introduction of heparin, supporting a defined role for heparin in bFGF dimerization. Screening a rat embryo cDNA library with bFGF in the yeast two hybrid system identified a short variant of FGF receptor 1, found most frequently in embryonal and tumor cells and which possesses affinity toward bFGF that is significantly greater than that of the more abundant, full-length receptor. We find the yeast two hybrid system, a most suitable alternative method for the analysis of growth factor-receptor interactions as well as for screening for novel interacting proteins and modulators of FGF and its receptors. PMID: 10503237 [PubMed - indexed for MEDLINE] 658: Proc Natl Acad Sci U S A 1999 Sep 28;96(20):11206-10 Chitin synthase III: synthetic lethal mutants and "stress related" chitin synthesis that bypasses the CSD3/CHS6 localization pathway. Osmond BC, Specht CA, Robbins PW. Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Delta, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that "stress response" chitin synthesis proceeds through an alternate Chs3p targeting pathway. PMID: 10500155 [PubMed - indexed for MEDLINE] 659: J Biol Inorg Chem 1999 Apr;4(2):209-19 X-ray crystal structures of active site mutants of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis. Macedo-Ribeiro S, Hemrika W, Renirie R, Wever R, Messerschmidt A. Max-Planck Institut fur Biochemie, Abteilung Strukturforschung, Martinsried, Germany. The X-ray structures of the chloroperoxidase from Curvularia inaequalis, heterologously expressed in Saccharomyces cerevisiae, have been determined both in its apo and in its holo forms at 1.66 and 2.11 A resolution, respectively. The crystal structures reveal that the overall structure of this enzyme remains nearly unaltered, particularly at the metal binding site. At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interactions and hydrogen bonds with positively charged residues involved in the interactions with the vanadate in the native protein. The vanadate binding pocket seems to form a very rigid frame stabilising oxyanion binding. The rigidity of this active site matrix is the result of a large number of hydrogen bonding interactions involving side chains and the main chain of residues lining the active site. The structures of single site mutants to alanine of the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed. Additionally we determined the structural effects of mutations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involved in forming a salt bridge with Arg490 which also interacts with the vanadate. The enzymatic chlorinating activity is drastically reduced to approximately 1% in mutants D292A, H404A and H496A. The structures of the mutants confirm the view of the active site of this chloroperoxidase as a rigid matrix providing an oxyanion binding site. No large changes are observed at the active site for any of the analysed mutants. The empty space left by replacement of large side chains by alanines is usually occupied by a new solvent molecule which partially replaces the hydrogen bonding interactions to the vanadate. The new solvent molecules additionally replace part of the interactions the mutated side chains were making to other residues lining the active site frame. When this is not possible, another side chain in the proximity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame. PMID: 10499093 [PubMed - indexed for MEDLINE] 660: J Biol Chem 1999 Oct 1;274(40):28803-7 Isolation of the protein kinase TAO2 and identification of its mitogen-activated protein kinase/extracellular signal-regulated kinase kinase binding domain. Chen Z, Hutchison M, Cobb MH. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA. We previously reported the cloning of the thousand and one-amino acid protein kinase 1 (TAO1), a rat homolog of the Saccharomyces cerevisiae protein kinase sterile 20 protein. Here we report the complete sequence and properties of a related rat protein kinase TAO2. Like TAO1, recombinant TAO2 selectively activated mitogen-activated protein/extracellular signal-regulated kinase kinases (MEKs) 3, 4, and 6 of the stress-responsive mitogen-activated protein kinase pathways in vitro and copurified with MEK3 endogenous to Sf9 cells. To examine TAO2 interactions with MEKs, the MEK binding domain of TAO2 was localized to an approximately 135-residue sequence just C-terminal to the TAO2 catalytic domain. In vitro this MEK binding domain associated with MEKs 3 and 6 but not MEKs 1, 2, or 4. Using chimeric MEK proteins, we found that the MEK N terminus was sufficient for binding to TAO2. Catalytic activity of full-length TAO2 enhanced its binding to MEKs. However, neither the autophosphorylation of the MEK binding domain of TAO2 nor the activity of MEK itself was required for MEK binding. These results suggest that TAO proteins lie in stress-sensitive kinase cascades and define a mechanism by which these kinases may organize downstream targets. PMID: 10497253 [PubMed - indexed for MEDLINE] 661: J Biol Chem 1999 Oct 1;274(40):28246-55 Molecular analysis of yeast and human type II topoisomerases. Enzyme-DNA and drug interactions. Strumberg D, Nitiss JL, Dong J, Kohn KW, Pommier Y. Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA. The DNA sequence selectivity of topoisomerase II (top2)-DNA cleavage complexes was examined for the human (top2alpha), yeast, and Escherichia coli (i.e. gyrase) enzymes in the absence or presence of anticancer or antibacterial drugs. Species-specific differences were observed for calcium-promoted DNA cleavage. Similarities and differences in DNA cleavage patterns and nucleic acid sequence preferences were also observed between the human, yeast, and E. coli top2 enzymes in the presence of the non-intercalators fluoroquinolone CP-115,953, etoposide, and azatoxin and the intercalators amsacrine and mitoxantrone. Additional base preferences were generally observed for the yeast when compared with the human top2alpha enzyme. Preferences in the immediate flanks of the top2-mediated DNA cleavage sites are, however, consistent with the drug stacking model for both enzymes. We also analyzed and compared homologous mutations in yeast and human top2, i.e. Ser(740) --> Trp and Ser(763) --> Trp, respectively. Both mutations decreased the reversibility of the etoposide-stabilized cleavage sites and produced consistent base sequence preference changes. These data demonstrate similarities and differences between human and yeast top2 enzymes. They also indicate that the structure of the enzyme/DNA interface plays a key role in determining the specificity of top2 poisons and cleavage sites for both the intercalating and non-intercalating drugs. PMID: 10497180 [PubMed - indexed for MEDLINE] 662: Mol Cell Biol 1999 Oct;19(10):6729-41 Regulation of cell cycle transcription factor Swi4 through auto-inhibition of DNA binding. Baetz K, Andrews B. Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8. In Saccharomyces cerevisiae, two transcription factors, SBF (SCB binding factor) and MBF (MCB binding factor), promote the induction of gene expression at the G(1)/S-phase transition of the mitotic cell cycle. Swi4 and Mbp1 are the DNA binding components of SBF and MBF, respectively. The Swi6 protein is a common subunit of both transcription factors and is presumed to play a regulatory role. SBF binding to its target sequences, the SCBs, is a highly regulated event and requires the association of Swi4 with Swi6 through their C-terminal domains. Swi4 binding to SCBs is restricted to the late M and G(1) phases, when Swi6 is localized to the nucleus. We show that in contrast to Swi6, Swi4 remains nuclear throughout the cell cycle. This finding suggests that the DNA binding domain of Swi4 is inaccessible in the full-length protein when not complexed with Swi6. To explore this hypothesis, we expressed Swi4 and Swi6 in insect cells by using the baculovirus system. We determined that partially purified Swi4 cannot bind SCBs in the absence of Swi6. However, Swi4 derivatives carrying point mutations or alterations in the extreme C terminus were able to bind DNA or activate transcription in the absence of Swi6, and the C terminus of Swi4 inhibited Swi4 derivatives from binding DNA in trans. Full-length Swi4 was determined to be monomeric in solution, suggesting an intramolecular mechanism for auto-inhibition of binding to DNA by Swi4. We detected a direct in vitro interaction between a C-terminal fragment of Swi4 and the N-terminal 197 amino acids of Swi4, which contain the DNA binding domain. Together, our data suggest that intramolecular interactions involving the C-terminal region of Swi4 physically prevent the DNA binding domain from binding SCBs. The interaction of the carboxy-terminal region of Swi4 with Swi6 alleviates this inhibition, allowing Swi4 to bind DNA. PMID: 10490612 [PubMed - indexed for MEDLINE] 663: Mol Cell Biol 1999 Oct;19(10):6642-51 The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and functionally separated from NOT2, NOT4, and NOT5. Bai Y, Salvadore C, Chiang YC, Collart MA, Liu HY, Denis CL. Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824, USA. The CCR4-NOT complex (1 mDa in size), consisting of the proteins CCR4, CAF1, and NOT1 to NOT5, regulates gene expression both positively and negatively and is distinct from other large transcriptional complexes in Saccharomyces cerevisiae such as SNF/SWI, TFIID, SAGA, and RNA polymerase II holoenzyme. The physical and genetic interactions between the components of the CCR4-NOT complex were investigated in order to gain insight into how this complex affects the expression of diverse genes and processes. The CAF1 protein was found to be absolutely required for CCR4 association with the NOT proteins, and CCR4 and CAF1, in turn, physically interacted with NOT1 through its central amino acid region from positions 667 to 1152. The NOT3, NOT4, and NOT5 proteins had no significant effect on the association of CCR4, CAF1, and NOT1 with each other. In contrast, the NOT2, NOT4, and NOT5 interacted with the C-terminal region (residues 1490 to 2108) of NOT1 in which NOT2 and NOT5 physically associated in the absence of CAF1, NOT3, and NOT4. These and other data indicate that the physical ordering of these proteins in the complex is CCR4-CAF1-NOT1-(NOT2, NOT5), with NOT4 and NOT3 more peripheral to NOT2 and NOT5. The physical separation of CCR4 and CAF1 from other components of the CCR4-NOT complex correlated with genetic analysis indicating partially separate functions for these two groups of proteins. ccr4 or caf1 deletion suppressed the increased 3-aminotriazole resistance phenotype conferred by not mutations, resulted in opposite effects on gene expression as compared to several not mutations, and resulted in a number of synthetic phenotypes in combination with not mutations. These results define the CCR4-NOT complex as consisting of at least two physically and functionally separated groups of proteins. PMID: 10490603 [PubMed - indexed for MEDLINE] 664: Mol Cell Biol 1999 Oct;19(10):6543-53 Genetic and physical interactions involving the yeast nuclear cap-binding complex. Fortes P, Kufel J, Fornerod M, Polycarpou-Schwarz M, Lafontaine D, Tollervey D, Mattaj IW. European Molecular Biology Laboratory, D-69117 Heidelberg, Germany. Yeast strains lacking the yeast nuclear cap-binding complex (yCBC) are viable, although impaired in growth. We have taken advantage of this observation to carry out a genetic screen for components that show synthetic lethality (SL) with a cbp20-Delta cbp80-Delta double mutation. One set of SL interactions was due to mutations that were complemented by components of U1 small nuclear RNP (snRNP) and the yeast splicing commitment complex. These interactions confirm the role of yCBC in commitment complex formation. Physical interaction of yCBC with the commitment complex components Mud10p and Mud2p, which may directly mediate yCBC function, was demonstrated. Unexpectedly, we identified multiple SL mutations that were complemented by Cbf5p and Nop58p. These are components of the two major classes of yeast small nucleolar RNPs, which function in the maturation of rRNA precursors. Mutants lacking yCBC were found to be defective in rRNA processing. Analysis of the yCBC deletion phenotype suggests that this is likely to be due to a defect in the splicing of a subset of ribosomal protein mRNA precursors. PMID: 10490594 [PubMed - indexed for MEDLINE] 665: Mol Cell 1999 Aug;4(2):153-66 Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. Rittinger K, Budman J, Xu J, Volinia S, Cantley LC, Smerdon SJ, Gamblin SJ, Yaffe MB. Divison of Protein Structure, National Institute for Medical Research, London, United Kingdom. We have solved the high-resolution X-ray structure of 14-3-3 bound to two different phosphoserine peptides, representing alternative substrate-binding motifs. These structures reveal an evolutionarily conserved network of peptide-protein interactions within all 14-3-3 isotypes, explain both binding motifs, and identify a novel intrachain phosphorylation-mediated loop structure in one of the peptides. A 14-3-3 mutation disrupting Raf signaling alters the ligand-binding cleft, selecting a different phosphopeptide-binding motif and different substrates than the wild-type protein. Many 14-3-3: peptide contacts involve a C-terminal amphipathic alpha helix containing a putative nuclear export signal, implicating this segment in both ligand and Crm1 binding. Structural homology between the 14-3-3 NES structure and those within I kappa B alpha and p53 reveals a conserved topology recognized by the Crm1 nuclear export machinery. PMID: 10488331 [PubMed - indexed for MEDLINE] 666: Mol Gen Genet 1999 Jul;261(6):967-76 Genetic interactions between a null allele of the RIT1 gene encoding an initiator tRNA-specific modification enzyme and genes encoding translation factors in Saccharomyces cerevisiae. Astrom SU, Nordlund ME, Erickson FL, Hannig EM, Bystrom AS. Department of Microbiology, Umea University, Sweden. The Saccharomyces cerevisiae gene RIT1 encodes a phospho-ribosyl transferase that exclusively modifies the initiator tRNA (tRNAMet(i)) by the addition of a 2'-O-ribosyl phosphate group to Adenosine 64. As a result, tRNAMet(i) is prevented from participating in the elongation steps of protein synthesis. We previously showed that the modification is not essential for the function of tRNAMet(i) in the initiation of translation, since rit1 null strains are viable and show no obvious growth defects. Here, we demonstrate that yeast strains in which a rit1 null allele is combined with mutations in any of the genes for the three subunits of eukaryotic initiation factor-2 (eIF-2), or with disruption alleles of two of the four initiator methionine tRNA (IMT) genes, show synergistic growth defects. A multicopy plasmid carrying an IMT gene can alleviate these defects. On the other hand, introduction of a high-copy-number plasmid carrying the TEF2 gene, which encodes the eukaryotic elongation factor 1alpha (eEF-1alpha), into rit1 null strains with two intact IMT genes had the opposite effect, indicating that increased levels of eEF-1alpha are deleterious to these strains, presumably due to sequestration of the unmodified met-tRNAMet(i) for elongation. Thus, under conditions in which the components of the ternary met-tRNAMet(i):GTP:eIF-2 complex become limiting or are functionally impaired, the presence of the 2'-O-ribosyl phosphate modification in tRNAMet(i) is important for the provision of adequate amounts of tRNAMet(i) for formation of this ternary complex. PMID: 10485288 [PubMed - indexed for MEDLINE] 667: Cell 1999 Aug 20;98(4):453-63 The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Wang SL, Hawkins CJ, Yoo SJ, Muller HA, Hay BA. Division of Biology MC 156-29, California Institute of Technology, Pasadena 91125, USA. Drosophila Reaper (RPR), Head Involution Defective (HID), and GRIM induce caspase-dependent cell death and physically interact with the cell death inhibitor DIAP1. Here we show that HID blocks DIAP1's ability to inhibit caspase activity and provide evidence suggesting that RPR and GRIM can act similarly. Based on these results, we propose that RPR, HID, and GRIM promote apoptosis by disrupting productive IAP-caspase interactions and that DIAP1 is required to block apoptosis-inducing caspase activity. Supporting this hypothesis, we show that elimination of DIAP1 function results in global early embryonic cell death and a large increase in DIAP1-inhibitable caspase activity and that DIAP1 is still required for cell survival when expression of rpr, hid, and grim is eliminated. PMID: 10481910 [PubMed - indexed for MEDLINE] 668: Mol Endocrinol 1999 Sep;13(9):1550-7 Coactivators for the orphan nuclear receptor RORalpha. Atkins GB, Hu X, Guenther MG, Rachez C, Freedman LP, Lazar MA. Department of Medicine, The Penn Diabetes Center, University of Pennsylvania School of Medicine, Philadelphia 19104-6149, USA. A mutation in the nuclear orphan receptor RORalpha results in a severe impairment of cerebellar development by unknown mechanisms. We have shown previously that RORalpha contains a strong constitutive activation domain in its C terminus. We therefore searched for mammalian RORalpha coactivators using the minimal activation domain as bait in a two-hybrid screen. Several known and putative coactivators were isolated, including glucocorticoid receptor-interacting protein-1 (GRIP-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205). These interactions were confirmed in vitro and require the intact activation domain of RORalpha although different requirements for interaction with GRIP-1 and PBP were detected. Even in the absence of exogenous ligand, RORalpha interacts with a complex or complexes of endogenous proteins, similar to those that bind to ligand-occupied thyroid hormone and vitamin D receptors. Both PBP and GRIP-1 were shown to be present in these complexes. Thus we have identified several potential RORalpha coactivators that, in contrast to the interactions with hormone receptors, interact with RORalpha in yeast, in bacterial extracts, and in mammalian cells in vivo and in vitro in the absence of exogenous ligand. GRIP-1 functioned as a coactivator for the RORalpha both in yeast and in mammalian cells. Thus, GRIP-1 is the first proven coactivator for RORalpha. PMID: 10478845 [PubMed - indexed for MEDLINE] 669: FEBS Lett 1999 Sep 3;457(3):363-8 Tpr1, a Schizosaccharomyces pombe protein involved in potassium transport. Lichtenberg H, Heyer M, Hofer M. Botanisches Institut der Universitat Bonn, Kirschallee 1, 53115, Bonn, Germany. H.Lichtenberg@uni-bonn.de The Schizosaccharomyces pombe Tpr1 was isolated as suppressor of the Saccharomyces cerevisiae Delta trk1,2 potassium uptake deficient phenotype. Tpr1, for tetratrico peptide repeat, encodes a 1039 amino acid residues protein with several reiterated TPR units displaying significant homology to p150(TSP), a recently identified phosphoprotein of mouse, to S. cerevisiae CTR9 and to related sequences of human, Caenorhabditis elegans, Methanoccocus jannaschii and Arabidopsis thaliana. Expression of Tpr1 restored growth on 0.2 mM K(+) media, induced K(+) transport with a K(T) of 4.6 mM and resumed inward currents of -90 pA at -250 mV (pH 7.2) conducting K(+) and other alkali-metal ions. The tetratrico peptide repeat is a degenerate motif of 34 amino acids that is repeated several times within TPR-containing proteins and has been suggested to mediate protein-protein interactions. The sequence and putative binding properties of Tpr1 suggest the protein unlikely as transporter but involved in the enhancement of K(+) uptake via conventional carriers. PMID: 10471809 [PubMed - indexed for MEDLINE] 670: Genetics 1999 Sep;153(1):81-94 Genetic study of interactions between the cytoskeletal assembly protein sla1 and prion-forming domain of the release factor Sup35 (eRF3) in Saccharomyces cerevisiae. Bailleul PA, Newnam GP, Steenbergen JN, Chernoff YO. School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230, USA. Striking similarities between cytoskeletal assembly and the "nucleated polymerization" model of prion propagation suggest that similar or overlapping sets of proteins may assist in both processes. We show that the C-terminal domain of the yeast cytoskeletal assembly protein Sla1 (Sla1C) specifically interacts with the N-terminal prion-forming domain (Sup35N) of the yeast release factor Sup35 (eRF3) in the two-hybrid system. Sla1C and several other Sup35N-interacting proteins also exhibit two-hybrid interactions with the poly-Gln-expanded N-proximal fragment of human huntingtin, which promotes Huntington disease-associated aggregation. The Sup35N-Sla1C interaction is inhibited by Sup35N alterations that make Sup35 unable to propagate the [PSI(+)] state and by the absence of the chaperone protein Hsp104, which is essential for [PSI] propagation. In a Sla1(-) background, [PSI] curing by dimethylsulfoxide or excess Hsp104 is increased, while translational readthrough and de novo [PSI] formation induced by excess Sup35 or Sup35N are decreased. These data show that, in agreement with the proposed function of Sla1 during cytoskeletal formation, Sla1 assists in [PSI] formation and propagation, but is not required for these processes. Sla1(-) strains are sensitive to some translational inhibitors, and some sup35 mutants, obtained in a Sla1(-) background, are sensitive to Sla1, suggesting that the interaction between Sla1 and Sup35 proteins may play a role in the normal function of the translational apparatus. We hypothesize that Sup35N is involved in regulatory interactions with intracellular structural networks, and [PSI] prion may be formed as a by-product of this process. PMID: 10471702 [PubMed - indexed for MEDLINE] 671: Nat Genet 1999 Sep;23(1):81-5 Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants. Chen C, Kolodner RD. Ludwig Institute for Cancer Research, Cancer Center and Department of Medicine, University of California-San Diego School of Medicine, La Jolla, California 92093, USA. Cancer progression is often associated with the accumulation of gross chromosomal rearrangements (GCRs), such as translocations, deletion of a chromosome arm, interstitial deletions or inversions. In many instances, GCRs inactivate tumour-suppressor genes or generate novel fusion proteins that initiate carcinogenesis. The mechanism underlying GCR formation appears to involve interactions between DNA sequences of little or no homology. We previously demonstrated that mutations in the gene encoding the largest subunit of the Saccharomyces cerevisiae single-stranded DNA binding protein (RFA1) increase microhomology-mediated GCR formation. To further our understanding of GCR formation, we have developed a novel mutator assay in S. cerevisiae that allows specific detection of such events. In this assay, the rate of GCR formation was increased 600-5, 000-fold by mutations in RFA1, RAD27, MRE11, XRS2 and RAD50, but was minimally affected by mutations in RAD51, RAD54, RAD57, YKU70, YKU80, LIG4 and POL30. Genetic analysis of these mutants suggested that at least three distinct pathways can suppress GCRs: two that suppress microhomology-mediated GCRs (RFA1 and RAD27) and one that suppresses non-homology-mediated GCRs (RAD50/MRE11/XRS2). PMID: 10471504 [PubMed - indexed for MEDLINE] 672: J Biol Chem 1999 Sep 3;274(36):25461-70 Helical interactions and membrane disposition of the 16-kDa proteolipid subunit of the vacuolar H(+)-ATPase analyzed by cysteine replacement mutagenesis. Harrison MA, Murray J, Powell B, Kim YI, Finbow ME, Findlay JB. School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom. m.a.harrison@leeds.ac.uk Theoretical mechanisms of proton translocation by the vacuolar H(+)-ATPase require that a transmembrane acidic residue of the multicopy 16-kDa proteolipid subunit be exposed at the exterior surface of the membrane sector of the enzyme, contacting the lipid phase. However, structural support for this theoretical mechanism is lacking. To address this, we have used cysteine mutagenesis to produce a molecular model of the 16-kDa proteolipid complex. Transmembrane helical contacts were determined using oxidative cysteine cross-linking, and accessibility of cysteines to the lipid phase was determined by their reactivity to the lipid-soluble probe N-(1-pyrenyl)maleimide. A single model for organization of the four helices of each monomeric proteolipid was the best fit to the experimental data, with helix 1 lining a central pore and helix 2 and helix 3 immediately external to it and forming the principal intermolecular contacts. Helix 4, containing the crucial acidic residue, is peripheral to the complex. The model is consistent not only with theoretical proton transport mechanisms, but has structural similarity to the dodecameric ring complex formed by the related 8-kDa proteolipid of the F(1)F(0)-ATPase. This suggests some commonality between the proton translocating mechanisms of the vacuolar and F(1)F(0)-ATPases. PMID: 10464277 [PubMed - indexed for MEDLINE] 673: J Cell Sci 1999 Sep;112 Pt 18:3103-14 The S. pombe zfs1 gene is required to prevent septation if mitotic progression is inhibited. Beltraminelli N, Murone M, Simanis V. Cell Cycle Control Laboratory, ISREC, Chemin des Boveresses, 1066 Epalinges, Switzerland. Schizosaccharomyces pombe cdc16p is required to limit the cell to forming a single division septum per cell cycle; the heat-sensitive loss-of-function mutant cdc16-116 completes mitosis, and then undergoes multiple rounds of septum formation without cell cleavage. cdc16p is a homologue of Saccharomyces cerevisiae BUB2p, and has also been implicated in the spindle assembly checkpoint function in S. pombe. To identify other proteins involved in regulating septum formation, we have screened for multicopy suppressors of the cdc16-116 mutation. In this paper, we describe one of these suppressors, zfs1. The null allele (zfs1-D1) is viable. However, at low temperatures it divides at a reduced size, while at higher temperatures, it partially suppresses heat sensitive mutants in genes signalling the onset of septum formation. Zfs1-D1 cells show an increased rate of chromosome loss during exponential growth. Moreover, if assembly of the spindle is prevented, zfs1-D1 cells do not arrest normally, but the activity of cdc2p kinase decays, and cells form a division septum without completing a normal mitosis. We conclude that zfs1 function is required to prevent septum formation and exit from mitosis if the mitotic spindle is not assembled. The suppression of cdc16-116 by zfs1 is independent of dma1 function and the spindle assembly checkpoint genes mad2 and mph1. The genetic interactions of zfs1 with genes regulating septum formation suggest that it may be a modulator of the signal transduction network controlling the onset of septum formation and exit from mitosis. PMID: 10462526 [PubMed - indexed for MEDLINE] 674: Mol Cell Biol 1999 Sep;19(9):6441-7 Trithorax and ASH1 interact directly and associate with the trithorax group-responsive bxd region of the Ultrabithorax promoter. Rozovskaia T, Tillib S, Smith S, Sedkov Y, Rozenblatt-Rosen O, Petruk S, Yano T, Nakamura T, Ben-Simchon L, Gildea J, Croce CM, Shearn A, Canaani E, Mazo A. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. Trithorax (TRX) and ASH1 belong to the trithorax group (trxG) of transcriptional activator proteins, which maintains homeotic gene expression during Drosophila development. TRX and ASH1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers. Based on genetic interactions between trx and ash1 and our previous observation that association of the TRX protein with polytene chromosomes is ash1 dependent, we investigated the possibility of a physical linkage between the two proteins. We found that the endogenous TRX and ASH1 proteins coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes. Furthermore, we demonstrated that TRX and ASH1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s). This suggests that ASH1 and TRX act on Ubx in relatively close proximity to each other. Finally, TRX and ASH1 appear to interact directly through their conserved SET domains, based on binding assays in vitro and in yeast and on coimmunoprecipitation assays with embryo extracts. Collectively, these results suggest that TRX and ASH1 are components that interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription. PMID: 10454589 [PubMed - indexed for MEDLINE] 675: Mol Cell Biol 1999 Sep;19(9):6110-9 The yeast trimeric guanine nucleotide-binding protein alpha subunit, Gpa2p, controls the meiosis-specific kinase Ime2p activity in response to nutrients. Donzeau M, Bandlow W. Institut fur Genetik und Mikrobiologie, Ludwig-Maximilians-Universitat Munchen, D-80638 Munich, Germany. Saccharomyces cerevisiae Gpa2p, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein (G protein), is involved in the regulation of vegetative growth and pseudohyphal development. Here we report that Gpa2p also controls sporulation by interacting with the regulatory domain of Ime2p (Sme1p), a protein kinase essential for entrance of meiosis and sporulation. Protein-protein interactions between Gpa2p and Ime2p depend on the GTP-bound state of Gpa2p and correlate with down-regulation of Ime2p kinase activity in vitro. Overexpression of Ime2p inhibits pseudohyphal development and enables diploid cells to sporulate even in the presence of glucose or nitrogen. In contrast, overexpression of Gpa2p in cells simultaneously overproducing Ime2p results in a drastic reduction of sporulation efficiency, demonstrating an inhibitory effect of Gpa2p on Ime2p function. Furthermore, deletion of GPA2 accelerates sporulation on low-nitrogen medium. These observations are consistent with the following model. In glucose-containing medium, diploid cells do not sporulate because Ime2p is inactive or expressed at low levels. Upon starvation, expression of Gpa2p and Ime2p is induced but sporulation is prevented as long as nitrogen is present in the medium. The negative control of Ime2p kinase activity is exerted at least in part through the activated form of Gpa2p and is released as soon as nutrients are exhausted. This model attributes a switch function to Gpa2p in the meiosis-pseudohyphal growth decision. PMID: 10454558 [PubMed - indexed for MEDLINE] 676: Mol Cell Biol 1999 Sep;19(9):6065-75 Interactions of TLC1 (which encodes the RNA subunit of telomerase), TEL1, and MEC1 in regulating telomere length in the yeast Saccharomyces cerevisiae. Ritchie KB, Mallory JC, Petes TD. Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280, USA. In the yeast Saccharomyces cerevisiae, chromosomes terminate with a repetitive sequence [poly(TG(1-3))] 350 to 500 bp in length. Strains with a mutation of TEL1, a homolog of the human gene (ATM) mutated in patients with ataxia telangiectasia, have short but stable telomeric repeats. Mutations of TLC1 (encoding the RNA subunit of telomerase) result in strains that have continually shortening telomeres and a gradual loss of cell viability; survivors of senescence arise as a consequence of a Rad52p-dependent recombination events that amplify telomeric and subtelomeric repeats. We show that a mutation in MEC1 (a gene related in sequence to TEL1 and ATM) reduces telomere length and that tel1 mec1 double mutant strains have a senescent phenotype similar to that found in tlc1 strains. As observed in tlc1 strains, survivors of senescence in the tel1 mec1 strains occur by a Rad52p-dependent amplification of telomeric and subtelomeric repeats. In addition, we find that strains with both tel1 and tlc1 mutations have a delayed loss of cell viability compared to strains with the single tlc1 mutation. This result argues that the role of Tel1p in telomere maintenance is not solely a direct activation of telomerase. PMID: 10454554 [PubMed - indexed for MEDLINE] 677: J Mol Biol 1999 Aug 27;291(4):761-73 Yeast aspartyl-tRNA synthetase residues interacting with tRNA(Asp) identity bases connectively contribute to tRNA(Asp) binding in the ground and transition-state complex and discriminate against non-cognate tRNAs. Eriani G, Gangloff J. UPR 9002 SMBMR du CNRS, Institut de Biologie Moleculaire et Cellulaire, 15, rue Rene Descartes, Strasbourg, 67084, France. Crystallographic studies of the aspartyl-tRNA synthetase-tRNA(Asp)complex from yeast identified on the enzyme a number of residues potentially able to interact with tRNA(Asp). Alanine replacement of these residues (thought to disrupt the interactions) was used in the present study to evaluate their importance in tRNA(Asp)recognition and acylation. The results showed that contacts with the acceptor A of tRNA(Asp)by amino acid residues interacting through their side-chain occur only in the acylation transition state, whereas those located near the G73 discriminator base occur also during initial binding of tRNA(Asp). Interactions with the anticodon bases provide the largest free energy contribution to stability of the enzyme-tRNA complex in its ground state. These contacts also favour catalysis, by acting connectively with each other and with those of G73, as shown by multiple mutant analysis. This implies structural communication transmitting the anticodon recognition signal to the distally located acylation site. This signal might be conveyed via tRNA(Asp)as suggested by the observed conformational change of this molecule upon interaction with AspRS. From binding free energy values corresponding to the different AspRS-tRNA(Asp)interaction domains, it might be concluded that upon complex formation, the anticodon interacts first. Finally, acylation efficiencies of AspRS mutants in the presence of pure tRNA(Asp)and non-fractionated tRNAs indicate that residues involved in the binding of identity bases also discriminate against non-cognate tRNAs. Copyright 1999 Academic Press. PMID: 10452887 [PubMed - indexed for MEDLINE] 678: Cell Biol Int 1998;22(9-10):709-14 Resumption of rapid proliferation from lag phase in cultures of Saccharomyces cerevisiae in poor nutrient conditions. Effect of surface and intracellular signalling mechanisms. Overgaard AK, Kierklo L, Christiansen H, Chrois P, Rasmussen L, Friis J. Department of Anatomy and Cytology, Odense University, Odense M, 5230, Denmark. Saccharomyces cerevisiae was inoculated into a dilute synthetic minimal medium with glycerol as the carbon source. The number of live cells in the cultures was determined by colony counts on agar plates. Untreated control cells had doubled in number about once at the end of the first week and had gone through eight doublings by the end of the second week. Addition of either 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP) or human recombinant insulin, made the cells go through 12 and 10 doublings, respectively, by the end of the first week. In contrast, 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP) had only slight stimulating effects on cell multiplication, but if it was combined with phorbol-12-myristate-13-acetate (PMA) the cells went through about 12 doublings during the first week. Addition of LY 83583, an inhibitor of soluble guanylate cyclase, prevented cell proliferation. Further addition of 8-bromo-cGMP bypassed this inhibition. Singly, bradykinin or PMA did not affect cell multiplication. However, when these two compounds were combined, the cells went through about 10 doublings during the first week. Neither bradykinin, nor PMA had any releasing effect on the inhibition of LY 83583. These results indicate the existence of several routes leading to cell proliferation in wildtype S. cerevisiae cells. Copyright 1998 Academic Press. PMID: 10452842 [PubMed - indexed for MEDLINE] 679: J Mol Biol 1999 Aug 20;291(3):715-25 Proteins can adopt totally different folded conformations. Damaschun G, Damaschun H, Gast K, Zirwer D. Humboldt-Universitat zu Berlin, Institut fur Biologie, c/o Max-Delbruck-Centrum fur Molekulare Medizin, Robert-Rossle-Strasse 10, Berlin, PF 740238, D-13092, Germany. gdamasc@mdc-berlin.de The three-dimensional structure of a protein is determined by interactions between its amino acids and by interactions of the amino acids with molecules of the environment. The great influence of the latter interactions is demonstrated for the enzyme phosphoglycerate kinase from yeast (PGK). In the native state, PGK is a compact, bilobal molecule; 35% and 13% of its amino acids are organised in the form of alpha-helices and beta-sheets, respectively. The molecules unfold at acidic pH and low ionic strength forming random-walk structures with a persistence length of 3 nm. More than 90% of the amino acid residues of the ensemble have phi,psi-angles corresponding to those of a straight beta-chain. Upon addition of 50% (v/v) trifluoroethanol to the acid-unfolded protein, the entire molecule is transformed into a rod-like, flexible alpha-helix. Addition of anions, such as chloride or trichloroacetate, to the acid-unfolded protein leads to the formation of amyloid-like fibres over a period of many hours when the anion concentration exceeds a critical limit. Half of the amino acid residues are then organised in beta-sheets. Both of the non-natively folded states of PGK contain more regular secondary structure than the native one. The misfolding starts in both cases from the acid-unfolded state, in which the molecules are essentially more expanded than in other denatured states, e.g. those effected by temperature or guanidine hydrochloride. Copyright 1999 Academic Press. PMID: 10448049 [PubMed - indexed for MEDLINE] 680: J Bioenerg Biomembr 1999 Apr;31(2):95-104 The role of the amino-terminal beta-barrel domain of the alpha and beta subunits in the yeast F1-ATPase. Yao B, Mueller DM. Department of Biochemistry and Molecular Biology, The Chicago Medical School, Illinois 60064, USA. The crystal structure of mitochondrial F1-ATPase indicates that the alpha and beta subunits fold into a structure defined by three domains: the top beta-barrel domain, the middle nucleotide-binding domain, and the C-terminal alpha-helix bundle domain (Abrahams et al., 1994); Bianchet et al., 1998). The beta-barrel domains of the alpha and beta subunits form a crown structure at the top of F1, which was suggested to stabilize it (Abrahams et al. 1994). In this study, the role of the beta-barrel domain in the alpha and beta subunits of the yeast Saccharomyces cerevisiae F1, with regard to its folding and assembly, was investigated. The beta-barrel domains of yeast F1alpha and beta subunits were expressed individually and together in Escherichia coli. When expressed separately, the beta-barrel domain of the beta subunit formed a large aggregate structure, while the domain of the alpha subunit was predominately a monomer or dimer. However, coexpression of the beta-barrel domain of alpha subunit with the beta-barrel domain of beta subunit, greatly reduced the aggregation of the beta subunit domain. Furthermore, the two domains copurified in complexes with the major portion of the complex found in a small molecular weight form. These results indicate that the beta-barrel domain of the alpha and beta subunits interact specifically with each other and that these interactions prevent the aggregation of the beta-barrel domain of the beta subunit. These results mimic in vivo results and suggest that the interactions of the beta-barrel domains may be critical during the folding and assembly of F1. PMID: 10449236 [PubMed - indexed for MEDLINE] 681: Curr Genet 1999 Aug;36(1-2):13-20 Characterization of the role played by the RAD59 gene of Saccharomyces cerevisiae in ectopic recombination. Jablonovich Z, Liefshitz B, Steinlauf R, Kupiec M. Department of Molecular Microbiology and Biotechnology, The RAD52 group of genes in the yeast Saccharomyces cerevisiae controls the repair of DNA damage by a recombinational mechanism. Despite the growing evidence for physical and biochemical interactions between the proteins of this repair group, mutations in individual genes show very different effects on various types of recombination. The RAD59 gene encodes a protein with similarity to Rad52p which plays a role in the repair of damage caused by ionizing radiation. In the present study we have examined the role played by the Rad59 protein in mitotic ectopic recombination and analyzed the genetic interactions with other members of the repair group. We found that Rad59p plays a role in ectopic gene conversion that depends on the presence of Rad52p but is independent of the function of the RecA homologue Rad51p and of the Rad57 protein. The RAD59 gene product also participates in the RAD1-dependent pathway of recombination between direct repeats. We propose that Rad59p may act in a salvage mechanism that operates when the Rad51 filament is not functional. PMID: 10447590 [PubMed - indexed for MEDLINE] 682: J Biol Chem 1999 Aug 20;274(34):23794-801 Half of Saccharomyces cerevisiae carbamoyl phosphate synthetase produces and channels carbamoyl phosphate to the fused aspartate transcarbamoylase domain. Serre V, Guy H, Penverne B, Lux M, Rotgeri A, Evans D, Herve G. Laboratoire de Biochimie des Signaux Regulateurs Cellulaires et Moleculaires, UMR 7631 CNRS-Universite Pierre et Marie Curie, 96 Bd Raspail 75006 Paris, France. The first two steps of the de novo pyrimidine biosynthetic pathway in Saccharomyces cerevisiae are catalyzed by a 240-kDa bifunctional protein encoded by the ura2 locus. Although the constituent enzymes, carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase) function independently, there are interdomain interactions uniquely associated with the multifunctional protein. Both CPSase and ATCase are feedback inhibited by UTP. Moreover, the intermediate carbamoyl phosphate is channeled from the CPSase domain where it is synthesized to the ATCase domain where it is used in the synthesis of carbamoyl aspartate. To better understand these processes, a recombinant plasmid was constructed that encoded a protein lacking the amidotransferase domain and the amino half of the CPSase domain, a 100-kDa chain segment. The truncated complex consisted of the carboxyl half of the CPSase domain fused to the ATCase domain via the pDHO domain, an inactive dihydroorotase homologue that bridges the two functional domains in the native molecule. Not only was the "half CPSase" catalytically active, but it was regulated by UTP to the same extent as the parent molecule. In contrast, the ATCase domain was no longer sensitive to the nucleotide, suggesting that the two catalytic activities are controlled by distinct mechanisms. Most remarkably, isotope dilution and transient time measurements showed that the truncated complex channels carbamoyl phosphate. The overall CPSase-ATCase reaction is much less sensitive than the parent molecule to the ATCase bisubstrate analogue, N-phosphonacetyl-L-aspartate (PALA), providing evidence that the endogenously produced carbamoyl phosphate is sequestered and channeled to the ATCase active site. PMID: 10446140 [PubMed - indexed for MEDLINE] 683: J Enzyme Inhib 1999;14(3):175-92 Molecular modelling of lanosterol 14 alpha-demethylase (CYP51) from Saccharomyces cerevisiae via homology with CYP102, a unique bacterial cytochrome P450 isoform: quantitative structure-activity relationships (QSARs) within two related series of antifungal azole derivatives. Lewis DF, Wiseman A, Tarbit MH. School of Biological Sciences, University of Surrey, Guildford, UK. d.lewis@surrey.ac.uk The construction of a three-dimensional molecular model of the fungal form of cytochrome P450 (CYP51) from Saccharomyces cerevisiae, based on homology with the haemoprotein domain of CYP102 from Bacillus megaterium (a unique bacterial P450 of known crystal structure) is described. It is found that the endogenous substrate, lanosterol, can readily occupy the putative active site of the CYP51 model such that the known mono-oxygenation reaction, leading to C14-demethylation of lanosterol, is the preferred route of metabolism for this particular substrate. Key amino acid contacts within the CYP51 active site appear to orientate lanosterol for oxidative attack at the C14-methyl group, and the position of the substrate relative to the haem moiety is consistent with the phenyl-iron complexation studies reported by Tuck et al. [J. Biol. Chem., 267, 13175-13179 (1992)]. Typical azole inhibitors, such as ketoconazole, are able to fit the putative active site of CYP51 by a combination of haem ligation, hydrogen bonding, pi-pi stacking and hydrophobic interactions within the enzyme's haem environment. The mode of action of azole antifungals, as described by the modelling studies, is supported by quantitative structure-activity relationship (QSAR) analyses on two groups of structurally related fungal inhibitors. Moreover, the results of molecular electrostatic isopotential (EIP) energy calculations are compatible with the proposed mode of binding between azole antifungal agents and the putative active site of CYP51, although membrane interactions may also have a role in the antifungal activity of azole derivatives. PMID: 10445042 [PubMed - indexed for MEDLINE] 684: Genes Dev 1999 Aug 1;13(15):1983-93 Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center. Siatecka M, Reyes JL, Konarska MM. The Rockefeller University, New York, New York 10021, USA. A U5 snRNP protein, hPrp8, interacts closely with the GU dinucleotide at the 5' splice site (5'SS), forming a specific UV-inducible cross-link. To test if this physical contact between the 5'SS and the carboxy-terminal region of Prp8 reflects a functional recognition of the 5'SS during spliceosome assembly, we mutagenized the corresponding region of yeast Prp8 and screened the resulting mutants for suppression of 5'SS mutations in vivo. All of the isolated prp8 alleles not only suppress 5'SS but also 3'SS mutations, affecting the second catalytic step. Suppression of the 5'SS mutations by prp8 alleles was also tested in the presence of U1-7U snRNA, a predicted suppressor of the U+2A mutation. As expected, U1-7U efficiently suppresses prespliceosome formation, and the first, but not the second, step of U+2A pre-mRNA splicing. Independently, Prp8 functionally interacts with both splice sites at the later stage of splicing, affecting the efficiency of the second catalytic step. The striking proximity of two of the prp8 suppressor mutations to the site of the 5'SS:hPrp8 cross-link suggests that some protein:5'SS contacts made before the first step may be subsequently extended to accommodate the 3'SS for the second catalytic step. Together, these results strongly implicate Prp8 in specific interactions at the catalytic center of the spliceosome. PMID: 10444596 [PubMed - indexed for MEDLINE] 685: Genes Dev 1999 Aug 1;13(15):1970-82 Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome. Collins CA, Guthrie C. Graduate Group in Biophysics, University of California San Francisco (UCSF), San Francisco, California 94143-0448, USA. The highly conserved spliceosomal protein Prp8 is known to cross-link the critical sequences at both the 5' (GU) and 3' (YAG) ends of the intron. We have identified prp8 mutants with the remarkable property of suppressing exon ligation defects due to mutations in position 2 of the 5' GU, and all positions of the 3' YAG. The prp8 mutants also suppress mutations in position A51 of the critical ACAGAG motif in U6 snRNA, which has been observed previously to cross-link position 2 of the 5' GU. Other mutations in the 5' splice site, branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed. Notably, the suppressed residues are specifically conserved from yeast to man, and from U2- to U12-dependent spliceosomes. We propose that Prp8 participates in a previously unrecognized tertiary interaction between U6 snRNA and both the 5' and 3' ends of the intron. This model suggests a mechanism for positioning the 3' splice site for catalysis, and assigns a fundamental role for Prp8 in pre-mRNA splicing. PMID: 10444595 [PubMed - indexed for MEDLINE] 686: Dev Genet 1999;25(2):168-79 Enhancer of split [E(spl)(D)] is a gro-independent, hypermorphic mutation in Drosophila. Nagel AC, Yu Y, Preiss A. Universitat Hohenheim, Institut fur Genetik, Stuttgart, Germany. Enhancer of split [E(spl)] refers to a gene complex in Drosophila melanogaster, which contains a number of target genes of the Notch signaling pathway. The complex was originally identified by a dominant mutation E(spl)(D) that displays allele-specific interactions with a recessive mutation in the Notch locus called split (N(spl)). The spl phenotype is characterized by smaller eyes with irregularly spaced ommatidia, and it is strongly enhanced by E(spl)(D). This enhancement is correlated with a truncation of one of the E(spl) bHLH genes, m8, causing an increased stability of the mutant transcripts and an altered C-terminus in the mutant M8* protein. Concurrently, an insertion of a middle repetitive element in the adjacent groucho (gro) gene was observed. In this work, three different E(spl)(D) revertants (BE22, BE25, BX37), which have lost the ability to enhance N(spl) completely, were analyzed at the molecular level. In each case, the structure of the mutant M8* protein was affected, suggesting a specific involvement of the aberrant protein in the enhancement of the spl phenotype. This hypothesis is supported by the finding that a perfect phenocopy of spl enhancement can be achieved with hybrid constructs, where the altered C-terminus of M8* was fused to other E(spl) bHLH proteins. Thus, the ability to interact with N(spl) is not restricted to M8* but instead can be induced by an appropriate mutation in other E(spl) bHLH genes within the context of N(spl). In a N(spl) background, E(spl)(D) behaves like a hyperactive M8 mutation. However, the mutant M8* protein has lost the ability of binding to the corepressor Gro, which is an essential feature for normal E(spl) activity. Yet, other protein interactions, notably those with other bHLH proteins of either E(spl) or proneural family, are still observed. These findings suggest that the structural changes associated with the E(spl)(D) mutant protein are the primary cause for the phenotypic interactions with the recessive Notch mutation N(spl). Copyright 1999 Wiley-Liss, Inc. PMID: 10440851 [PubMed - indexed for MEDLINE] 687: Solid State Nucl Magn Reson 1999 Jul;14(2):117-36 Long-distance rotational echo double resonance measurements for the determination of secondary structure and conformational heterogeneity in peptides. Arshava B, Breslav M, Antohi O, Stark RE, Garbow JR, Becker JM, Naider F. Department of Chemistry, College of Staten Island and the Graduate School of the City University of New York, 10314, USA. The utility of rotational echo double resonance (REDOR) NMR spectroscopy for determining the conformations of linear peptides has been examined critically using a series of crystalline and amorphous samples. The focus of the present work was the evaluation of long-distance (> 5 A) interactions using 13C-15N dephasing. Detailed studies of specifically labeled melanostatin and synthetic analogs of the alpha-factor yeast mating hormone show that nitrogen-dephased, carbon-observe REDOR measurements are reliable for distances up to 6.0 A, and that dipolar interactions can be detected for distances up to 7 A. By contrast, nitrogen-observe REDOR gives reliable results only for distances shorter than 5.0 A. To measure distances accurately, REDOR data must be corrected for the effects of natural-abundance spins. These corrections are particularly important for measuring long distances, which are of the greatest value for determining peptide secondary structure. We have developed a spherical shell model for calculating the effect of these background spins. The REDOR studies also indicate that in a lyophilized powder, the tridecapeptide alpha-factor mating pheromone from Saccharomyces cerevisiae (WHWLQLKPGQPMY) probably exists as a distribution of different turn structures around the KPGQ region. This finding revises previous solid-state NMR studies on this peptide, which concluded alpha-factor assumes a distorted type-I beta-turn in the Pro-Gly central region of the molecule [J.R. Garbow, M. Breslav, O. Antohi, F. Naider, Biochemistry, 33 (1994) 10094]. PMID: 10437665 [PubMed - indexed for MEDLINE] 688: Mol Biol Cell 1999 Aug;10(8):2559-72 Multiple sex pheromones and receptors of a mushroom-producing fungus elicit mating in yeast. Fowler TJ, DeSimone SM, Mitton MF, Kurjan J, Raper CA. Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA. The mushroom-producing fungus Schizophyllum commune has thousands of mating types defined, in part, by numerous lipopeptide pheromones and their G protein-linked receptors. Compatible combinations of pheromones and receptors encoded by different mating types regulate a pathway of sexual development leading to mushroom formation and meiosis. A complex set of pheromone-receptor interactions maximizes the likelihood of outbreeding; for example, a single pheromone can activate more than one receptor and a single receptor can be activated by more than one pheromone. The current study demonstrates that the sex pheromones and receptors of Schizophyllum, when expressed in Saccharomyces cerevisiae, can substitute for endogenous pheromone and receptor and induce the yeast pheromone response pathway through the yeast G protein. Secretion of active Schizophyllum pheromone requires some, but not all, of the biosynthetic machinery used by the yeast lipopeptide pheromone a-factor. The specificity of interaction among pheromone-receptor pairs in Schizophyllum was reproduced in yeast, thus providing a powerful system for exploring molecular aspects of pheromone-receptor interactions for a class of seven-transmembrane-domain receptors common to a wide range of organisms. PMID: 10436012 [PubMed - indexed for MEDLINE] 689: Biochemistry 1999 Aug 3;38(31):9992-10003 Role of glutamate 91 in information transfer during substrate activation of yeast pyruvate decarboxylase. Li H, Furey W, Jordan F. Department of Chemistry and Biological Sciences and Program in Cellular and Molecular Biodynamics, Rutgers, the State University, Newark, New Jersey 07102, USA. Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at E91, located on the putative substrate activation pathway and linking the alpha and gamma domains of the enzyme. While C221 on the beta domain is the residue at which substrate activation is triggered [Baburina, I., et al. (1994) Biochemistry 33, 5630-5635; Baburina, I., et al. (1996) Biochemistry 35, 10249-10255], that information, via the substrate bound at C221, is transmitted to H92 on the alpha domain, across the domain divide from C221 [Baburina, I. , et al. (1998) Biochemistry 37, 1235-1244], thence to E91 on the alpha domain, and then on to W412 on the gamma domain [Li, H., and Jordan, F. (1999) Biochemistry 38, 10004-10012] and to the active site thiamin diphosphate located at the interface of the alpha and gamma domains [Arjunan, D., et al. (1996) J. Mol. Biol. 256, 590-600]. Substitution at E91 with Q, D, or A led to modest reductions in the specific activity (4-, 5-, and 30-fold), as well as in both the turnover number and the catalytic efficiency, in that order. Interestingly, the Hill coefficient was only slightly reduced for the E91D variant, but cooperativity was virtually abolished for the E91Q and E91A variants. The thermal stability of the variants was reduced in the following order: wild type > E91Q > E91D > E91A; circular dichroism and fluorescence experiments also demonstrated that the tertiary structure of the enzyme was affected by these substitutions. The variants could be purified as apoenzymes, demonstrating their impaired ability to bind thiamin diphosphate. Apparently, the charge at residue 91 is quite important for maintaining optimal cooperativity. To maintain strong domain-domain interactions, the length of the side chain at position 91 with hydrogen bonding potential to W412 is sufficient. PMID: 10433706 [PubMed - indexed for MEDLINE] 690: Biochem J 1999 Aug 15;342 ( Pt 1):27-32 Production in vitro by the cytochrome P450 CYP94A1 of major C18 cutin monomers and potential messengers in plant-pathogen interactions: enantioselectivity studies. Pinot F, Benveniste I, Salaun JP, Loreau O, Noel JP, Schreiber L, Durst F. Institut de Biologie Moleculaire des Plantes-CNRS UPR406, Departement d'Enzymologie Cellulaire et Moleculaire, 28 rue Goethe, F-67083 Strasbourg Cedex, France. franck.pinot@bota-ulp.u-strasbg.fr The major C(18) cutin monomers are 18-hydroxy-9,10-epoxystearic and 9,10,18-trihydroxystearic acids. These compounds are also known messengers in plant-pathogen interactions. We have previously shown that their common precursor 9,10-epoxystearic acid was formed by the epoxidation of oleic acid in Vicia sativa microsomes (Pinot, Salaun, Bosch, Lesot, Mioskowski and Durst (1992) Biochem. Biophys. Res. Commun. 184, 183-193). Here we determine the chirality of the epoxide produced as (9R,10S) and (9S,10R) in the ratio 90:10 respectively. We further show that microsomes from yeast expressing the cytochrome P450 CYP94A1 are capable of hydroxylating the methyl terminus of 9,10-epoxystearic and 9,10-dihydroxystearic acids in the presence of NADPH to form the corresponding 18-hydroxy derivatives. The reactions were not catalysed by microsomes from yeast transformed with a void plasmid or in absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid with microsomes of yeast expressing CYP94A1, the chirality of the residual epoxide was shifted to 66:34 in favour of the (9S,10R) enantiomer. Both enantiomers were incubated separately and V(max)/K(m) values of 16 and 3.42 ml/min per nmol of P450 for (9R, 10S) and (9S,10R) respectively were determined, demonstrating that CYP94A1 is enantioselective for the (9R,10S) enantiomer, which is preferentially formed in V. sativa microsomes. Compared with the epoxide, the diol 9,10-dihydroxystearic acid was a much poorer substrate for the omega-hydroxylase, with a measured V(max)/K(m) of 0.33 ml/min per nmol of P450. Our results indicate that the activity of CYP94A1 is strongly influenced by the stereochemistry of the 9, 10-epoxide and the nature of substituents on carbons 9 and 10, with V(max)/K(m) values for epoxide>>oleic acid>diol. PMID: 10432296 [PubMed - indexed for MEDLINE] 691: Proc Natl Acad Sci U S A 1999 Aug 3;96(16):9033-8 Binding of elongin A or a von Hippel-Lindau peptide stabilizes the structure of yeast elongin C. Botuyan MV, Koth CM, Mer G, Chakrabartty A, Conaway JW, Conaway RC, Edwards AM, Arrowsmith CH, Chazin WJ. Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, ON, Canada M5G 2M9. Elongin is a heterotrimeric transcription elongation factor composed of subunits A, B, and C in mammals. Elongin A and C are F-box-containing and SKP1 homologue proteins, respectively, and are therefore of interest for their potential roles in cell cycle-dependent proteolysis. Mammalian elongin C interacts with both elongin A and elongin B, as well as with the von Hippel-Lindau tumor suppressor protein VHL. To investigate the corresponding interactions in yeast, we have utilized NMR spectroscopy combined with ultracentrifugal sedimentation experiments to examine complexes of yeast elongin C (Elc1) with yeast elongin A (Ela1) and two peptides from homologous regions of Ela1 and human VHL. Elc1 alone is a homotetramer composed of subunits with a structured N-terminal region and a dynamically unstable C-terminal region. Binding of a peptide fragment of the Elc1-interaction domain of Ela1 or with a homologous peptide from VHL promotes folding of the C-terminal region of Elc1 into two regular helical structures and dissociates Elc1 into homodimers. Moreover, analysis of the complex of Elc1 with the full Elc1-interaction domain of Ela1 reveals that the Elc1 homodimer is dissociated to preferentially form an Ela1/Elc1 heterodimer. Thus, elongin C is found to oligomerize in solution and to undergo significant structural rearrangements upon binding of two different partner proteins. These results suggest a structural basis for the interaction of an F-box-containing protein with a SKP1 homologue and the modulation of this interaction by the tumor suppressor VHL. PMID: 10430890 [PubMed - indexed for MEDLINE] 692: Proc Natl Acad Sci U S A 1999 Aug 3;96(16):8890-4 Domain structure and lipid interaction of recombinant yeast Tim44. Weiss C, Oppliger W, Vergeres G, Demel R, Jeno P, Horst M, de Kruijff B, Schatz G, Azem A. Department of Biochemistry, Tel-Aviv University, Tel-Aviv 69978, Israel. Tim44 is an essential component of the machinery that mediates the translocation of nuclear-encoded proteins across the mitochondrial inner membrane. It functions as a membrane anchor for the ATP-driven protein import motor whose other subunits are the mitochondrial 70-kDa heat-shock protein (mhsp70) and its nucleotide exchange factor, mGrpE. To understand how this motor is anchored to the inner membrane, we have overexpressed Tim44 in Escherichia coli and studied the properties of the pure protein and its interaction with model lipid membranes. Limited proteolysis and analytical ultracentrifugation indicate that Tim44 is an elongated monomer with a stably folded C-terminal domain. The protein binds strongly to liposomes composed of phosphatidylcholine and cardiolipin but only weakly to liposomes containing phosphatidylcholine alone. Studies with phospholipid monolayers suggest that Tim44 binds to phospholipids of the mitochondrial inner membrane both by electrostatic interactions and by penetrating the polar head group region. PMID: 10430866 [PubMed - indexed for MEDLINE] 693: Genetics 1999 Aug;152(4):1543-56 Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers. Jones S, Jedd G, Kahn RA, Franzusoff A, Bartolini F, Segev N. Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois 60637, USA. Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport. While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment. Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well. Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation. Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases. Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function. Sec7p itself has an important role in the yeast secretory pathway. However, its mechanism of action is not yet understood. We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes. Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt GTPases. The Sec7-domain of Sec7p is sufficient for this activity. Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a fungal metabolite that inhibits some of the Arf-GEFs, indicating that this domain is a target for BFA. These results demonstrate that the ability to act as Arf-GEFs is a general property of all Sec7-domain proteins in yeast. The genetic interactions observed between Arf GEFs and Ypt GTPases suggest the existence of a Ypt-Arf GTPase cascade in the secretory pathway. PMID: 10430582 [PubMed - indexed for MEDLINE] 694: Eur J Biochem 1999 Jul;263(1):118-27 ATP-regulation of cytochrome oxidase in yeast mitochondria: role of subunit VIa. Beauvoit B, Bunoust O, Guerin B, Rigoulet M. Institut de Biochimie et Genetique Cellulaires du CNRS, Universite Victor Segalen, Bordeaux, France. bertrand.beauvoit@ibgc.u-bordeaux2.fr The role of the nuclear-encoded subunit VIa in the regulation of cytochrome oxidase by ATP was investigated in isolated yeast mitochondria. As the subunit VIa-null strain possesses a fully active and assembled cytochrome oxidase, multiple ATP-regulating sites were characterized with respect to their location and their kinetic effect: (a) intra-mitochondrial ATP inhibited the complex IV activity of the null strain, whereas the prevailing effect of ATP on the wild-type strain, at low ionic strength, was activation on the cytosolic side of complex IV, mediated by subunit VIa. However, at physiological ionic strength (i.e. approximately 200 mM), activation by ATP was absent but inhibition was not impaired; (b) in ethanol-respiring mitochondria, when the electron flux was modulated using a protonophoric uncoupler, the redox state of aa3 cytochromes varied with respect to activation (wild-type) or inhibition (null-mutant) of the cytochrome oxidase by ATP; (c) consequently, the control coefficient of cytochrome oxidase on respiratory flux, decreased (wild-type) or increased (null-mutant) in the presence of ATP; (d) considering electron transport from cytochrome c to oxygen, the response of cytochrome oxidase to its thermodynamic driving force was increased by ATP for the wild-type but not for the mutant subunit. Taken together, these findings indicate that at physiological concentration, ATP regulates yeast cytochrome oxidase via subunit-mediated interactions on both sides of the inner membrane, thus subtly tuning the thermodynamic and kinetic control of respiration. This study opens up new prospects for understanding the feedback regulation of the respiratory chain by ATP. PMID: 10429195 [PubMed - indexed for MEDLINE] 695: Plant J 1999 Jun;18(5):541-50 Arabidopsis thaliana proteins related to the yeast SIP and SNF4 interact with AKINalpha1, an SNF1-like protein kinase. Bouly JP, Gissot L, Lessard P, Kreis M, Thomas M. Laboratoire de Biologie du Dveloppement des Plantes, Institut de Biotechnologie des Plantes, UMR CNRS 8618, Universite de Paris-Sud, Orsay, France. AKINalpha1, a Ser/Thr kinase from Arabidopsis thaliana belongs to the highly conserved SNF1 family of protein kinases in eukaryotes. Recent data suggest that the plant SNF1-related kinases (SnRK1 family) are key enzymes implicated in the regulation of carbohydrate and lipid metabolism. In Saccharomyces cerevisiae and mammals, the SNF1 and AMPKalpha protein kinases interact with two other families of proteins, namely SNF4/AMPKgamma and SIP1/SIP2/GAL83/AMPKbeta, to form active heterotrimeric complexes. In this paper, we describe the characterisation of three novel cDNAs. AKINbeta1 and AKINbeta2 encode proteins similar to SIP1, SIP2 and GAL83 and AKINgamma codes for a protein showing similarity with SNF4. Using the two-hybrid system, specific interactions have been shown between A. thaliana AKINbeta1/beta2, AKINgamma and AKINgamma as well as between the A. thaliana and S. cerevisiae subunits. Interestingly, AKINbeta1, AKINbeta2 and AKINgamma mRNAs accumulate differentially in A. thaliana tissues and are modulated during development and under different growth conditions. These data suggest the presence in higher plants of a conserved heterotrimeric complex. Moreover, the differential transcription of different non-catalytic subunits can constitute a first level of regulation of the SNF1-like complex in plants. PMID: 10417704 [PubMed - indexed for MEDLINE] 696: Biochemistry 1999 Jul 20;38(29):9242-53 Ubiquitin binding interface mapping on yeast ubiquitin hydrolase by NMR chemical shift perturbation. Rajesh S, Sakamoto T, Iwamoto-Sugai M, Shibata T, Kohno T, Ito Y. Laboratory of Cellular and Molecular Biology, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan. The interaction between the 26 kDa yeast ubiquitin hydrolase (YUH1), involved in maintaining the monomeric ubiquitin pool in cells, and the 8.5 kDa yeast ubiquitin protein has been studied by heteronuclear multidimensional NMR spectroscopy. Chemical shift perturbation of backbone (1)H(N), (15)N, and (13)C(alpha) resonances of YUH1, in a YUH1-ubiquitin mixture and in a 35 kDa covalent complex with ubiquitin (a stable analogue of the tetrahedral reaction intermediate), was employed to identify the ubiquitin binding interface of YUH1. This interface mapped on the secondary structure of YUH1 suggests a wide area of contact for ubiquitin, encompassing the N-terminus, alpha1, alpha4, beta2, beta3, and beta6, coincident with the high specificity of YUH1 for ubiquitin. The presence of several hydrophobic clusters in the ubiquitin binding interface of YUH1 suggests that hydrophobic interactions are equally important as ionic interactions in contacting ubiquitin. The residues in the binding interface exhibit a high percentage of homology among the members of the ubiquitin C-terminal hydrolase family, indicating the well-conserved nature of the ubiquitin binding interface reported in this study. The secondary structure of YUH1, from our NMR studies, was similar to the recently determined structure of its human homologue ubiquitin C-terminal hydrolase L3 (UCH-L3), except for the absence of the helix H3 of UCH-L3. This region in YUH1 (helix H3 of UCH-L3) was least perturbed upon ubiquitin binding. Therefore, the binding interface was mapped onto the corresponding residues in the UCH-L3 crystal structure. A model for ubiquitin binding to YUH1 is proposed, in which a good correlation was observed for the lateral binding of ubiquitin to UCH-L3 (YUH1), stabilized by the electrostatic and hydrophobic interactions. PMID: 10413498 [PubMed - indexed for MEDLINE] 697: Biochemistry 1999 Jul 20;38(29):9198-208 Glutamic acid 472 and lysine 480 of the sodium pump alpha 1 subunit are essential for activity. Their conservation in pyrophosphatases suggests their involvement in recognition of ATP phosphates. Scheiner-Bobis G, Schreiber S. Institut fur Biochemie und Endokrinologie, Fachbereich Veterinarmedizin, Justus-Liebig-Universitat Giessen, Germany. Georgios.Scheiner-Bobis@vetmed.uni-giessen.de P-type ATPases such as the Na+,K+-ATPase (sodium pump) hydrolyze ATP to pump ions through biological membranes against their electrochemical gradients. The mechanisms that couple ATP hydrolysis to the vectorial ion transport are not yet understood, but unveiling structures that participate in ATP binding and in the formation of the ionophore might help to gain insight into this process. Looking at the alpha- and beta-phosphates of ATP as a pyrophosphate molecule, we found that peptides highly conserved among all soluble inorganic pyrophosphatases are also present in ion-transporting ATPases. Included therein are Glu48 and Lys56 of the Saccharomyces cerevisiae pyrophosphatase (SCE1-PPase) that are essential for the activity of this enzyme and have been shown in crystallographic analysis to interact with phosphate molecules. To test the hypothesis that equivalent amino acids are also essential for the activity of ion-transporting ATPases, Glu472 and Lys480 of the sodium pump alpha 1 subunit corresponding to Glu48 and Lys56 of SCE1-PPase were mutated to various amino acids. Mutants of the sodium pump alpha1 subunit were expressed in yeast and analyzed for their ATPase activity and their ability to bind ouabain in the presence of either ATP, Mg2+, and Na+ or phosphate and Mg2+. All four mutants investigated, Glu472Ala, Glu472Asp, Lys480Ala, and Lys480Arg, display only a fraction of the ATPase activity obtained with the wild-type enzyme. The same applies with respect to their ability to bind ouabain, where maximum ouabain binding to the mutants accounts for only about 10% of the binding obtained with the wild-type enzyme. On the basis of our results, we conclude that Glu472 and Lys480 are essential for the activity of the sodium pump. Their function is probably to arrest the alpha- and beta-phosphate groups of ATP in a proper position prior to hydrolysis of the gamma-phosphate group. The identification of these amino acids as essential components of the ATP-recognizing mechanism of the pump has resulted in a testable hypothesis for the initial interactions of the sodium pump, and possibly of other P-type ATPases, with ATP. PMID: 10413494 [PubMed - indexed for MEDLINE] 698: Biochemistry 1999 Jul 13;38(28):8961-71 Spt16 and Pob3 of Saccharomyces cerevisiae form an essential, abundant heterodimer that is nuclear, chromatin-associated, and copurifies with DNA polymerase alpha. Wittmeyer J, Joss L, Formosa T. Department of Biochemistry, University of Utah, Salt Lake City 84132, USA. Previously we showed that the yeast proteins Spt16 (Cdc68) and Pob3 are physically associated, and interact physically and genetically with the catalytic subunit of DNA polymerase alpha, Pol1 [Wittmeyer and Formosa (1997) Mol. Cell. Biol. 17, 4178-4190]. Here we show that purified Spt16 and Pob3 form a stable, abundant, elongated heterodimer and provide evidence that this is the functional form of these proteins. Genetic interactions between mutations in SPT16 and POB3 support the importance of the Spt16-Pob3 interaction in vivo. Spt16, Pob3, and Pol1 proteins were all found to localize to the nucleus in S. cerevisiae. A portion of the total cellular Spt16-Pob3 was found to be chromatin-associated, consistent with the proposed roles in modulating chromatin function. Some of the Spt16-Pob3 complex was found to copurify with the yeast DNA polymerase alpha/primase complex, further supporting a connection between Spt16-Pob3 and DNA replication. PMID: 10413469 [PubMed - indexed for MEDLINE] 699: Proc Natl Acad Sci U S A 1999 Jul 20;96(15):8567-72 "Mutagenesis" by peptide aptamers identifies genetic network members and pathway connections. Geyer CR, Colman-Lerner A, Brent R. The Molecular Sciences Institute, 2168 Shattuck Avenue, Berkeley, CA 94704, USA. We selected peptide aptamers from combinatorial libraries that disrupted cell-cycle arrest caused by mating pheromone in yeast. We used these aptamers as baits in two-hybrid hunts to identify genes involved in cell-cycle arrest. These experiments identified genes known to function in the pathway, as well as a protein kinase, the CBK1 product, whose function was not known. We used a modified two-hybrid system to identify specific interactions disrupted by these aptamers. These experiments demonstrate a means to perform "genetics" on the protein complement of a cell without altering its genetic material. Peptide aptamers can be identified that disrupt a process. These aptamers can then be used as affinity reagents to identify individual proteins and protein interactions needed for the process. Forward genetic analysis with peptide aptamer "mutagens" should be particularly useful in elucidating genetic networks in organisms and processes for which classical genetics is not feasible. PMID: 10411916 [PubMed - indexed for MEDLINE] 700: Mol Cell Biol 1999 Aug;19(8):5417-28 Hec1p, an evolutionarily conserved coiled-coil protein, modulates chromosome segregation through interaction with SMC proteins. Zheng L, Chen Y, Lee WH. Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center San Antonio, San Antonio, Texas 78245, USA. hsHec1p, a Homo sapiens coiled-coil-enriched protein, plays an important role in M-phase progression in mammalian cells. A Saccharomyces cerevisiae protein, identical to Tid3p/Ndc80p and here designated scHec1p, has similarities in structure and biological function to hsHec1p. Budding yeast cells deleted in the scHEC1/NDC80 allele are not viable, but this lethal phenotype can be rescued by hsHEC1 under control of the endogenous scHEC1 promoter. At the nonpermissive temperature, significant mitotic delay, chromosomal missegregation, and decreased viability were observed in yeast cells with temperature-sensitive (ts) alleles of hsHEC1. In the hshec1-113 ts mutant, we found a single-point mutation changing Trp395 to a stop codon, which resulted in the expression of a C-terminally truncated 45-kDa protein. The binding of this mutated protein, hshec1-113p, to five identified hsHec1p-associated proteins was unchanged, while its binding to human SMC1 protein and yeast Smc1p was ts. Hec1p also interacts with Smc2p, and the binding of the mutated hshec1-113p to Smc2p was not ts. Overexpression of either hsHEC1 or scHEC1 suppressed the lethal phenotype of smc1-2 and smc2-6 at nonpermissive temperatures, suggesting that the interactions between Hec1p and Smc1p and -2p are biologically significant. These results suggest that Hec1 proteins play a critical role in modulating chromosomal segregation, in part, through their interactions with SMC proteins. PMID: 10409732 [PubMed - indexed for MEDLINE] 701: Mol Cell Biol 1999 Aug;19(8):5279-88 Chromatin opening and transactivator potentiation by RAP1 in Saccharomyces cerevisiae. Yu L, Morse RH. Molecular Genetics Program, Wadsworth Center, New York State Department of Health, and State University of New York School of Public Health, Albany, New York 12201-2002, USA. Transcriptional activators function in vivo via binding sites that may be packaged into chromatin. Here we show that whereas the transcriptional activator GAL4 is strongly able to perturb chromatin structure via a nucleosomal binding site in yeast, GCN4 does so poorly. Correspondingly, GCN4 requires assistance from an accessory protein, RAP1, for activation of the HIS4 promoter, whereas GAL4 does not. The requirement for RAP1 for GCN4-mediated HIS4 activation is dictated by the DNA-binding domain of GCN4 and not the activation domain, suggesting that RAP1 assists GCN4 in gaining access to its binding site. Consistent with this, overexpression of GCN4 partially alleviates the requirement for RAP1, whereas HIS4 activation via a weak GAL4 binding site requires RAP1. RAP1 is extremely effective at interfering with positioning of a nucleosome containing its binding site, consistent with a role in opening chromatin at the HIS4 promoter. Furthermore, increasing the spacing between binding sites for RAP1 and GCN4 by 5 or 10 bp does not impair HIS4 activation, indicating that cooperative protein-protein interactions are not involved in transcriptional facilitation by RAP1. We conclude that an important role of RAP1 is to assist activator binding by opening chromatin. PMID: 10409719 [PubMed - indexed for MEDLINE] 702: J Biol Chem 1999 Jul 23;274(30):21297-304 The Cap-binding protein eIF4E promotes folding of a functional domain of yeast translation initiation factor eIF4G1. Hershey PE, McWhirter SM, Gross JD, Wagner G, Alber T, Sachs AB. Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA. The association of eucaryotic translation initiation factor eIF4G with the cap-binding protein eIF4E establishes a critical link between the mRNA and the ribosome during translation initiation. This association requires a conserved seven amino acid peptide within eIF4G that binds to eIF4E. Here we report that a 98-amino acid fragment of S. cerevisiae eIF4G1 that contains this eIF4E binding peptide undergoes an unfolded to folded transition upon binding to eIF4E. The folding of the eIF4G1 domain was evidenced by the eIF4E-dependent changes in its protease sensitivity and (1)H-(15)N HSQC NMR spectrum. Analysis of a series of charge-to-alanine mutations throughout the essential 55.4-kDa core of yeast eIF4G1 also revealed substitutions within this 98-amino acid region that led to reduced eIF4E binding in vivo and in vitro. These data suggest that the association of yeast eIF4E with eIF4G1 leads to the formation of a structured domain within eIF4G1 that could serve as a specific site for interactions with other components of the translational apparatus. They also suggest that the stability of the native eIF4E-eIF4G complex is determined by amino acid residues outside of the conserved seven-residue consensus sequence. PMID: 10409688 [PubMed - indexed for MEDLINE] 703: Biotechniques 1999 Jul;27(1):86-8, 92-4 Application of the green fluorescent protein as a reporter for Ace1-based, two-hybrid studies. Mayer G, Launhardt H, Munder T. Hans-Knoll-Institut fur Naturstoff-Forschung e.V. Jena, Germany. The two-hybrid system in Saccharomyces cerevisiae is a genetic approach for the detection of of protein-protein interactions in vivo. This technology relies on the the activity of separated DNA-binding and transactivation domains of specific transcription factors to reconstitute an active transcription factor complex if interacting proteins are fused to these domains. Interactions are consequently detected through the activity of reporter genes. The two-hybrid technology has been successfully applied for the determination of interactions between numerous proteins of several organisms. Conventional reporter systems, such as the beta-galacatosidase from Escherichia coli, suffer from a variety of drawbacks, including the requirement for external substrates. In this report, we describe an alternative version of the two hybrid system using the combined advantages of the copper-inducible transcription factor Acel together with the yeast metallothionein gene CUP1 and the green fluorescence protein from aquatic invertebrates as reporters. This technique allows the copper-dependent monitoring of protein-protein interactions in living yeast cells. PMID: 10407670 [PubMed - indexed for MEDLINE] 704: Yeast 1999 Jul;15(10B):963-72 Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Knop M, Siegers K, Pereira G, Zachariae W, Winsor B, Nasmyth K, Schiebel E. The Beatson Institute for Cancer Research, CRC Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, U.K. Epitope tagging of proteins as a strategy for the analysis of function, interactions and the subcellular distribution of proteins has become widely used. In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR-based strategy to introduce epitope tags to chromosomal loci (Wach et al., 1994). To further employ the power of this strategy, a variety of novel tags was constructed. These tags were combined with different selectable marker genes, resulting in PCR amplificable modules. Only one set of primers is required for the amplification of any module. Furthermore, convenient laboratory techniques are described that facilitate the genetic manipulations of yeast strains, as well as the analysis of the epitope-tagged proteins. Copyright 1999 John Wiley & Sons, Ltd. PMID: 10407276 [PubMed - indexed for MEDLINE] 705: Yeast 1999 Jul;15(10A):865-72 A systematic nomenclature for new translation initiation factor genes from S. pombe and other fungi. Linder P, Vornlocher HP, Hershey JW, McCarthy JE. Departement de Biochimie Medicale, Centre Medical Universitaire, 1, rue Michel Servet 1211 Geneve 4, Switzerland. Eukaryotic translation initiation factors and their corresponding genes have been characterized using biochemical and genetic methods from a variety of different organisms. The designations of the factors relate to their apparent roles in the biochemical process. Many gene names indicate genetic interactions with other genes or the functional attributes used to identify them. On the other hand, progress in systematic sequencing of the genomes of organisms like Saccharomyces cerevisiae and Schizosaccharomyces pombe has revealed many genes homologous to known translation initiation factor genes. The genes defined by the systematic sequencing approach are assigned numerical designations completely unrelated to their biological function. So far there have been publications on only three genes encoding translation initiation factors from Schizosaccharomyces pombe. We therefore see this an an ideal opportunity to propose a systematic and logical nomenclature for genes encoding translation initiation factor genes that can be applied to all further genes of this type that are characterized in this fission yeast. Copyright 1999 John Wiley & Sons, Ltd. PMID: 10407266 [PubMed - indexed for MEDLINE] 706: EMBO J 1999 Jul 15;18(14):4068-75 Repressor binding to a dorsal regulatory site traps human eIF4E in a high cap-affinity state. Ptushkina M, von der Haar T, Karim MM, Hughes JM, McCarthy JE. Posttranscriptional Control Group, Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester M60 1QD, UK. Eukaryotic translation initiation involves recognition of the 5' end of cellular mRNA by the cap-binding complex known as eukaryotic initiation factor 4F (eIF4F). Initiation is a key point of regulation in gene expression in response to mechanisms mediated by signal transduction pathways. We have investigated the molecular interactions underlying inhibition of human eIF4E function by regulatable repressors called 4E-binding proteins (4E-BPs). Two essential components of eIF4F are the cap-binding protein eIF4E, and eIF4G, a multi-functional protein that binds both eIF4E and other essential eIFs. We show that the 4E-BPs 1 and 2 block the interaction between eIF4G and eIF4E by competing for binding to a dorsal site on eIF4E. Remarkably, binding of the 4E-BPs at this dorsal site enhances cap-binding via the ventral cap-binding slot, thus trapping eIF4E in inactive complexes with high affinity for capped mRNA. The binding contacts and affinities for the interactions between 4E-BP1/2 and eIF4E are distinct (estimated K(d) values of 10(-8) and 3x10(-9) for 4E-BP1 and 2, respectively), and the differences in these properties are determined by three amino acids within an otherwise conserved motif. These data provide a quantitative framework for a new molecular model of translational regulation. PMID: 10406811 [PubMed - indexed for MEDLINE] 707: Biochem Biophys Res Commun 1999 Jul 14;260(3):799-805 Oligomerized Ced-4 kills budding yeast through a caspase-independent mechanism. Tao W, Walke DW, Morgan JI. Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, Tennessee, 38105-2794, USA. In Caenorhabdtis elegans, Ced-3, Ced-4, and Ced-9 are components of a cell suicide program. Ced-4 facilitates the proteolytic activation of the caspase, Ced-3, while Ced-9 opposes Ced-3/Ced-4 killing. To examine the interactions among these proteins they were expressed in Saccharomyces cerevisiae. Ced-3 and Ced-4 were lethal when expressed alone, revealing an intrinsic Ced-4 killing activity. Coexpression of Ced-9 blocked Ced-3- and Ced-4-induced killing, showing Ced-9 can independently antagonize the action of both proteins. Ced-3- but not Ced-4-toxicity was attenuated by coexpression of the caspase inhibitors, CrmA and p35. Thus, besides its Ced-3- and Ced-9-dependent action in C. elegans, Ced-4 has an additional Ced-9-dependent, Ced-3-independent killing mechanism in yeast. Two-hybrid analysis confirmed that Ced-4 formed heteromers with Ced-9. In addition, Ced-4 formed homomers and mutation of its nucleoside triphosphate binding motif eliminated both homomerization and cell killing. We suggest the caspase-independent lethality of Ced-4 in yeast is mediated by a Ced-4 homomer. Copyright 1999 Academic Press. PMID: 10403845 [PubMed - indexed for MEDLINE] 708: J Biol Chem 1999 Jul 16;274(29):20235-43 A small region in HMG I(Y) is critical for cooperation with NF-kappaB on DNA. Zhang XM, Verdine GL. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA. The high mobility group HMG I(Y) protein has been reported to promote the expression of several NF-kappaB-dependent genes by enhancing the binding of NF-kappaB to DNA. The molecular origins of cooperativity in the binding of NF-kappaB and HMG I(Y) to DNA are not well understood. Here we have examined the determinants of specificity in the binding of HMG I(Y), both alone and in cooperation with NF-kappaB, to two different DNA elements, PRDII from the interferon-beta enhancer and IgkappaB from the immunoglobulin kappa light chain enhancer. Of particular interest was the influence of a flanking AT-rich sequence on binding by HMG I(Y). Utilizing yeast one-hybrid screening assays together with alanine-scanning mutagenesis, we have identified mutations of residues in HMG I(Y) that decrease cooperative binding of NF-kappaB to PRDII and IgkappaB sites. These same mutations similarly decreased the binding of HMG I(Y) alone to DNA, and paradoxically, decreased the strength of protein-protein interactions between HMG I(Y) and NF-kappaB. Of the three tandemly repeated basic regions that represent putative DNA-binding motifs in HMG I(Y), the residues within the second repeat are most important for recognition of core NF-kappaB sites, whereas the second and third repeats both appear to be involved in binding to sites that are flanked by AT-rich sequences. Overall, the second repeat of HMG I(Y) is primarily responsible for the stimulatory effect of this protein on the binding of NF-kappaB to PRDII and IgkappaB elements. PMID: 10400641 [PubMed - indexed for MEDLINE] 709: Yeast 1999 Jun 30;15(9):721-40 Specific negative effects resulting from elevated levels of the recombinational repair protein Rad54p in Saccharomyces cerevisiae. Clever B, Schmuckli-Maurer J, Sigrist M, Glassner BJ, Heyer WD. Institute for General Microbiology, University of Bern, Bern, Switzerland. RAD54 is an important gene in the RAD52 group that controls recombinational repair of DNA damage in Saccharomyces cerevisiae. Rad54p is a DNA-dependent ATPase and shares seven conserved sequence motifs with proteins of the Swi2p/Snf2p family. Genetic analysis of mutations in motif IA, the putative ATP-binding fold of Rad54p, demonstrated the functional importance of this motif. Overexpression of these mutant proteins resulted in strong, dominant-negative effects on cell survival. High levels of full-length wild-type Rad54p or specific parts of Rad54p also resulted in negative effects, dependent on the ploidy of the host cell. This differential effect was not under a/alpha mating-type control. Deletion of the RAD54 gene led to a small but significant increase in the mutation rate. However, the negative overexpression effects in haploid cells could not be explained by an accumulation of (recessive) lethal mutations. All negative overexpression effects were found to be enhanced under genotoxic stress. We suggest that the negative overexpression effects are the result of unbalanced protein-protein interactions, indicating that Rad54p is involved in multiple interactions, dependent on the physiological situation. Diploid wild-type cells contained an estimated 7000 Rad54p molecules/cell, whereas haploid cells about 3500/cell. Rad54p levels were highest in actively growing cells compared to stationary phase cells. Rad54 protein levels were found to be elevated after DNA damage. Copyright 1999 John Wiley & Sons, Ltd. PMID: 10398342 [PubMed - indexed for MEDLINE] 710: Mol Gen Genet 1999 Jun;261(4-5):788-95 Genetic evidence for interactions between yeast importin alpha (Srp1p) and its nuclear export receptor, Cse1p. Schroeder AJ, Chen XH, Xiao Z, Fitzgerald-Hayes M. Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003, USA. The yeast Srp1p protein functions as an import receptor for proteins bearing basic nuclear localization signals. Cse1p, the yeast homolog of mammalian CAS, recycles Srp1p back to the cytoplasm after import substrates have been released into the nucleoplasm. In this report we describe genetic interactions between SRP1 and CSE1. Results from genetic suppression and synthetic lethality studies demonstrate that these gene products interact to ensure accurate chromosome segregation. We also describe new mutant alleles of CSE1 and analyze a new temperature-sensitive allele of CSE1, cse1-2. This allele causes high levels of chromosome missegregation and cell cycle arrest during mitosis at the nonpermissive temperature. PMID: 10394916 [PubMed - indexed for MEDLINE] 711: Proc Natl Acad Sci U S A 1999 Jul 6;96(14):7791-6 Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription. Bieniasz PD, Grdina TA, Bogerd HP, Cullen BR. Howard Hughes Medical Institute and Department of Genetics, Box 3025, Duke University Medical Center, Durham, NC 27710, USA. Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR) promoter element by the viral Tat protein is an essential step in the HIV-1 life cycle. Tat function is mediated by the TAR RNA target element encoded within the LTR and is known to require the recruitment of a complex consisting of Tat and the cyclin T1 (CycT1) component of positive transcription elongation factor b (P-TEFb) to TAR. Here, we demonstrate that both TAR and Tat become entirely dispensable for activation of the HIV-1 LTR promoter when CycT1/P-TEFb is artificially recruited to a heterologous promoter proximal RNA target. The level of activation observed was indistinguishable from the level induced by Tat and was neither inhibited nor increased when Tat was expressed in trans. Activation by artificially recruited CycT1 depended on the ability to bind the CDK9 component of P-TEFb. In contrast, although binding to both Tat and TAR was essential for the ability of CycT1 to act as a Tat cofactor, these interactions became dispensable when CycT1 was directly recruited to the LTR. Importantly, activation of the LTR both by Tat and by directly recruited CycT1 was found to be at the level of transcription elongation. Together, these data demonstrate that recruitment of CycT1/P-TEFb to the HIV-1 LTR is fully sufficient to activate this promoter element and imply that the sole role of the Tat/TAR axis in viral transcription is to permit the recruitment of CycT1/P-TEFb. PMID: 10393900 [PubMed - indexed for MEDLINE] 712: J Biol Chem 1999 Jul 9;274(28):19617-22 N-terminal tail export from the mitochondrial matrix. Adherence to the prokaryotic "positive-inside" rule of membrane protein topology. Rojo EE, Guiard B, Neupert W, Stuart RA. Institut fur Physiologische Chemie der Universitat Munchen, Goethestrasse 33, 80336 Munchen, Germany. Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N- and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for sec-independent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery. PMID: 10391898 [PubMed - indexed for MEDLINE] 713: Genetics 1999 Jul;152(3):881-93 In vivo analysis of the domains of yeast Rvs167p suggests Rvs167p function is mediated through multiple protein interactions. Colwill K, Field D, Moore L, Friesen J, Andrews B. Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Morphological changes during cell division in the yeast Saccharomyces cerevisiae are controlled by cell-cycle regulators. The Pcl-Pho85p kinase complex has been implicated in the regulation of the actin cytoskeleton at least in part through Rvs167p. Rvs167p consists of three domains called BAR, GPA, and SH3. Using a two-hybrid assay, we demonstrated that each region of Rvs167p participates in protein-protein interactions: the BAR domain bound the BAR domain of another Rvs167p protein and that of Rvs161p, the GPA region bound Pcl2p, and the SH3 domain bound Abp1p. We identified Rvs167p as a Las17p/Bee1p-interacting protein in a two-hybrid screen and showed that Las17p/Bee1p bound the SH3 domain of Rvs167p. We tested the extent to which the Rvs167p protein domains rescued phenotypes associated with deletion of RVS167: salt sensitivity, random budding, and endocytosis and sporulation defects. The BAR domain was sufficient for full or partial rescue of all rvs167 mutant phenotypes tested but not required for the sporulation defect for which the SH3 domain was also sufficient. Overexpression of Rvs167p inhibits cell growth. The BAR domain was essential for this inhibition and the SH3 domain had only a minor effect. Rvs167p may link the cell cycle regulator Pcl-Pho85p kinase and the actin cytoskeleton. We propose that Rvs167p is activated by phosphorylation in its GPA region by the Pcl-Pho85p kinase. Upon activation, Rvs167p enters a multiprotein complex, making critical contacts in its BAR domain and redundant or minor contacts with its SH3 domain. PMID: 10388809 [PubMed - indexed for MEDLINE] 714: J Mol Biol 1999 Jul 2;290(1):331-45 Equilibrium folding properties of the yeast prion protein determinant Ure2. Perrett S, Freeman SJ, Butler PJ, Fersht AR. Centre for Protein Engineering, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK. The yeast non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The [URE3] phenotype is equivalent to loss of function of Ure2, a protein involved in regulation of nitrogen metabolism. The prion-like behaviour of Ure2 in vivo is dependent on the first 65 amino acid residues of its N-terminal region which contains a highly repetitive sequence rich in asparagine. This region has been termed the prion-determining domain (PrD). Removal of as little as residues 2-20 of the protein is sufficient to prevent occurrence of the [URE3] phenotype. Removal of the PrD does not affect the regulatory activity of Ure2. The C-terminal portion of the protein has homology to glutathione S -transferases, which are dimeric proteins. We have produced the Ure2 protein to high yield in Escherichia coli from a synthetic gene. The recombinant purified protein is shown to be a dimer. The stability, folding and oligomeric state of Ure2 and a series of N-terminally truncated or deleted variants were studied and compared. The stability of Ure2, DeltaGD-N, H2O, determined by chemical denaturation and monitored by fluorescence, is 12.1(+/-0.4) kcal mol-1at 25 degrees C and pH 8.4. A range of structural probes show a single, coincident unfolding transition, which is invariant over a 550-fold change in protein concentration. The stability is the same within error for Ure2 variants lacking all or part of the prion-determining domain. The data indicate that in the folded protein the PrD is in an unstructured conformation and does not form specific intra- or intermolecular interactions at micromolar protein concentrations. This suggests that the C-terminal domain may stabilise the PrD against prion formation by steric means, and implies that the PrD does not induce prion formation by altering the thermodynamic stability of the folded protein. Copyright 1999 Academic Press. PMID: 10388576 [PubMed - indexed for MEDLINE] 715: Biochemistry 1999 Jun 22;38(25):8138-49 Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability. Ibarra-Molero B, Loladze VV, Makhatadze GI, Sanchez-Ruiz JM. Facultad de Ciencias, Departamento de Quimica Fisica, Universidad de Granada, Spain. We have characterized the guanidine-induced unfolding of both yeast and bovine ubiquitin at 25 degrees C and in the acidic pH range on the basis of fluorescence and circular dichroism measurements. Unfolding Gibbs energy changes calculated by linear extrapolation from high guanidine unfolding data are found to depend very weakly on pH. A simple explanation for this result involves the two following assumptions: (1) charged atoms of ionizable groups are exposed to the solvent in native ubiquitin (as supported by accessible surface area calculations), and Gibbs energy contributions associated with charge desolvation upon folding (a source of pK shifts) are small; (2) charge-charge interactions (another source of pK shifts upon folding) are screened out in concentrated guanidinium chloride solutions. We have also characterized the thermal unfolding of both proteins using differential scanning calorimetry. Unfolding Gibbs energy changes calculated from the calorimetric data do depend strongly on pH, a result that we attribute to the pH dependence of charge-charge interactions (not eliminated in the absence of guanidine). In fact, we find good agreement between the difference between the two series of experimental unfolding Gibbs energy changes (determined from high guanidine unfolding data by linear extrapolation and from thermal denaturation data in the absence of guanidine) and the theoretical estimates of the contribution from charge-charge interactions to the Gibbs energy change for ubiquitin unfolding obtained by using the solvent-accessibility-corrected Tanford-Kirkwood model, together with the Bashford-Karplus (reduced-set-of-sites) approximation. This contribution is found to be stabilizing at neutral pH, because most charged groups on the native protein interact mainly with groups of the opposite charge, a fact that, together with the absence of large charge-desolvation contributions, may explain the high stability of ubiquitin at neutral pH. In general, our analysis suggests the possibility of enhancing protein thermal stability by adequately redesigning the distribution of solvent-exposed, charged residues on the native protein surface. PMID: 10387059 [PubMed - indexed for MEDLINE] 716: Protein Sci 1999 Jun;8(6):1250-6 The Schiff base complex of yeast 5-aminolaevulinic acid dehydratase with laevulinic acid. Erskine PT, Newbold R, Roper J, Coker A, Warren MJ, Shoolingin-Jordan PM, Wood SP, Cooper JB. Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, United Kingdom. The X-ray structure of the complex formed between yeast 5-aminolaevulinic acid dehydratase (ALAD) and the inhibitor laevulinic acid has been determined at 2.15 A resolution. The inhibitor binds by forming a Schiff base link with one of the two invariant lysines at the catalytic center: Lys263. It is known that this lysine forms a Schiff base link with substrate bound at the enzyme's so-called P-site. The carboxyl group of laevulinic acid makes hydrogen bonds with the side-chain-OH groups of Tyr329 and Ser290, as well as with the main-chain >NH group of Ser290. The aliphatic moiety of the inhibitor makes hydrophobic interactions with surrounding aromatic residues in the protein including Phe219, which resides in the flap covering the active site. Our analysis strongly suggests that the same interactions will be made by P-side substrate and also indicates that the substrate that binds at the enzyme's A-site will interact with the enzyme's zinc ion bound by three cysteines (133, 135, and 143). Inhibitor binding caused a substantial ordering of the active site flap (residues 217-235), which was largely invisible in the native electron density map and indicates that this highly conserved yet flexible region has a specific role in substrate binding during catalysis. PMID: 10386874 [PubMed - indexed for MEDLINE] 717: Neuroreport 1999 May 14;10(7):1409-15 A novel candidate presenilin-1 interacting protein containing tetratricopeptide repeats. Prihar G, Gonzalez de Chavez F, Baker M, Crook R, McGowan E, Grover A, Hardy J, Hutton M. Neurogenetics Laboratory, The Mayo Clinic, Jacksonville, FL 32224, USA. The yeast two-hybrid system, immunofluorescence and co-immunoprecipitation techniques were used to identify a novel candidate protein with which presenilin-1 (PS-1) interacts. This interacting protein, the gene of which is encoded on chromosome 16, contains two tetratricopeptide repeats (TPR) that are known to mediate interactions between proteins, appears to be primarily localized to the cytoplasm of transfected HEK293 cells, and is expressed in brain. Preliminary yeast two-hybrid data suggests this candidate may interact with both heat shock protein-90 and heat shock protein-70 and thus may be a novel member of TPR-containing proteins which interact with this complex. PMID: 10380955 [PubMed - indexed for MEDLINE] 718: Microbiology 1999 May;145 ( Pt 5):1115-22 Changes in Aspergillus nidulans gene expression induced by bafilomycin, a Streptomyces-produced antibiotic. Melin P, Schnurer J, Wagner EG. Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala. Petter.Melin@mikrob.slu.se In natural environments bacteria and filamentous fungi often compete for the same resources. Consequently, production of antibiotic secondary metabolites and defence mechanisms against these compounds have evolved in these organisms. An experimental model has been developed to study the response in fungi exposed to one such antibiotic. The filamentous fungus Aspergillus nidulans was treated with bafilomycin B1, a Streptomyces-produced antibiotic which reduces radial growth rate and induces morphological changes in fungi. mRNA differential display was used to study changes in fungal gene expression. For five genes, changes in abundance of the corresponding mRNAs, directly or indirectly caused by bafilomycin, were observed. Of these, three were up-regulated and two repressed. With four of these the change in mRNA abundance measured ranged from 10- to 60-fold. However, for one gene the mRNA was only detected after bafilomycin treatment. One of the down-regulated mRNAs encodes ASPND1, a glycoprotein that belongs to a known family of antigens identified in aspergilloma patients. One up-regulated mRNA shows sequence similarities, at the amino acid level, with a cell-wall protein of Saccharomyces cerevisiae. The remaining three genes were also cloned and sequenced; their sequences do not correspond to known genes in A. nidulans, and no similarities with published nucleotide or protein sequences in other organisms were found. These results indicate the feasibility of using mRNA differential display to study interactions between bacteria and filamentous fungi. PMID: 10376827 [PubMed - indexed for MEDLINE] 719: Mol Cell Biol 1999 Jul;19(7):4561-71 Std1 and Mth1 proteins interact with the glucose sensors to control glucose-regulated gene expression in Saccharomyces cerevisiae. Schmidt MC, McCartney RR, Zhang X, Tillman TS, Solimeo H, Wolfl S, Almonte C, Watkins SC. Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA. mcs2@pop.pitt.edu The Std1 protein modulates the expression of glucose-regulated genes, but its exact molecular role in this process is unclear. A two-hybrid screen for Std1-interacting proteins identified the hydrophilic C-terminal domains of the glucose sensors, Snf3 and Rgt2. The homologue of Std1, Mth1, behaves differently from Std1 in this assay by interacting with Snf3 but not Rgt2. Genetic interactions between STD1, MTH1, SNF3, and RGT2 suggest that the glucose signaling is mediated, at least in part, through interactions of the products of these four genes. Mutations in MTH1 can suppress the raffinose growth defect of a snf3 mutant as well as the glucose fermentation defect present in cells lacking both glucose sensors (snf3 rgt2). Genetic suppression by mutations in MTH1 is likely to be due to the increased and unregulated expression of hexose transporter genes. In media lacking glucose or with low levels of glucose, the hexose transporter genes are subject to repression by a mechanism that requires the Std1 and Mth1 proteins. An additional mechanism for glucose sensing must exist since a strain lacking all four genes (snf3 rgt2 std1 mth1) is still able to regulate SUC2 gene expression in response to changes in glucose concentration. Finally, studies with green fluorescent protein fusions indicate that Std1 is localized to the cell periphery and the cell nucleus, supporting the idea that it may transduce signals from the plasma membrane to the nucleus. PMID: 10373505 [PubMed - indexed for MEDLINE] 720: FEMS Microbiol Rev 1999 Jun;23(3):277-95 3'-End processing of pre-mRNA in eukaryotes. Wahle E, Ruegsegger U. Institut fur Biochemie, Martin-Luther-Universitat Halle-Wittenberg, Germany. ewahle@biochemtech.uni-halle.de 3'-Ends of almost all eukaryotic mRNAs are generated by endonucleolytic cleavage and addition of a poly(A) tail. In mammalian cells, the reaction depends on the sequence AAUAAA upstream of the cleavage site, a degenerate GU-rich sequence element downstream of the cleavage site and stimulatory sequences upstream of AAUAAA. Six factors have been identified that carry out the two reactions. With a single exception, they have been purified to homogeneity and cDNAs for 11 subunits have been cloned. Some of the cooperative RNA-protein and protein-protein interactions within the processing complex have been analyzed, but many details, including the identity of the endonuclease, remain unknown. Several examples of regulated polyadenylation are being analyzed at the molecular level. In the yeast Saccharomyces cerevisiae, sequences directing cleavage and polyadenylation are more degenerate than in metazoans, and a downstream element has not been identified. The list of processing factors may be complete now with approximately a dozen polypeptides, but their functions in the reaction are largely unknown. 3'-Processing is known to be coupled to transcription. This connection is thought to involve interactions of processing factors with the mRNA cap as well as with RNA polymerase II. Publication Types: Review Review, Tutorial PMID: 10371034 [PubMed - indexed for MEDLINE] 721: J Mol Biol 1999 Jun 18;289(4):691-9 Polyanionic inhibitors of phosphoglycerate mutase: combined structural and biochemical analysis. Rigden DJ, Walter RA, Phillips SE, Fothergill-Gilmore LA. School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, England. The effects that the inhibitors inositol hexakisphosphate and benzene tri-, tetra- and hexacarboxylates have on the phosphoglycerate mutases from Saccharomyces cerevisiae and Schizosaccharomyces pombe have been determined. Their Kivalues have been calculated, and the ability of the inhibitors to protect the enzymes against limited proteolysis investigated. These biochemical data have been placed in a structural context by the solution of the crystal structures of S. cerevisiae phosphoglycerate mutase soaked with inositol hexakisphosphate or benzene hexacarboxylate. These large polyanionic compounds bind to the enzyme so as to block the entrance to the active-site cleft. They form multiple interactions with the enzyme, consistent with their low Kivalues, and afford good protection against limited proteolysis of the C-terminal region by thermolysin. The inositol compound is more efficacious because of its greater number of negative charges. The S. pombe phosphoglycerate mutase that is inherently lacking a comparable C-terminal region has higher Kivalues for the compounds tested. Moreover, the S. pombe enzyme is less sensititive to proteolysis, and the presence or absence of the inhibitor molecules has little effect on susceptibility to proteolysis. Copyright 1999 Academic Press. PMID: 10369755 [PubMed - indexed for MEDLINE] 722: Cell 1999 May 28;97(5):657-66 Crystal structure of a phosphatidylinositol 3-phosphate-specific membrane-targeting motif, the FYVE domain of Vps27p. Misra S, Hurley JH. Laboratory of Molecular Biology, National Institute of Digestive, Diabetes, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0580, USA. Phosphatidylinositol 3-phosphate regulates membrane trafficking and signaling pathways by interacting with the FYVE domains of target proteins. The 1.15 A structure of the Vps27p FYVE domain reveals two antiparallel beta sheets and an alpha helix stabilized by two Zn2+-binding clusters. The core secondary structures are similar to a rabphilin-3A Zn2+-binding domain and to the C1 and LIM domains. Phosphatidylinositol 3-phosphate binds to a pocket formed by the (R/K)(R/K)HHCR motif. A lattice contact shows how anionic ligands can interact with the phosphatidylinositol 3-phosphate-binding site. The tip of the FYVE domain has basic and hydrophobic surfaces positioned so that nonspecific interactions with the phospholipid bilayer can abet specific binding to phosphatidylinositol 3-phosphate. PMID: 10367894 [PubMed - indexed for MEDLINE] 723: Virology 1999 Jun 5;258(2):271-81 Activation of Ste20 by Nef from human immunodeficiency virus induces cytoskeletal rearrangements and downstream effector functions in Saccharomyces cerevisiae. Plemenitas A, Lu X, Geyer M, Veranic P, Simon MN, Peterlin BM. Institute of Biochemistry, Medical Faculty, Vrazov trg2, Ljubljana, 1000, Slovenia. The negative factor (Nef) from human and simian immunodeficiency viruses is important for the pathogenesis of acquired immune deficiency syndrome. Among other targets, it activates the Nef-associated kinase, which is related to the p21-activated kinase. In this study, we demonstrate that Nef activates Ste20, the homolog of p21-activated kinase in Saccharomyces cerevisiae. Nef binds to the adaptor proteins Bem1 and Ste20 via its proline-rich (PXXP) and diarginine (RR) motifs, respectively. These interactions induce the mitogen-activated protein kinase and increase the rates of budding, sizes of cells, and patterns of mating projections. These effects of Nef depend on the small GTPase Cdc42 and guanine nucleotide exchange factor Cdc24. Thus, studies in S. cerevisiae identified specific interactions between Nef and cellular proteins and their associated signaling cascade. Copyright 1999 Academic Press. PMID: 10366564 [PubMed - indexed for MEDLINE] 724: Biochem Biophys Res Commun 1999 Jun 7;259(2):391-400 RNase treatment of yeast and mammalian cell extracts affects in vitro substrate methylation by type I protein arginine N-methyltransferases. Frankel A, Clarke S. Department of Chemistry & Biochemistry and Molecular Biology Institute, UCLA, Los Angeles, California 90095-1569, USA. Type I protein arginine N-methyltransferases catalyze the formation of omega-NG-monomethylarginine and asymmetric omega-NG, NG-dimethylarginine residues using S-adenosyl-l-methionine as the methyl donor. In vitro these enzymes can modify a number of soluble methyl-accepting substrates in yeast and mammalian cell extracts including several species that interact with RNA. We treated normal and hypomethylated Saccharomyces cerevisiae and RAT1 cell extracts with RNase prior to in vitro methylation by recombinant protein N-arginine methyltransferases and found that the methylation of certain polypeptides is enhanced up to 12-fold whereas that of others is diminished. 2-D gel electrophoresis of RNase-treated yeast extracts allowed us to tentatively identify the glycine- and arginine-rich (GAR) domain-containing proteins Gar1, Nop1, Sbp1, and Npl3 as major methyl-acceptors based on their known isoelectric points and apparent molecular weights. These results suggest that the methylation and RNA-binding of GAR domain-containing proteins in vivo may regulate protein-nucleic acid or protein-protein interactions. Copyright 1999 Academic Press. PMID: 10362520 [PubMed - indexed for MEDLINE] 725: J Biol Chem 1999 Jun 11;274(24):17219-25 Modulation of human heat shock factor trimerization by the linker domain. Liu PC, Thiele DJ. Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA. dthiele@umich.edu Heat shock transcription factors (HSFs) are stress-responsive proteins that activate the expression of heat shock genes and are highly conserved from bakers' yeast to humans. Under basal conditions, the human HSF1 protein is maintained as an inactive monomer through intramolecular interactions between two coiled-coil domains and interactions with heat shock proteins; upon environmental, pharmacological, or physiological stress, HSF1 is converted to a homotrimer that binds to its cognate DNA binding site with high affinity. To dissect regions of HSF1 that make important contributions to the stability of the monomer under unstressed conditions, we have used functional complementation in bakers' yeast as a facile assay system. Whereas wild-type human HSF1 is restrained as an inactive monomer in yeast that is unable to substitute for the essential yeast HSF protein, mutations in the linker region between the DNA binding domain and the first coiled-coil allow HSF1 to homotrimerize and rescue the viability defect of a hsfDelta strain. Fine mapping by functional analysis of HSF1-HSF2 chimeras and point mutagenesis revealed that a small region in the amino-terminal portion of the HSF1 linker is required for maintenance of HSF1 in the monomeric state in both yeast and in transfected human 293 cells. Although linker regions in transcription factors are known to modulate DNA binding specificity, our studies suggest that the human HSF1 linker plays no role in determining HSF1 binding preferences in vivo but is a critical determinant in regulating the HSF1 monomer-trimer equilibrium. PMID: 10358080 [PubMed - indexed for MEDLINE] 726: J Biol Chem 1999 Jun 11;274(24):17080-7 A two-hybrid dual bait system to discriminate specificity of protein interactions. Serebriiskii I, Khazak V, Golemis EA. Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to which observed interactions are specific. We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, fused to DNA binding domain B (lambda cI). Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systematically compared LexA- and cI-fused baits and reporters. The LexA and cI baitr reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background. These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners. PMID: 10358061 [PubMed - indexed for MEDLINE] 727: J Biol Chem 1999 Jun 11;274(24):16861-70 The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating the apical-isotropic switch and nuclear division in yeast. Richman TJ, Sawyer MM, Johnson DI. Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA. The Cdc42p GTPase is involved in the signal transduction cascades controlling bud emergence and polarized cell growth in S. cerevisiae. Cells expressing the cdc42(V44A) effector domain mutant allele displayed morphological defects of highly elongated and multielongated budded cells indicative of a defect in the apical-isotropic switch in bud growth. In addition, these cells contained one, two, or multiple nuclei indicative of a G2/M delay in nuclear division and also a defect in cytokinesis and/or cell separation. Actin and chitin were delocalized, and septin ring structure was aberrant and partially delocalized to the tips of elongated cdc42(V44A) cells; however, Cdc42(V44A)p localization was normal. Two-hybrid protein analyses showed that the V44A mutation interfered with Cdc42p's interactions with Cla4p, a p21(Cdc42/Rac)-activated kinase (PAK)-like kinase, and the novel effectors Gic1p and Gic2p, but not with the Ste20p or Skm1p PAK-like kinases, the Bni1p formin, or the Iqg1p IQGAP homolog. Furthermore, the cdc42(V44A) morphological defects were suppressed by deletion of the Swe1p cyclin-dependent kinase inhibitory kinase and by overexpression of Cla4p, Ste20p, the Cdc12 septin protein, or the guanine nucleotide exchange factor Cdc24p. In sum, these results suggest that proper Cdc42p function is essential for timely progression through the apical-isotropic switch and G2/M transition and that Cdc42(V44A)p differentially interacts with a number of effectors and regulators. PMID: 10358031 [PubMed - indexed for MEDLINE] 728: J Biol Chem 1999 Jun 11;274(24):16747-53 Homo- and heterodimerization of synapsins. Hosaka M, Sudhof TC. Center for Basic Neuroscience and Department of Molecular Genetics, Howard Hughes Medical Institute, The University of Texas Southwestern Medical School, Dallas, Texas 75235, USA. In vertebrates, synapsins constitute a family of synaptic vesicle proteins encoded by three genes. Synapsins contain a central ATP-binding domain, the C-domain, that is highly homologous between synapsins and evolutionarily conserved in invertebrates. The crystal structure of the C-domain from synapsin I revealed that it constitutes a large (>300 amino acids), independently folded domain that forms a tight dimer with or without bound ATP. We now show that the C-domains of all synapsins form homodimers, and that in addition, C-domains from different synapsins associate into heterodimers. This conclusion is based on four findings: 1) in yeast two-hybrid screens with full-length synapsin IIa as a bait, the most frequently isolated prey cDNAs encoded the C-domain of synapsins; 2) quantitative yeast two-hybrid protein-protein binding assays demonstrated pairwise strong interactions between all synapsins; 3) immunoprecipitations from transfected COS cells confirmed that synapsin II heteromultimerizes with synapsins I and III in intact cells, and similar results were obtained with bacterial expression systems; and 4) quantification of the synapsin III level in synapsin I/II double knockout mice showed that the level of synapsin III is decreased by 50%, indicating that heteromultimerization of synapsin III with synapsins I or II occurs in vivo and is required for protein stabilization. These data suggest that synapsins coat the surface of synaptic vesicles as homo- and heterodimers in which the C-domains of the various subunits have distinct regulatory properties and are flanked by variable C-terminal sequences. The data also imply that synapsin III does not compensate for the loss of synapsins I and II in the double knockout mice. PMID: 10358015 [PubMed - indexed for MEDLINE] 729: EMBO J 1999 Jun 1;18(11):3139-52 Post-termination ribosome interactions with the 5'UTR modulate yeast mRNA stability. Vilela C, Ramirez CV, Linz B, Rodrigues-Pousada C, McCarthy JE. Post-transcriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester M60 1QD, UK. A novel form of post-transcriptional control is described. The 5' untranslated region (5'UTR) of the Saccharomyces cerevisiae gene encoding the AP1-like transcription factor Yap2 contains two upstream open reading frames (uORF1 and uORF2). The YAP2-type of uORF functions as a cis-acting element that attenuates gene expression at the level of mRNA turnover via termination-dependent decay. Release of post-termination ribosomes from the YAP2 5'UTR causes accelerated decay which is largely independent of the termination modulator gene UPF1. Both of the YAP2 uORFs contribute to the destabilization effect. A G/C-rich stop codon context, which seems to promote ribosome release, allows an uORF to act as a transferable 5'UTR-destabilizing element. Moreover, termination-dependent destabilization is potentiated by stable secondary structure 3' of the uORF stop codon. The potentiation of uORF-mediated destabilization is eliminated if the secondary structure is located further downstream of the uORF, and is also influenced by a modulatory mechanism involving eIF2. Destabilization is therefore linked to the kinetics of acquisition of reinitiation-competence by post-termination ribosomes in the 5'UTR. Our data explain the destabilizing properties of YAP2-type uORFs and also support a more general model for the mode of action of other known uORFs, such as those in the GCN4 mRNA. PMID: 10357825 [PubMed - indexed for MEDLINE] 730: Scand J Immunol 1999 Jun;49(6):620-8 Protein-protein interactions between native Ro52 and immunoglobulin G heavy chain. Yang Y, Eversole T, Lee DJ, Sontheimer RD, Capra JD. Department of Internal Medicine, Pulmonary and Critical Care Division, UT Southwestern Medical Center, 5323 Harry Hines Blvd. Dallas, TX 75235-9034, USA. Using a yeast two-hybrid system to search for proteins interacting with Ro52 autoantigen, we identified a novel protein-protein interaction. Two different cDNA clones, which interacted with Ro52 in the yeast two-hybrid system, were identified and isolated from a human B-cell library. Surprisingly, both clones encoded the heavy chain of human IgG1. The expression of both HIS3 and beta-galactosidase reporter genes in yeast suggested that the interaction between Ro52 and IgG occurred in vivo. In vitro studies utilizing recombinant Ro52 and purified immunoglobulins indicated that the interaction was immunoglobulin class and subclass specific. Ro52 interacted with IgG1 and IgG4, but not with IgG2, IgG3, IgA or IgM. Ro52 could also precipitate IgG directly from serum. The identified cDNA clones did not include the variable region of IgG, which suggested a non-classical interaction independent of antibody specificity. We further mapped the domain of Ro52 responsible for this interaction to the C-terminus rfp-like region. In conclusion, our data support an unusual interaction between native Ro52 and IgG. The potential biological significance of this unusual protein-protein interaction is discussed. PMID: 10354373 [PubMed - indexed for MEDLINE] 731: J Cell Biol 1999 May 31;145(5):933-50 The unstable F-box protein p58-Ctf13 forms the structural core of the CBF3 kinetochore complex. Russell ID, Grancell AS, Sorger PK. Massachusetts Institute of Technology, Department of Biology, Cambridge, Massachusetts 02139, USA. Kinetochores are smaller and more accessible experimentally in budding yeast than in any other eukaryote. Believing that simple and complex kinetochores have important structural and functional properties in common, we characterized the structure of CBF3, the essential centromere-binding complex that initiates kinetochore formation in Saccharomyces cerevisiae. We find that the four subunits of CBF3 are multimeric in solution: p23(Skp1) and p58(Ctf13) form a heterodimer, and p64(Cep3) and p110(Ndc10) form homodimers. Subcomplexes involving p58 and each of the other CBF3 subunits can assemble in the absence of centromeric DNA. In these subcomplexes, p58 appears to function as a structural core mediating stable interactions among other CBF3 proteins. p58 has a short half-life in yeast, being subject to ubiquitin-dependent proteolysis, but we find that it is much more stable following association with p64. We propose that p23(Skp1)-p58-p64 complexes constitute the primary pool of active p58 in yeast cells. These complexes can either dissociate, reexposing p58 to the degradation pathway, or can bind to p110 and centromeric DNA, forming a functional CBF3 complex in which p58 is fully protected from degradation. This pathway may constitute an editing mechanism preventing the formation of ectopic kinetochores and ensuring the fidelity of chromosome segregation. PMID: 10352012 [PubMed - indexed for MEDLINE] 732: Methods Enzymol 1999;303:422-50 Screening for protein-protein interactions. Germino FJ, Moskowitz NK. University of Medical Dentistry of New Jersey, Clinical Institute of New Jersey, New Brunswick 08901, USA. The assays described above can be used to screen for cellular proteins that can interact with a protein of interest, to screen for mutant proteins that retain the ability to bind to its partner, and to identify the domains and amino acids involved in known protein-protein interactions. Nonetheless, the biological significance of some of these interactions needs to be confirmed by appropriate cellular studies. PMID: 10349658 [PubMed - indexed for MEDLINE] 733: J Biol Chem 1999 Jun 4;274(23):16508-12 Species barrier to RNA recognition overcome with nonspecific RNA binding domains. Wang CC, Schimmel P. The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA. We show here that nonspecific RNA-protein interactions can significantly enhance the biological activity of an essential RNA. protein complex. Bacterial glutaminyl-tRNA synthetase poorly aminoacylates yeast tRNA and, as a consequence, cannot rescue a knockout allele of the gene for the yeast homologue. In contrast to the bacterial protein, the yeast enzyme has an extra appended domain at the N terminus. Previously, we showed that fusion of this yeast-specific domain to the bacterial protein enabled it to function as a yeast enzyme in vivo and in vitro. We suggested that the novel yeast-specific domain contributed to RNA interactions in a way that compensated for the poor fit between the yeast tRNA and bacterial enzyme. Here we establish that the novel appended domain by itself binds nonspecifically to different RNA structures. In addition, we show that fusion of an unrelated yeast protein, Arc1p, to the bacterial enzyme also converts it into a functional yeast enzyme in vivo and in vitro. A small C-terminal segment of Arc1p is necessary and sufficient for this conversion. This segment was shown by others to have nonspecific tRNA binding properties. Thus, nonspecific RNA binding interactions in general can compensate for barriers to formation of a specific and essential RNA.protein complex. PMID: 10347214 [PubMed - indexed for MEDLINE] 734: J Biol Chem 1999 Jun 4;274(23):16363-9 Structure/function of the beta-barrel domain of F1-ATPase in the yeast Saccharomyces cerevisiae. Bakhtiari N, Lai-Zhang J, Yao B, Mueller DM. Department of Biochemistry and Molecular Biology, Chicago Medical School, North Chicago, Illinois 60064, USA. The first 90 amino acids of the alpha- and beta-subunits of mitochondrial F1-ATPase are folded into beta-barrel domains and were postulated to be important for stabilizing the enzyme (Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). The role of the domains was studied by making chimeric enzymes, replacing the domains from the yeast Saccharomyces cerevisiae enzyme with the corresponding domains from the enzyme of the thermophilic bacterium Bacillus PS3. The enzymes containing the chimeric alpha-, beta-, or alpha- and beta-subunits were not functional. However, gain-of-function mutations were obtained from the strain containing the enzyme with the chimeric PS3/yeast beta-subunit. The gain-of-function mutations were all in codons encoding the beta-barrel domain of the beta-subunit, and the residues appear to map out a region of subunit-subunit interactions. Gain-of-function mutations were also obtained that provided functional expression of the chimeric PS3/yeast alpha- and beta-subunits together. Biochemical analysis of this active chimeric enzyme indicated that it was not significantly more thermostable or labile than the wild type. The results of this study indicate that the beta-barrel domains form critical contacts (distinct from those between the alpha- and beta-subunits) that are important for the assembly of the ATP synthase. PMID: 10347195 [PubMed - indexed for MEDLINE] 735: J Biol Chem 1999 Jun 4;274(23):16242-8 Physical and functional interactions of neuronal growth suppressor necdin with p53. Taniura H, Matsumoto K, Yoshikawa K. Division of Regulation of Macromolecular Functions, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan. hideo@protein.osaka-u.ac.jp Necdin is expressed in virtually all postmitotic neurons, and ectopic expression of this protein suppresses cell proliferation. Necdin, like the retinoblastoma protein, interacts with cell cycle promoting proteins such as simian virus 40 large T antigen, adenovirus E1A, and the transcription factor E2F1. Here we demonstrate that necdin interacts with the tumor suppressor protein p53 as well. The yeast two-hybrid and in vitro binding analyses revealed that necdin bound to a narrow region (amino acids 35-62) located between the MDM2-binding site and the proline-rich region in the amino-terminal domain of p53. The electrophoretic mobility shift assay showed that necdin supershifted a complex between p53 and its binding DNA, implying that the p53-necdin complex is competent for DNA binding. In p53-deficient osteosarcoma SAOS-2 cells, necdin markedly suppressed p53-dependent activation of the p21/WAF promoter. Necdin and p53 inhibited cell growth in an additive manner as assessed by the colony formation of SAOS-2 cells, suggesting that necdin does not affect p53-mediated growth suppression. On the other hand, necdin inhibited p53-induced apoptosis of osteosarcoma U2OS cells. Thus, necdin can be a growth suppressor that targets p53 and modulates its biological functions in postmitotic neurons. PMID: 10347180 [PubMed - indexed for MEDLINE] 736: Proc Natl Acad Sci U S A 1999 May 25;96(11):6523-8 Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Zhang Y, Fan W, Kinkema M, Li X, Dong X. Developmental, Cell, and Molecular Biology Group, Department of Botany, Box 91000, Duke University, Durham, NC 27708-1000, USA. The Arabidopsis thaliana NPR1 has been shown to be a key regulator of gene expression during the onset of a plant disease-resistance response known as systemic acquired resistance. The npr1 mutant plants fail to respond to systemic acquired resistance-inducing signals such as salicylic acid (SA), or express SA-induced pathogenesis-related (PR) genes. Using NPR1 as bait in a yeast two-hybrid screen, we identified a subclass of transcription factors in the basic leucine zipper protein family (AHBP-1b and TGA6) and showed that they interact specifically in yeast and in vitro with NPR1. Point mutations that abolish the NPR1 function in A. thaliana also impair the interactions between NPR1 and the transcription factors in the yeast two-hybrid assay. Furthermore, a gel mobility shift assay showed that the purified transcription factor protein, AHBP-1b, binds specifically to an SA-responsive promoter element of the A. thaliana PR-1 gene. These data suggest that NPR1 may regulate PR-1 gene expression by interacting with a subclass of basic leucine zipper protein transcription factors. PMID: 10339621 [PubMed - indexed for MEDLINE] 737: Genes Genet Syst 1998 Dec;73(6):365-75 Isolation and characterization of the yeast las21 mutants, which are sensitive to a local anestheticum, tetracaine. Tohe A, Oguchi T. Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan. We isolated and characterized yeast mutants whose growth is sensitive to a local anestheticum tetracaine and, at the same time, temperature sensitive. These mutants were collectively called las mutants (local anestheticum sensitive). The las21 mutants were analyzed in this study. The wild type LAS21 gene was cloned by exploiting temperature sensitivity of the las21 mutants and we found that LAS21 encodes ORF YJL062w which has not been analyzed before. Las21p is putative membrane protein belonging to the major facilitator super family containing plural membrane spanning domains. Complete elimination of the LAS21 ORF did not kill the cells but made their growth temperature sensitive. Interestingly, the complete loss of the LAS21 gene canceled the sensitivity to tetracaine. The ability of the las21 mutants to grow at a higher temperature was recovered in the various media containing an osmotic stabilizer or salts. Furthermore, temperature sensitivity of the las21 mutants was partially suppressed by introduction of PKC1, encoding protein kinase C, on a high copy vector. We found some genetic interactions between LAS21 and Ras/cAMP cascade genes. These results suggest that LAS21 defines unknown pathway regulating the stress response of yeast. PMID: 10333567 [PubMed - indexed for MEDLINE] 738: Hum Mol Genet 1999 Jun;8(6):947-57 Dentatorubral-pallidoluysian atrophy protein interacts through a proline-rich region near polyglutamine with the SH3 domain of an insulin receptor tyrosine kinase substrate. Okamura-Oho Y, Miyashita T, Ohmi K, Yamada M. Department of Genetics, National Children's Memorial Medical Research Center, Taishido, Setagaya, Tokyo, Japan. Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neuro degrees enerative disorder associated with CAG/glutamine repeat expansion. While the DRPLA gene is ubiquitously expressed, neuron death occurs in specific anatomical areas of the brain. This predicts that the DRPLA protein interacts with other proteins and that these interactions may play a role in pathogenesis. Here, we describe a protein that binds to the DRPLA product. One of the clones isolated with a yeast two-hybrid system was identified as a human homolog of the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). The gene produced two mRNA forms by differential splicing and encoded 552 and 521 amino acids, respectively. The longer form was mainly expressed in the brain and the shorter one in other tissues. The products were phosphorylated upon stimulation of cultured cells with insulin or insulin-like growth factor 1. Binding of the DRPLA protein to IRSp53 was ascertained by co-immunoprecipitation with antibodies and also by co-localization in perinuclear oval dots in cells expressing engineered constructs. A proline-rich region near the polyglutamine tract of the DRPLA protein and the SH3 domain of IRSp53 were involved in the binding. An extended polyglutamine tract significantly reduced binding ability in yeast cells, but not in in vitro binding assays. The identification of IRSp53 and other proteins detected by the yeast hybrid system predicts that DRPLA functions in a signal transduction pathway coupled with insulin/IGF-1. PMID: 10332026 [PubMed - indexed for MEDLINE] 739: J Cell Biol 1999 May 17;145(4):659-72 LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum. Roberg KJ, Crotwell M, Espenshade P, Gimeno R, Kaiser CA. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. In Saccharomyces cerevisiae, vesicles that carry proteins from the ER to the Golgi compartment are encapsulated by COPII coat proteins. We identified mutations in ten genes, designated LST (lethal with sec-thirteen), that were lethal in combination with the COPII mutation sec13-1. LST1 showed synthetic-lethal interactions with the complete set of COPII genes, indicating that LST1 encodes a new COPII function. LST1 codes for a protein similar in sequence to the COPII subunit Sec24p. Like Sec24p, Lst1p is a peripheral ER membrane protein that binds to the COPII subunit Sec23p. Chromosomal deletion of LST1 is not lethal, but inhibits transport of the plasma membrane proton-ATPase (Pma1p) to the cell surface, causing poor growth on media of low pH. Localization by both immunofluorescence microscopy and cell fractionation shows that the export of Pma1p from the ER is impaired in lst1Delta mutants. Transport of other proteins from the ER was not affected by lst1Delta, nor was Pma1p transport found to be particularly sensitive to other COPII defects. Together, these findings suggest that a specialized form of the COPII coat subunit, with Lst1p in place of Sec24p, is used for the efficient packaging of Pma1p into vesicles derived from the ER. PMID: 10330397 [PubMed - indexed for MEDLINE] 740: Mol Cell Biol 1999 Jun;19(6):4414-22 DIX domains of Dvl and axin are necessary for protein interactions and their ability to regulate beta-catenin stability. Kishida S, Yamamoto H, Hino S, Ikeda S, Kishida M, Kikuchi A. Department of Biochemistry, Hiroshima University School of Medicine, Minami-ku, Hiroshima 734-8551, Japan. The N-terminal region of Dvl-1 (a mammalian Dishevelled homolog) shares 37% identity with the C-terminal region of Axin, and this related region is named the DIX domain. The functions of the DIX domains of Dvl-1 and Axin were investigated. By yeast two-hybrid screening, the DIX domain of Dvl-1 was found to interact with Dvl-3, a second mammalian Dishevelled relative. The DIX domains of Dvl-1 and Dvl-3 directly bound one another. Furthermore, Dvl-1 formed a homo-oligomer. Axin also formed a homo-oligomer, and its DIX domain was necessary. The N-terminal region of Dvl-1, including its DIX domain, bound to Axin directly. Dvl-1 inhibited Axin-promoted glycogen synthase kinase 3beta-dependent phosphorylation of beta-catenin, and the DIX domain of Dvl-1 was required for this inhibitory activity. Expression of Dvl-1 in L cells induced the nuclear accumulation of beta-catenin, and deletion of the DIX domain abolished this activity. Although expression of Axin in SW480 cells caused the degradation of beta-catenin and reduced the cell growth rate, expression of an Axin mutant that lacks the DIX domain did not affect the level of beta-catenin or the growth rate. These results indicate that the DIX domains of Dvl-1 and Axin are important for protein-protein interactions and that they are necessary for the ability of Dvl-1 and Axin to regulate the stability of beta-catenin. PMID: 10330181 [PubMed - indexed for MEDLINE] 741: Mol Cell Biol 1999 Jun;19(6):4324-33 A new class of repression modules is critical for heme regulation of the yeast transcriptional activator Hap1. Hach A, Hon T, Zhang L. Department of Biochemistry, NYU Medical Center, New York, New York 10016, USA. Heme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen. In the yeast Saccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain. To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated. Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation. All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme. More importantly, we show that a novel class of repression modules-RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244-is critical for Hap1 repression in the absence of heme. Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC. These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme. PMID: 10330173 [PubMed - indexed for MEDLINE] 742: Mol Cell Biol 1999 Jun;19(6):4167-81 GCD14p, a repressor of GCN4 translation, cooperates with Gcd10p and Lhp1p in the maturation of initiator methionyl-tRNA in Saccharomyces cerevisiae. Calvo O, Cuesta R, Anderson J, Gutierrez N, Garcia-Barrio MT, Hinnebusch AG, Tamame M. Instituto de Microbiologia Bioquimica del CSIC/Universidad de Salamanca, 37007 Salamanca, Spain. Gcd10p and Gcd14p were first identified genetically as repressors of GCN4 mRNA translation in Saccharomyces cerevisiae. Recent findings indicate that Gcd10p and Gcd14p reside in a nuclear complex required for the presence of 1-methyladenosine in tRNAs. Here we show that Gcd14p is an essential protein with predicted binding motifs for S-adenosylmethionine, consistent with a direct function in tRNA methylation. Two different gcd14 mutants exhibit defects in cell growth and accumulate high levels of initiator methionyl-tRNA (tRNAiMet) precursors containing 5' and 3' extensions, suggesting a defect in processing of the primary transcript. Dosage suppressors of gcd10 mutations, encoding tRNAiMet (hcIMT1 to hcIMT4; hc indicates that the gene is carried on a high-copy-number plasmid) or a homologue of human La protein implicated in tRNA 3'-end formation (hcLHP1), also suppressed gcd14 mutations. In fact, the lethality of a GCD14 deletion was suppressed by hcIMT4, indicating that the essential function of Gcd14p is required for biogenesis of tRNAiMet. A mutation in GCD10 or deletion of LHP1 exacerbated the defects in cell growth and expression of mature tRNAiMet in gcd14 mutants, consistent with functional interactions between Gcd14p, Gcd10p, and Lhp1p in vivo. Surprisingly, the amounts of NME1 and RPR1, the RNA components of RNases P and MRP, were substantially lower in gcd14 lhp1::LEU2 double mutants than in the corresponding single mutants, whereas 5S rRNA was present at wild-type levels. Our findings suggest that Gcd14p and Lhp1p cooperate in the maturation of a subset of RNA polymerase III transcripts. PMID: 10330157 [PubMed - indexed for MEDLINE] 743: Mol Cell Biol 1999 Jun;19(6):4101-12 Rapamycin antifungal action is mediated via conserved complexes with FKBP12 and TOR kinase homologs in Cryptococcus neoformans. Cruz MC, Cavallo LM, Gorlach JM, Cox G, Perfect JR, Cardenas ME, Heitman J. Departments of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA. Cryptococcus neoformans is a fungal pathogen that causes meningitis in patients immunocompromised by AIDS, chemotherapy, organ transplantation, or high-dose steroids. Current antifungal drug therapies are limited and suffer from toxic side effects and drug resistance. Here, we defined the targets and mechanisms of antifungal action of the immunosuppressant rapamycin in C. neoformans. In the yeast Saccharomyces cerevisiae and in T cells, rapamycin forms complexes with the FKBP12 prolyl isomerase that block cell cycle progression by inhibiting the TOR kinases. We identified the gene encoding a C. neoformans TOR1 homolog. Using a novel two-hybrid screen for rapamycin-dependent TOR-binding proteins, we identified the C. neoformans FKBP12 homolog, encoded by the FRR1 gene. Disruption of the FKBP12 gene conferred rapamycin and FK506 resistance but had no effect on growth, differentiation, or virulence of C. neoformans. Two spontaneous mutations that confer rapamycin resistance alter conserved residues on TOR1 or FKBP12 that are required for FKBP12-rapamycin-TOR1 interactions or FKBP12 stability. Two other spontaneous mutations result from insertion of novel DNA sequences into the FKBP12 gene. Our observations reveal that the antifungal activities of rapamycin and FK506 are mediated via FKBP12 and TOR homologs and that a high proportion of spontaneous mutants in C. neoformans result from insertion of novel DNA sequences, and they suggest that nonimmunosuppressive rapamycin analogs have potential as antifungal agents. PMID: 10330150 [PubMed - indexed for MEDLINE] 744: J Biol Chem 1999 May 21;274(21):15262-70 Interaction of insulin receptor substrate 3 with insulin receptor, insulin receptor-related receptor, insulin-like growth factor-1 receptor, and downstream signaling proteins. Xu P, Jacobs AR, Taylor SI. Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA. Insulin receptor substrates (IRS) mediate biological actions of insulin, growth factors, and cytokines. All four mammalian IRS proteins contain pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains at their N termini. However, the molecules diverge in their C-terminal sequences. IRS3 is considerably shorter than IRS1, IRS2, and IRS4, and is predicted to interact with a distinct group of downstream signaling molecules. In the present study, we investigated interactions of IRS3 with various signaling molecules. The PTB domain of mIRS3 is necessary and sufficient for binding to the juxtamembrane NPXpY motif of the insulin receptor in the yeast two-hybrid system. This interaction is stronger if the PH domain or the C-terminal phosphorylation domain is retained in the construct. As determined in a modified yeast two-hybrid system, mIRS3 bound strongly to the p85 subunit of phosphatidylinositol 3-kinase. Although high affinity interaction required the presence of at least two of the four YXXM motifs in mIRS3, there was not a requirement for specific YXXM motifs. mIRS3 also bound to SHP2, Grb2, Nck, and Shc, but less strongly than to p85. Studies in COS-7 cells demonstrated that deletion of either the PH or the PTB domain abolished insulin-stimulated phosphorylation of mIRS3. Insulin stimulation promoted the association of mIRS3 with p85, SHP2, Nck, and Shc. Despite weak association between mIRS3 and Grb2, this interaction was not increased by insulin, and may not be mediated by the SH2 domain of Grb2. Thus, in contrast to other IRS proteins, mIRS3 appears to have greater specificity for activation of the phosphatidylinositol 3-kinase pathway rather than the Grb2/Ras pathway. PMID: 10329736 [PubMed - indexed for MEDLINE] 745: J Biol Chem 1999 May 21;274(21):14685-91 Identification of rabbit reticulocyte E217K as a UBC7 homologue and functional characterization of its core domain loop. Lin H, Wing SS. Department of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada. The structural basis by which ubiquitin (Ub)-conjugating enzymes (E2s) determine substrate specificity remains unclear. We cloned rabbit reticulocyte E217K because unlike the similarly sized class I E2s, E214K and UBC4, it is unable to support ubiquitin-protein ligase (E3)-dependent conjugation to endogenous proteins. RNA analysis revealed that this E2 was expressed in all tissues tested, with higher levels in the testis. Analysis of testis RNA from rats of different ages showed that E217K mRNA was induced from days 15 to 30. The predicted amino acid sequence indicates that E217K is a 19. 5-kDa class I E2 but differs from other class I enzymes in possessing an insertion of 13 amino acids distal to the active site cysteine. E217K shows 74% amino acid identity with Saccharomyces cerevisiae UBC7, and therefore, we rename it mammalian UBC7. Yeast UBC7 crystal structure indicates that this insertion forms a loop out of the otherwise conserved folding structure. Sequence analysis of E2s had previously suggested that this loop is a hypervariable region and may play a role in substrate specificity. We created mutant UBC7 lacking the loop (ubc7Deltaloop) and a mutant E214k with an inserted loop (E214k+loop) and characterized their biochemical functions. Ubc7Deltaloop had higher affinity for the E1-Ub thiol ester than native UBC7 and permitted conjugation of Ub to selected proteins in the testis but did not permit the broad spectrum E3-dependent conjugation to endogenous reticulocyte proteins. Surprisingly, E214k+loop was unable to accept Ub from ubiquitin-activating enzyme (E1) but was able to accept NEDD8 from E1. E214k+loop was able to support conjugation of NEDD8 to endogenous reticulocyte proteins but with much lower efficiency than E214k. Thus, the loop can influence interactions of the E2 with charged E1 as well as with E3s or substrates, but the exact nature of these interactions depends on divergent sequences in the remaining conserved core domain. PMID: 10329663 [PubMed - indexed for MEDLINE] 746: Virology 1999 May 25;258(1):95-9 Potyvirus helper component-proteinase self-interaction in the yeast two-hybrid system and delineation of the interaction domain involved. Urcuqui-Inchima S, Walter J, Drugeon G, German-Retana S, Haenni AL, Candresse T, Bernardi F, Le Gall O. Institut Jacques Monod, 2 place Jussieu-Tour 43, Paris Cedex 05, 75251, France. Using the yeast two-hybrid system, a screen was performed for possible interactions between the proteins encoded by the 5' region of potyviral genomes [P1, helper component-proteinase (HC-Pro), and P3]. A positive self-interaction involving HC-Pro was detected with lettuce mosaic virus (LMV) and potato virus Y (PVY). The possibility of heterologous interaction between the HC-Pro of LMV and of PVY was also demonstrated. No interaction involving either the P1 or the P3 proteins was detected. A series of ordered deletions from either the N- or C-terminal end of the LMV HC-Pro was used to map the domain involved in interaction to the 72 N-terminal amino acids of the protein, a region known to be dispensable for virus viability but necessary for aphid transmission. A similar but less detailed analysis mapped the interacting domain to the N-terminal half of the PVY HC-Pro. Copyright 1999 Academic Press. PMID: 10329571 [PubMed - indexed for MEDLINE] 747: J Mol Biol 1999 May 7;288(3):337-52 Characterization of KLBCK1, encoding a MAP kinase kinase kinase of Kluyveromyces lactis. Jacoby JJ, Kirchrath L, Gengenbacher U, Heinisch JJ. Institut fur Mikrobiologie, Heinrich-Heine-Universitat Dusseldorf, Universitatsstr.1 Geb.: 26.12, Dusseldorf, D-40225, FRG. The cellular integrity and response to hypoosmotic conditions in the yeast Saccharomyces cerevisiae are ensured by a MAP kinase signal transduction pathway mediated by the yeast homolog of mammalian protein kinase C. Bck1p functions as the MAP kinase kinase kinase of this pathway. Here we report on the cloning and analysis of the BCK1 homolog from the milk yeast Kluyveromyces lactis (KlBCK1). The deduced protein sequences display three highly conserved domains with the serine/threonine kinase domain containing 89 % identical amino acid residues. Interestingly, a region identified in KlBck1p as a putative SAM domain, mediating protein-protein interactions, is also conserved in ScBck1p. Yet, two-hybrid analyses indicate that this region may not be involved in dimerization of KlBck1p in contrast to its S. cerevisiae counterpart. Expression of KlBCK1 fully complements the defects in a Scbck1 null mutant and is capable of activating the pathway as indicated by a reporter system based on the transcription factor Rlm1p. However, deletion from the haploid K. lactis genome does not result in a loss of cellular integrity under a variety of conditions tested. Thus, despite the functional conservation in this component of the MAP kinase pathway in both yeast, cellular integrity in K. lactis may depend at least in part on different signalling mechanisms when compared with S. cerevisiae. Copyright 1999 Academic Press. PMID: 10329146 [PubMed - indexed for MEDLINE] 748: Trends Biochem Sci 1999 Apr;24(4):146-50 Interactions among pathways for phosphatidylcholine metabolism, CTP synthesis and secretion through the Golgi apparatus. Kent C, Carman GM. Dept of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA. Phosphatidylcholine is the major phospholipid in eukaryotic cells. It serves as a structural component of cell membranes and a reservoir of several lipid messengers. Recent studies in yeast and mammalian systems have revealed interrelationships between the two pathways of phosphatidylcholine metabolism, and between these pathways and those for CTP synthesis and secretion via the Golgi. These processes involve the regulation of the CDP-choline and phosphatidylethanolamine-methylation pathways of phosphatidylcholine synthesis, CTP synthetase, phospholipase D and the phospholipid-transfer protein Sec14p. Publication Types: Review Review, Tutorial PMID: 10322420 [PubMed - indexed for MEDLINE] 749: J Cell Biochem 1999 Jun 1;73(3):390-9 Human p120ctn catenin: tissue-specific expression of isoforms and molecular interactions with BP180/type XVII collagen. Aho S, Rothenberger K, Uitto J. Department of Dermatology, Jefferson Medical College, and Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. aho1@mail.tju.edu Catenins, a family of structurally related proteins, are involved in epidermal keratinocyte cell-cell adhesion by interacting through their central Armadillo repeats with the intracellular domains of cadherins, transmembrane components of the adhesion junctions. p120ctn is a catenin expressed in different isoforms due to alternative splicing and multiple translation start sites. BP180 is a collagenous transmembrane protein (type XVII collagen) localized to hemidesmosomal attachment complexes in basal keratinocytes. In this study, we have delineated the molecular interaction between these two proteins utilizing the yeast two-hybrid system, which was confirmed by an in vitro protein-protein interaction assay. Specifically, it was shown that an amino-terminal segment of BP180 (aa. 13-25) contains the information necessary for binding to p120ctn isoforms 1-3, but not to the isoform 4, suggesting that the interacting domain is located immediately upstream from the Armadillo repeats and is encoded by exons 5 and 6, which are subject to alternative splicing only in a minority of transcripts. In addition to epidermal keratinocytes, p120ctn was shown to be expressed in a variety of adult and fetal tissues as well as in a number of human tumors. The expression pattern of various p120ctn transcripts, reflecting alternative splicing of the 5' exons, was strikingly similar between the corresponding adult and fetal tissues, while the expression patterns were discordant between certain tumors and their normal parental tissues, suggesting a functional role for the tissue-specific expression of the p120ctn isoforms. Finally, the tissue-specific expression of BP180 was shown to partially overlap with that of p120ctn, suggesting that the interaction of these two proteins may contribute to the modulation of cell-cell/matrix interactions in such tissues. PMID: 10321838 [PubMed - indexed for MEDLINE] 750: Yeast 1999 Apr;15(6):481-96 Analysis of genetic interactions between DHH1, SSD1 and ELM1 indicates their involvement in cellular morphology determination in Saccharomyces cerevisiae. Moriya H, Isono K. Graduate School of Science and Technology, Kobe University, Japan. The DHH1 gene of Saccharomyces cerevisiae belongs to a family of genes that encode highly conserved DEAD-box proteins commonly present in various eukaryotic organisms. Its precise function in yeast has not yet been well documented. To investigate its role in vivo, we constructed a DHH1 disruptant, characterized it genetically and searched for genes the mutations in which would cause synthetic lethality in combination with the DHH1 disruption. CDC28, ELM1 and SSD1 were thus found to be such candidates and we subsequently analysed their interactions. Mutations in ELM1 were previously reported to result in the elongation of cells. We confirmed this phenotype and observed in addition elongated bud formation in an Elm1p overproducing strain. Also, Elm1p fused with the green fluorescent protein (GFP) was found to be localized at the bud neck. These and other observations seem to suggest that Elm1p plays a role during cytokinesis in S. cerevisiae. The phenotypes of strains harbouring either delta dhh1 delta elm1 or ssd1-d delta elm1 were very similar to each other, showing abnormal cellular morphology and defects in cytokinesis and mitosis. Furthermore, DHH1 and SSD1 could functionally complement each other in the ade2 red colour pigment formation, hypersensitivity to SDS, growth on synthetic media and at high temperature. A triple mutant, delta dhh1 ssd1-d delta elm1, apparently had very fragile cell walls and could grow only in a medium supplemented with 1 M sorbitol. PMID: 10234786 [PubMed - indexed for MEDLINE] 751: EMBO J 1999 May 4;18(9):2424-34 A Bub2p-dependent spindle checkpoint pathway regulates the Dbf2p kinase in budding yeast. Fesquet D, Fitzpatrick PJ, Johnson AL, Kramer KM, Toyn JH, Johnston LH. Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Exit from mitosis in all eukaroytes requires inactivation of the mitotic kinase. This occurs principally by ubiquitin-mediated proteolysis of the cyclin subunit controlled by the anaphase-promoting complex (APC). However, an abnormal spindle and/or unattached kinetochores activates a conserved spindle checkpoint that blocks APC function. This leads to high mitotic kinase activity and prevents mitotic exit. DBF2 belongs to a group of budding yeast cell cycle genes that when mutated prevent cyclin degradation and block exit from mitosis. DBF2 encodes a protein kinase which is cell cycle regulated, peaking in metaphase-anaphase B/telophase, but its function remains unknown. Here, we show the Dbf2p kinase activity to be a target of the spindle checkpoint. It is controlled specifically by Bub2p, one of the checkpoint components that is conserved in fission yeast and higher eukaroytic cells. Significantly, in budding yeast, Bub2p shows few genetic or biochemical interactions with other members of the spindle checkpoint. Our data now point to the protein kinase Mps1p triggering a new parallel branch of the spindle checkpoint in which Bub2p blocks Dbf2p function. PMID: 10228157 [PubMed - indexed for MEDLINE] 752: Anticancer Res 1999 Jan-Feb;19(1A):317-27 Expression, purification and DNA-cleavage activity of recombinant 68-kDa human topoisomerase I-target for antitumor drugs. Bronstein IB, Wynne-Jones A, Sukhanova A, Fleury F, Ianoul A, Holden JA, Alix AJ, Dodson GG, Jardillier JC, Nabiev I, Wilkinson AJ. Department of Chemistry, University of York, United Kingdom. igor@yorvic.york.ac.uk The gene encoding human DNA topoisomerase (topo) I, the target of numerous anticancer drugs, has been subcloned into bacterial, yeast and baculovirus-based expression systems in attempts to overexpress the enzyme for extensive structural and functional characterisation. Expression in E.coli produced a protein which was not suitable for structural studies. Expression in the yeast system was more successful enabling the enzyme to be purified and characterised. However, the resulting yield was modest for our requirements and the full-length protein was found to be susceptible to proteolysis when expressed in this system. As it is known that topo I from human placental tissue contains significant quantities of a 68kDa proteolytic fragment which retains both DNA relaxation and cleavage activity, we have isolated this fragment and shown by N-terminal sequence analysis that it starts at Lysine-191. This information was used to construct vectors which direct the overexpression of this fragment in baculovirus infected insect cells. The recombinant protein has been purified to homogeneity in a yield of 5-10mg/l of cell culture. The fragment is stable and retains all of the DNA driving activities of the intact enzyme. We have characterised the interactions of the topo I fragment with synthetic DNA substrates and identified oligonucleotides and conditions that allow covalent complexes between 68kDa topo I and DNA to be formed with high efficiency and in large quantity. A flow linear dichroism technique has been further developed and applied for real-time monitoring of supercoiled (sc) DNA relaxation by the enzyme and for comparative analysis of inhibition of 68kDa topo I by camptothecin (CPT). PMID: 10226561 [PubMed - indexed for MEDLINE] 753: J Cell Biol 1999 May 3;145(3):447-55 Phosphorylation regulates in vivo interaction and molecular targeting of serine/arginine-rich pre-mRNA splicing factors. Yeakley JM, Tronchere H, Olesen J, Dyck JA, Wang HY, Fu XD. Division of Cellular and Molecular Medicine, Department and School of Medicine, University of California, San Diego, La Jolla, California 92093-0651, USA. The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain-mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein-specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain-mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells. PMID: 10225947 [PubMed - indexed for MEDLINE] 754: Genetics 1999 May;152(1):153-66 Genetic and biochemical interactions involving tricarboxylic acid cycle (TCA) function using a collection of mutants defective in all TCA cycle genes. Przybyla-Zawislak B, Gadde DM, Ducharme K, McCammon MT. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA. The eight enzymes of the tricarboxylic acid (TCA) cycle are encoded by at least 15 different nuclear genes in Saccharomyces cerevisiae. We have constructed a set of yeast strains defective in these genes as part of a comprehensive analysis of the interactions among the TCA cycle proteins. The 15 major TCA cycle genes can be sorted into five phenotypic categories on the basis of their growth on nonfermentable carbon sources. We have previously reported a novel phenotype associated with mutants defective in the IDH2 gene encoding the Idh2p subunit of the NAD+-dependent isocitrate dehydrogenase (NAD-IDH). Null and nonsense idh2 mutants grow poorly on glycerol, but growth can be enhanced by extragenic mutations, termed glycerol suppressors, in the CIT1 gene encoding the TCA cycle citrate synthase and in other genes of oxidative metabolism. The TCA cycle mutant collection was utilized to search for other genes that can suppress idh2 mutants and to identify TCA cycle genes that display a similar suppressible growth phenotype on glycerol. Mutations in 7 TCA cycle genes were capable of functioning as suppressors for growth of idh2 mutants on glycerol. The only other TCA cycle gene to display the glycerol-suppressor-accumulation phenotype was IDH1, which encodes the companion Idh1p subunit of NAD-IDH. These results provide genetic evidence that NAD-IDH plays a unique role in TCA cycle function. PMID: 10224250 [PubMed - indexed for MEDLINE] 755: Trends Cell Biol 1999 Apr;9(4):150-3 Targeting vesicles to specific sites on the plasma membrane: the role of the sec6/8 complex. Hsu SC, Hazuka CD, Foletti DL, Scheller RH. Howard Hughes Medical Institute and the Dept of Molecular and Cellular Physiology, Stanford University Medical School, Stanford, CA 94305, USA. The delivery of secretory vesicles to appropriate docking and fusion sites on the plasma membrane is crucial for many cellular functions, including formation of synapses, exocytosis of neurotransmitter, establishment and maintenance of cell polarity, cell growth and plasma membrane wound healing. Cell-biological, genetic and biochemical approaches have identified crucial proteins and protein interactions important for vesicle docking and fusion. However, a description of the molecular mechanisms underlying vesicle targeting to specific membrane-fusion sites remains elusive. This review discusses a set of proteins that might direct vesicles to specific domains of the plasma membrane. Publication Types: Review Review, Tutorial PMID: 10203793 [PubMed - indexed for MEDLINE] 756: Biochemistry 1999 Apr 27;38(17):5401-11 Assembly of the type 1 procollagen molecule: selectivity of the interactions between the alpha 1(I)- and alpha 2(I)-carboxyl propeptides. Alvares K, Siddiqui F, Malone J, Veis A. Division of Oral Biology, Northwestern University Dental School, Chicago, Illinois 60611, USA. Assembly of the heterotrimeric procollagen I molecule is initiated by interactions between the carboxyl propeptide domains of the completed nascent pro alpha chains. The [pro alpha 1(I)]2[pro alpha 2(I)] heterotrimer is the predominant molecule, with much smaller amounts of stable [pro alpha 1(I)]3 homotrimer also being formed. However, the [pro alpha 2(1)]3 homotrimer has not been detected, raising questions as to the mechanism of chain assembly and why [pro alpha2(1)]3 homotrimers are not formed. These questions have been examined here by expressing the intact and amino- or carboxyl-terminal truncated C-propeptides of the pro alpha chains recombinantly in bacteria and in a coupled transcription/translation reticulocyte lysate system. Their interactions were studied in vitro by binding analyses and in vivo by using the yeast two-hybrid system. The C-pro alpha 1(I) interacted with itself, and with C-pro alpha 2(I), as expected. Surprisingly, the C-pro alpha 2(I) also interacted with itself, both in vitro and in vivo. While the interaction of C-pro alpha 2(I) with itself and C-pro alpha 1(I) in vitro was strong, these interactions were weaker in vivo. Deletion of 36 amino acids from the C-terminal domain of C-pro alpha 1 had no effect on its binding to intact self or intact C-pro alpha 2, but the same deletion in C-pro alpha 2 completely abolished its binding to intact C-pro alpha 2 and to C-pro alpha 1. Comparable N-terminal deletions in C-pro alpha 1 or C-pro alpha 2 diminished, but did not abolish, their binding to intact C-pro alpha 1 and C-pro alpha 2. In the yeast two-hybrid system, C-pro alpha 2 interacted with itself more weakly than with C-pro alpha 1. Molecular modeling and circular dichroism analyses showed that C-pro alpha 1 and C-pro alpha 2 have different folded structures and stability. Studies with antibodies specific to the C-pro alpha1 and alpha2 peptides showed them to precipitate different, specific, and distinct cell proteins from fibroblast lysates. The C-pro alpha 2(I) antibody complexed with more cell proteins. We hypothesize that the lack of pro alpha 2(I) homotrimers is not due to the inability of the C-pro alpha 2(I) to interact with itself, but rather to the competing presence of other cell proteins. The specificity of these interactions may reside in conformational differences in N- and C-terminal sequences of the two propeptides or in their different folding patterns. PMID: 10220327 [PubMed - indexed for MEDLINE] 757: Nucleic Acids Res 1999 May 15;27(10):2181-8 A Saccharomyces cerevisiae RNA 5'-triphosphatase related to mRNA capping enzyme. Rodriguez CR, Takagi T, Cho EJ, Buratowski S. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: the RNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Using computer homology searching, a S. cerevisiae gene was identified that encodes a protein resembling the C-terminal region of Cet1. Accordingly, we designated this gene CTL1 (capping enzyme RNAtriphosphatase-like 1). CTL1 is not essential for cell viability and no genetic or physical interactions with the capping enzyme genes were observed. The protein is found in both the nucleus and cytoplasm. Recombinant Ctl1 protein releases gamma-phosphate from the 5'-end of RNA to produce a diphosphate terminus. The enzyme is specific for polynucleotide RNA in the presence of magnesium, but becomes specific for nucleotide triphosphates in the presence of manganese. Ctl1 is the second member of the yeast RNA triphosphatase family, but is probably involved in an RNA processing event other than mRNA capping. PMID: 10219091 [PubMed - indexed for MEDLINE] 758: Nucleic Acids Res 1999 May 15;27(10):2165-74 Isolation of Ku70-binding proteins (KUBs). Yang CR, Yeh S, Leskov K, Odegaard E, Hsu HL, Chang C, Kinsella TJ, Chen DJ, Boothman DA. Department of Radiation Oncology and Department of Pharmacology and the Ireland Cancer Center,Laboratory of Molecular Stress Responses, Case Western Reserve University, BRB-326 East,10900 Euclid Avenue, Cleveland, OH 44106-4942, USA. DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80. KUB3 and KUB4 are unknown, >10 kb trans-cripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/XIP8 with Ku70 was confirmed by far-western analyses. Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation. PMID: 10219089 [PubMed - indexed for MEDLINE] 759: Nucleic Acids Res 1999 May 15;27(10):2126-34 A role for Ctr9p and Paf1p in the regulation G1 cyclin expression in yeast. Koch C, Wollmann P, Dahl M, Lottspeich F. Institut fur Genetik der Universitat Munchen, Maria-Ward-Strasse 1a, D-80638 Munchen, Germany. c.koch@lrz.uni-muenchen.de Entry into the cell cycle in budding yeast involves transcriptional activation of G1cyclin genes and DNA synthesis genes when cells reach a critical size in late G1. Expression of G1cyclins CLN1 and CLN2 is regulated by the transcription factor SBF (composed of Swi4p and Swi6p) and depends on the cyclin-dependent Cdc28 protein kinase and cyclin Cln3p. To identify novel regulators of SBF-dependent gene expression we screened for mutants that fail to activate transcription of G1cyclins. We found mutations in a gene called CTR9. ctr9 mutants are inviable at 37 degrees C and accumulate large cells. CTR9 is identical to CDP1. CTR9 encodes a conserved nuclear protein of 125 kDa containing several TPR repeats implicated in protein-protein interactions. We show that Ctr9p is a component of a high molecular weight protein complex. Using immuno-affinity chromatography we found that Ctr9p associates with polypeptides of 50 and 65 kDa. By mass spectrometry these were identified as Cdc73p and Paf1p. We show that Paf1p, like Ctr9p, is required for efficient CLN2 transcription, whereas Cdc73p is not. Paf1p and Cdc73p were previously reported to be RNA poly-merase II-associated proteins, suggesting that the Ctr9p complex may interact with the general transcription apparatus. PMID: 10219085 [PubMed - indexed for MEDLINE] 760: Nucleic Acids Res 1999 May 15;27(10):2072-9 Formation of the yeast Mre11-Rad50-Xrs2 complex is correlated with DNA repair and telomere maintenance. Chamankhah M, Xiao W. Department of Microbiology and Immunology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada. The yeast Mre11 is a multi-functional protein and is known to form a protein complex with Rad50 and Xrs2. In order to elucidate the relationship between Mre11 complex formation and its mitotic functions, and to determine domain(s) required for Mre11 protein interactions, we performed yeast two-hybrid and functional analyses with respect to Mre11 DNA repair and telomere maintenance. Evidence presented in this study indicates that the N-terminal region of Mre11 constitutes the core homo-dimerization and hetero-dimerization domain and is sufficient for Mre11 DNA repair and maintaining the wild-type telomere length. In contrast, a stretch of 134 amino acids from the extreme C-terminus, although essential for achieving a full level of self-association, is not required for the aforementioned Mre11 mitotic functions. Interestingly, deletion of these same 134 amino acids enhanced the interaction of Mre11 with Rad50 and Xrs2, which is consistent with the notion that this region is specific for meiotic functions. While Mre11 self-association alone is insufficient to provide the above mitotic activities, our results are consistent with a strong correlation between Mre11-Rad50-Xrs2 complex formation, mitotic DNA repair and telomere maintenance. This correlation was further strengthened by analyzing two mre11 phosphoesterase motif mutants ( mre11-2 and rad58S ), which are defective in DNA repair, telomere maintenance and protein interactions, and a rad50S mutant, which is normal in both complex formation and mitotic functions. Together, these results support and extend a current model regarding Mre11 structure and functions in mitosis and meiosis. PMID: 10219078 [PubMed - indexed for MEDLINE] 761: FEBS Lett 1999 Mar 19;447(1):115-20 The presumed potassium carrier Trk2p in Saccharomyces cerevisiae determines an H+-dependent, K+-independent current. Bihler H, Gaber RF, Slayman CL, Bertl A. Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, USA. Ionic currents related to the major potassium uptake systems in Saccharomyces cerevisiae were examined by whole cell patch-clamping, under K+ replete conditions. Those currents have the following properties. They (1) are inward under all conditions investigated, (2) arise instantaneously with appropriate voltage steps, (3) depend solely upon the moderate affinity transporter Trk2p, not upon the high affinity transporter Trk1p. They (4) appear to be independent of the extracellular K+ concentration, (5) are also independent of extracellular Ca2+, Mg2+ and Cl- but (6) are strongly dependent on extracellular pH, being large at low pH (up to several hundred pA at -200 mV and pH 4) and near zero at high pH (above 7.5). They (7) increase in proportion to log[H+]o, rather than directly in proportion to the proton concentration and (8) behave kinetically as if each transporter cycle moved one proton plus one (high pH) or two (low pH) other ions, as yet unidentified. In view of background knowledge on K+ transport related to Trk2p, the new results suggest that the K+ status of yeast cells modulates both the kinetics of Trk2p-mediated transport and the identity of ions involved. That modulation could act either on the Trk2 protein itself or on interactions of Trk2 with other proteins in a hypothetical transporter complex. Structural considerations suggest a strong analogy to the KtrAB system in Vibrio alginolyticus and/or the TrkH system in Escherichia coli. PMID: 10218594 [PubMed - indexed for MEDLINE] 762: J Bacteriol 1999 May;181(9):2963-5 The C-terminal domain of dnaQ contains the polymerase binding site. Taft-Benz SA, Schaaper RM. Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA. The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective alpha binding mutant. The data are consistent with the existence of two functional domains in epsilon, with the C-terminal domain responsible for polymerase binding. PMID: 10217794 [PubMed - indexed for MEDLINE] 763: J Biomol Struct Dyn 1999 Feb;16(4):757-74 The effect of queuosine on tRNA structure and function. Morris RC, Brown KG, Elliott MS. Department of Biochemistry and Chemistry, Old Dominion University, Norfolk, VA 23529, USA. Computational modeling was performed to determine the potential function of the queuosine modification of tRNA found in wobble position 34 of tRNAasp, tRNAasn, tRNAhis, and tRNAtyr. Using the crystal structure of tRNAasp and a tRNA-tRNA-mRNA complex model, we show that the queuosine modification serves as a structurally restrictive base for tRNA anticodon loop flexibility. An extended intraresidue and intramolecular hydrogen bonding network is established by queuosine. The quaternary amine of the 7-aminomethyl side chain hydrogen bonds with the base's carbonyl oxygen. This positions the dihydroxycyclopentenediol ring of queuosine in proper orientation for hydrogen bonding with the backbone of the neighboring uridine 33 residue. The interresidue association stabilizes the formation of a cross-loop hydrogen bond between the uridine 33 base and the phosphoribosyl backbone of the cytosine at position 36. Additional interactions between RNAs in the translation complex were studied with regard to potential codon context and codon bias effects. Neither steric nor electrostatic interaction occurs between aminoacyl- and peptidyl-site tRNA anticodon loops that are modified with queuosine. However, there is a difference in the strength of anticodon/codon associations (codon bias) based on the presence or lack of queuosine in the wobble position of the tRNA. Unmodified (guanosine-containing) tRNAasp forms a very stable association with cytosine (GAC), but is much less stable in complex with a uridine-containing codon (GAU). Queuosine-modified tRNAasp exhibits no bias for either of cognate codons GAC or GAU and demonstrates a lower binding energy similar to the wobble pairing of guanosine-containing tRNA with a GAU codon. This is proposed to be due to the inflexibility of the queuosine-modified anticodon loop to accommodate proper positioning for optimal Watson-Crick type associations. A preliminary survey of codon usage patterns in oncodevelopmental versus housekeeping gene transcripts suggests a significant difference in bias for the queuosine-associated codons. Therefore, the queuosine modification may have the potential to influence cellular growth and differentiation by codon bias-based regulation of protein synthesis for discrete mRNA transcripts. PMID: 10217448 [PubMed - indexed for MEDLINE] 764: Biochimie 1999 Jan-Feb;81(1-2):87-105 The contribution of homologous recombination in preserving genome integrity in mammalian cells. Thompson LH, Schild D. Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551-0808, USA. Although it is clear that mammalian somatic cells possess the enzymatic machinery to perform homologous recombination of DNA molecules, the importance of this process in mitigating DNA damage has been uncertain. An initial genetic framework for studying homologous recombinational repair (HRR) has come from identifying relevant genes by homology or by their ability to correct mutants whose phenotypes are suggestive of recombinational defects. While yeast has been an invaluable guide, higher eukaryotes diverge in the details and complexity of HRR. For eliminating DSBs, HRR and end-joining pathways share the burden, with HRR contributing critically during S and G2 phases. It is likely that the removal of interstrand cross-links is absolutely dependent on efficient HRR, as suggested by the extraordinary sensitivity of the ercc1, xpf/ercc4, xrcc2, and xrcc3 mutants to cross-linking chemicals. Similarly, chromosome stability in untreated cells requires intact HRR, which may eliminate DSBs arising during DNA replication and thereby prevent chromosome aberrations. Complex regulation of HRR by cell cycle checkpoint and surveillance functions is suggested not only by direct interactions between human Rad51 and p53, c-Abl, and BRCA2, but also by very high recombination rates in p53-deficient cells. Publication Types: Review Review, Academic PMID: 10214914 [PubMed - indexed for MEDLINE] 765: J Biol Chem 1999 Apr 30;274(18):12480-7 Genetic analysis and enzyme activity suggest the existence of more than one minimal functional unit capable of synthesizing phosphoribosyl pyrophosphate in Saccharomyces cerevisiae. Hernando Y, Carter AT, Parr A, Hove-Jensen B, Schweizer M. Genetics and Microbiology Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom. The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 or PRS3; simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3 together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their phosphoribosyl pyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions of PRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast phosphoribosyl pyrophosphate synthetase exists in vivo as multimeric complex(es). PMID: 10212224 [PubMed - indexed for MEDLINE] 766: Curr Biol 1999 Mar 25;9(6):325-8 Mouse Rad54 affects DNA conformation and DNA-damage-induced Rad51 foci formation. Tan TL, Essers J, Citterio E, Swagemakers SM, de Wit J, Benson FE, Hoeijmakers JH, Kanaar R. Department of Cell Biology and Genetics, Erasmus University, Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands. Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4] [5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6]. Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed. Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules. Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA [2] [3] [4]. PMID: 10209103 [PubMed - indexed for MEDLINE] 767: Mol Cell Biol 1999 May;19(5):3580-7 Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70. Robinson NG, Guo L, Imai J, Toh-E A, Matsui Y, Tamanoi F. Department of Microbiology and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, California 90095-1489, USA. The Rho3 protein plays a critical role in the budding yeast Saccharomyces cerevisiae by directing proper cell growth. Rho3 appears to influence cell growth by regulating polarized secretion and the actin cytoskeleton, since rho3 mutants exhibit large rounded cells with an aberrant actin cytoskeleton. To gain insights into how Rho3 influences these events, we have carried out a yeast two-hybrid screen using an S. cerevisiae cDNA library to identify proteins interacting with Rho3. Two proteins, Exo70 and Myo2, were identified in this screen. Interactions with these two proteins are greatly reduced or abolished when mutations are introduced into the Rho3 effector domain. In addition, a type of mutation known to produce dominant negative mutants of Rho proteins abolished the interaction with both of these proteins. In contrast, Rho3 did not interact with protein kinase C (Pkc1), an effector of another Rho family protein, Rho1, nor did Rho1 interact with Exo70 or Myo2. Rho3 did interact with Bni1, another effector of Rho1, but less efficiently than with Rho1. The interaction between Rho3 and Exo70 and between Rho3 and Myo2 was also demonstrated with purified proteins. The interaction between Exo70 and Rho3 in vitro was dependent on the presence of GTP, since Rho3 complexed with guanosine 5'-O-(3-thiotriphosphate) interacted more efficiently with Exo70 than Rho3 complexed with guanosine 5'-O-(3-thiodiphosphate). Overlapping subcellular localization of the Rho3 and Exo70 proteins was demonstrated by indirect immunofluorescence. In addition, patterns of localization of both Exo70 and Rho3 were altered when a dominant active allele of RHO3, RHO3(E129,A131), which causes a morphological abnormality, was expressed. These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton. PMID: 10207081 [PubMed - indexed for MEDLINE] 768: EMBO J 1999 Apr 15;18(8):2229-40 Role of the essential yeast protein PSU1 in p6anscriptional enhancement by the ligand-dependent activation function AF-2 of nuclear receptors. Gaudon C, Chambon P, Losson R. Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, College de France, BP 163, 67404 Illkirch Cedex, France. Nuclear receptors (NRs) can function as ligandinducible transregulators in both mammalian and yeast cells, indicating that important features of transcriptional control have been conserved throughout evolution. We report here the isolation and characterization of an essential yeast protein of unknown function, PSU1, which exhibits properties expected for a co-activator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of NRs. PSU1 interacts in a ligand-dependent manner with the LBD of several NRs, including retinoic acid (RARalpha), retinoid X (RXRalpha), thyroid hormone (TRalpha), vitamin D3 (VDR) and oestrogen (ERalpha) receptors. Importantly, both in yeast and in vitro, these interactions require the integrity of the AF-2 activating domain. When tethered to a heterologous DNA-binding domain, PSU1 can activate transcription on its own. By using yeast reporter cells that express PSU1 conditionally, we show that PSU1 is required for transactivation by the AF-2 of ERalpha. Taken together these data suggest that in yeast, PSU1 is involved in ligand-dependent transactivation by NRs. Sequence analysis revealed that in addition to a highly conserved motif found in a family of MutT-related proteins, PSU1 contains several alpha-helical leucine-rich motifs sharing the consensus sequence LLxPhiL (x, any amino acid; Phi, hydrophobic amino acid) in regions that elicit either transactivation or NR-binding activity. PMID: 10205176 [PubMed - indexed for MEDLINE] 769: J Immunol 1999 Apr 15;162(8):5019-24 Mutations that cause the Wiskott-Aldrich syndrome impair the interaction of Wiskott-Aldrich syndrome protein (WASP) with WASP interacting protein. Stewart DM, Tian L, Nelson DL. Immunophysiology Section, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, immune deficiency, and a proclivity toward lymphoid malignancy. Lymphocytes of affected individuals show defects of activation, motility, and cytoskeletal structure. The disease gene encodes a 502-amino acid protein named the WAS protein (WASP). Studies have identified a number of important interactions that place WASP in a role of integrating signaling pathways with cytoskeletal function. We performed a two-hybrid screen to identify proteins interacting with WASP and cloned a proline-rich protein as a specific WASP interactor. Our clone of this protein, termed WASP interacting protein (WIP) by others, shows a difference in seven amino acid residues, compared with the previously published sequence revealing an additional profilin binding motif. Deletion mutant analysis reveals that WASP residues 101-151 are necessary for WASP-WIP interaction. Point mutant analyses in the two-hybrid system and in vitro show impairment of WASP-WIP interaction with three WASP missense mutants known to cause WAS. We conclude that impaired WASP-WIP interaction may contribute to WAS. PMID: 10202051 [PubMed - indexed for MEDLINE] 770: Nucleic Acids Res 1999 May 1;27(9):1978-84 The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G-G paired DNAs. Sun H, Bennett RJ, Maizels N. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA. The Saccharomyces cerevisiae Sgs1p helicase localizes to the nucleolus and is required to maintain the integrity of the rDNA repeats. Sgs1p is a member of the RecQ DNA helicase family, which also includes Schizo-saccharomyces pombe Rqh1, and the human BLM and WRN genes. These genes encode proteins which are essential to maintenance of genomic integrity and which share a highly conserved helicase domain. Here we show that recombinant Sgs1p helicase efficiently unwinds guanine-guanine (G-G) paired DNA. Unwinding of G-G paired DNA is ATP- and Mg2+-dependent and requires a short 3' single-stranded tail. Strikingly, Sgs1p unwinds G-G paired substrates more efficiently than duplex DNAs, as measured either in direct assays or by competition experiments. Sgs1p efficiently unwinds G-G paired telomeric sequences, suggesting that one function of Sgs1p may be to prevent telomere-telomere interactions which can lead to chromosome non-disjunction. The rDNA is G-rich and has considerable potential for G-G pairing. Diminished ability to unwind G-G paired regions may also explain the deleterious effect of mutation of Sgs1 on rDNA stability, and the accelerated aging characteristic of yeast strains that lack Sgs1 as well as humans deficient in the related WRN helicase. PMID: 10198430 [PubMed - indexed for MEDLINE] 771: Genetics 1999 Apr;151(4):1459-70 Dna2 mutants reveal interactions with Dna polymerase alpha and Ctf4, a Pol alpha accessory factor, and show that full Dna2 helicase activity is not essential for growth. Formosa T, Nittis T. Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA. formosa@medschool.med.utah.edu Mutations in the gene for the conserved, essential nuclease-helicase Dna2 from the yeast Saccharomyces cerevisiae were found to interact genetically with POL1 and CTF4, which encode a DNA Polymerase alpha subunit and an associated protein, suggesting that Dna2 acts in a process that involves Pol alpha. DNA2 alleles were isolated that cause either temperature sensitivity, sensitivity to alkylation damage, or both. The alkylation-sensitive alleles clustered in the helicase domain, including changes in residues required for helicase activity in related proteins. Additional mutations known or expected to destroy the ATPase and helicase activities of Dna2 were constructed and found to support growth on some media but to cause alkylation sensitivity. Only damage-sensitive alleles were lethal in combination with a ctf4 deletion. Full activity of the Dna2 helicase function is therefore not needed for viability, but is required for repairing damage and for tolerating loss of Ctf4. Arrest of dna2 mutants was RAD9 dependent, but deleting this checkpoint resulted in either no effect or suppression of defects, including the synthetic lethality with ctf4. Dna2 therefore appears to act in repair or lagging strand synthesis together with Pol alpha and Ctf4, in a role that is optimal with, but does not require, full helicase activity. PMID: 10101169 [PubMed - indexed for MEDLINE] 772: Genetics 1999 Apr;151(4):1353-63 Suppression of a nuclear aep2 mutation in Saccharomyces cerevisiae by a base substitution in the 5'-untranslated region of the mitochondrial oli1 gene encoding subunit 9 of ATP synthase. Ellis TP, Lukins HB, Nagley P, Corner BE. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168, Australia. Mutations in the nuclear AEP2 gene of Saccharomyces generate greatly reduced levels of the mature form of mitochondrial oli1 mRNA, encoding subunit 9 of mitochondrial ATP synthase. A series of mutants was isolated in which the temperature-sensitive phenotype resulting from the aep2-ts1 mutation was suppressed. Three strains were classified as containing a mitochondrial suppressor: these lost the ability to suppress aep2-ts1 when their mitochondrial genome was replaced with wild-type mitochondrial DNA (mtDNA). Many other isolates were classified as containing dominant nuclear suppressors. The three mitochondrion-encoded suppressors were localized to the oli1 region of mtDNA using rho- genetic mapping techniques coupled with PCR analysis; DNA sequencing revealed, in each case, a T-to-C nucleotide transition in mtDNA 16 nucleotides upstream of the oli1 reading frame. It is inferred that the suppressing mutation in the 5' untranslated region of oli1 mRNA restores subunit 9 biosynthesis by accommodating the modified structure of Aep2p generated by the aep2-ts1 mutation (shown here to cause the substitution of proline for leucine at residue 413 of Aep2p). This mode of mitochondrial suppression is contrasted with that mediated by heteroplasmic rearranged rho- mtDNA genomes bypassing the participation of a nuclear gene product in expression of a particular mitochondrial gene. In the present study, direct RNA-protein interactions are likely to form the basis of suppression. PMID: 10101162 [PubMed - indexed for MEDLINE] 773: Genetics 1999 Apr;151(4):1273-85 Analysis of mutations in the yeast mRNA decapping enzyme. Tharun S, Parker R. Departments of Molecular and Cellular Biology and Biochemistry and the Howard Hughes Medical Institute, University of Arizona, Tucson, Arizona 85721-0106, USA. A major mechanism of mRNA decay in yeast is initiated by deadenylation, followed by mRNA decapping, which exposes the transcript to 5' to 3' exonucleolytic degradation. The decapping enzyme that removes the 5' cap structure is encoded by the DCP1 gene. To understand the function of the decapping enzyme, we used alanine scanning mutagenesis to create 31 mutant versions of the enzyme, and we examined the effects of the mutations both in vivo and in vitro. Two types of mutations that affected mRNA decapping in vivo were identified, including a temperature-sensitive allele. First, two mutants produced decapping enzymes that were defective for decapping in vitro, suggesting that these mutated residues are required for enzymatic activity. In contrast, several mutants that moderately affected mRNA decapping in vivo yielded decapping enzymes that had at least the same specific activity as the wild-type enzyme in vitro. Combination of alleles within this group yielded decapping enzymes that showed a strong loss of function in vivo, but that still produced fully active enzymes in vitro. This suggested that interactions of the decapping enzyme with other factors may be required for efficient decapping in vivo, and that these particular mutations may be disrupting such interactions. Interestingly, partial loss of decapping activity in vivo led to a defect in normal deadenylation-dependent decapping, but it did not affect the rapid deadenylation-independent decapping triggered by early nonsense codons. This observation suggested that these two types of mRNA decapping differ in their requirements for the decapping enzyme. PMID: 10101156 [PubMed - indexed for MEDLINE] 774: Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3572-7 Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein. Jiang J, Zhang Y, Krainer AR, Xu RM. W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. Human p32 (also known as SF2-associated p32, p32/TAP, and gC1qR) is a conserved eukaryotic protein that localizes predominantly in the mitochondrial matrix. It is thought to be involved in mitochondrial oxidative phosphorylation and in nucleus-mitochondrion interactions. We report the crystal structure of p32 determined at 2.25 A resolution. The structure reveals that p32 adopts a novel fold with seven consecutive antiparallel beta-strands flanked by one N-terminal and two C-terminal alpha-helices. Three monomers form a doughnut-shaped quaternary structure with an unusually asymmetric charge distribution on the surface. The implications of the structure on previously proposed functions of p32 are discussed and new specific functional properties are suggested. PMID: 10097078 [PubMed - indexed for MEDLINE] 775: Gene 1999 Mar 18;229(1-2):183-91 Translation elongation factor 2 is encoded by a single essential gene in Candida albicans. Mendoza A, Serramia MJ, Capa L, Garcia-Bustos JF. Research Department, Glaxo Wellcome, S.A., Severo Ochoa 2, E-28760, Tres Cantos, Spain. Translation elongation factor 2 (eEF2) is a large protein of more than 800 amino acids which establishes complex interactions with the ribosome in order to catalyze the conformational changes needed for translation elongation. Unlike other yeasts, the pathogenic fungus Candida albicans was found to have a single gene encoding this factor per haploid genome, located on chromosome 2. Expression of this locus is essential for vegetative growth, as evidenced by placing it under the control of a repressible promoter. This C. albicans gene, named EFT2, was cloned and sequenced (EMBL accession number Y09664). Genomic and cDNA sequence analysis identified common transcription initiation and termination signals and an 842 amino acid open reading frame (ORF), which is interrupted by a single intron. Despite some genetic differences, CaEFT2 was capable of complementing a Saccharomyces cerevisiae Deltaeft1 Deltaeft2 null mutant, which lacks endogenous eEF2, indicating that CaEFT2 can be expressed from its own promoter and its intron can be correctly spliced in S. cerevisiae. PMID: 10095118 [PubMed - indexed for MEDLINE] 776: Eur J Biochem 1999 Feb;259(3):939-45 Site-directed mutagenesis of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase. McHarg J, Kelly SM, Price NC, Cooper A, Littlechild JA. School of Chemistry, University of Exeter, UK. Site-specific mutants have been produced in order to investigate the role of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis-proline in the high resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T. maritima PGK, and may therefore have a role in the independent folding of the two domains or in the 'hinge' bending of the molecule during catalysis. The residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe), and the resulting proteins characterised by differential scanning calorimetry (DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic analysis. Although the secondary and tertiary structure of the Pro204His protein is generally similar to that of the wild-type enzyme as assessed by CD, the enzyme is less stable to heat and guanidinium chloride denaturation than the wild-type. In the denaturation experiments two transitions were observed for both the wild-type and the Pro204His mutant, as have been previously reported for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990) Biochemistry 29, 8683-8689]. The first transition is accompanied by an increase in fluorescence intensity leading to a hyperfluorescent state, followed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild-type protein, suggesting that sequence-distant interactions are more rapidly disrupted in this mutant enzyme than in the wild-type enzyme, while sequence-local interactions are disrupted in a similar way. The Michaelis constants (K(m)) for both 3-phospho-D-glycerate and ATP are increased only by three or fourfold, which confirms that, as expected, the substrate binding sites are largely unaffected by the mutation. However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of 'hinge' bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the wild-type and Pro204His enzymes, with considerable loss of both secondary and tertiary structure. It is proposed that the proline residue at 204 in the 'hinge' region of PGK plays a role in the stability and catalytic mechanism of the enzyme. PMID: 10092885 [PubMed - indexed for MEDLINE] 777: J Biol Chem 1999 Apr 2;274(14):9455-62 Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing. Tsai WY, Chow YT, Chen HR, Huang KT, Hong RI, Jan SP, Kuo NY, Tsao TY, Chen CH, Cheng SC. Institute of Microbiology and Immunology, National Yang-Ming University Shih-Pai, Taiwan, Republic of China. The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and protein-protein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex. PMID: 10092627 [PubMed - indexed for MEDLINE] 778: J Gen Virol 1999 Mar;80 ( Pt 3):607-15 A cellular protein which binds hepatitis B virus but not hepatitis B surface antigen. Harvey TJ, Macnaughton TB, Park DS, Gowans EJ. Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston, Queensland, Australia. The envelope of hepatitis B virus (HBV) consists of three related proteins known as the large (L), middle (M) and small (S) hepatitis B surface antigens (HBsAg). L-HBsAg has a 108-119 amino acid extension at the N terminus compared with M-HBsAg and contains the preS1 sequence of the HBV envelope. Previous research has identified this region as the likely virus attachment protein which is thought to interact with the cellular receptor for the virus. However, as the receptor has still not been identified unequivocally, we used the preS1 region of L-HBsAg to screen a human liver cDNA library by the yeast two-hybrid system. Several positive clones were isolated which encoded cellular proteins that interacted with the HBV preS1 protein. The specificity was examined in an independent manner in experiments in which baculovirus-derived glutathione S-transferase (GST)-preS1 was incubated with 35S-labelled protein expressed by in vitro translation from the positive clones. The intensity of the interactions using this alternative approach mirrored those observed in the yeast two-hybrid system and two proteins (an unidentified protein and a mitochondrial protein) were selected for further study. The specificity of the binding reaction between the preS1 protein and these two proteins was further confirmed in a competition assay; HBV purified from serum, but not purified HBsAg, was able to compete with preS1 and thus block GST-preS1 binding to the unidentified protein but not to the mitochondrial protein. The unidentified protein was then expressed as a fusion protein with GST and this was able to bind HBV virions in a direct manner. PMID: 10091999 [PubMed - indexed for MEDLINE] 779: Genes Dev 1999 Mar 15;13(6):686-97 Characterization of the imitation switch subfamily of ATP-dependent chromatin-remodeling factors in Saccharomyces cerevisiae. Tsukiyama T, Palmer J, Landel CC, Shiloach J, Wu C. Laboratory of Molecular Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. ttsukiya@fhcrc.org We have identified and characterized two Imitation Switch genes in Saccharomyces cerevisiae, ISW1 and ISW2, which are highly related to Drosophila ISWI, encoding the putative ATPase subunit of three ATP-dependent chromatin remodeling factors. Purification of ISW1p reveals a four-subunit complex with nucleosome-stimulated ATPase activity, as well as ATP-dependent nucleosome disruption and spacing activities. Purification of ISW2p reveals a two-subunit complex also with nucleosome-stimulated ATPase and ATP-dependent nucleosome spacing activities but no detectable nucleosome disruption activity. Null mutations of ISW1, ISW2, and CHD1 genes cause synthetic lethality in various stress conditions in yeast cells, revealing the first in vivo functions of the ISWI subfamily of chromatin-remodeling complexes and demonstrating their genetic interactions. A single point mutation within the ATPase domain of both ISW1p and ISW2p inactivated all ATP-dependent biochemical activities of the complexes, as well as the ability of the genes to rescue the mutant phenotypes. This demonstrates that the ATP-dependent chromatin-remodeling activities are essential for the in vivo functions of both ISW1 and ISW2 complexes. PMID: 10090725 [PubMed - indexed for MEDLINE] 780: J Cell Biol 1999 Mar 22;144(6):1187-202 A Cdc24p-Far1p-Gbetagamma protein complex required for yeast orientation during mating. Nern A, Arkowitz RA. Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, CB2 2QH, United Kingdom. Oriented cell growth requires the specification of a site for polarized growth and subsequent orientation of the cytoskeleton towards this site. During mating, haploid Saccharomyces cerevisiae cells orient their growth in response to a pheromone gradient overriding an internal landmark for polarized growth, the bud site. This response requires Cdc24p, Far1p, and a heterotrimeric G-protein. Here we show that a two- hybrid interaction between Cdc24p and Gbeta requires Far1p but not pheromone-dependent MAP-kinase signaling, indicating Far1p has a role in regulating the association of Cdc24p and Gbeta. Binding experiments demonstrate that Cdc24p, Far1p, and Gbeta form a complex in which pairwise interactions can occur in the absence of the third protein. Cdc24p localizes to sites of polarized growth suggesting that this complex is localized. In the absence of CDC24-FAR1-mediated chemotropism, a bud site selection protein, Bud1p/Rsr1p, is essential for morphological changes in response to pheromone. These results suggest that formation of a Cdc24p-Far1p-Gbetagamma complex functions as a landmark for orientation of the cytoskeleton during growth towards an external signal. PMID: 10087263 [PubMed - indexed for MEDLINE] 781: J Cell Biol 1999 Mar 8;144(5):963-75 The cortical localization of the microtubule orientation protein, Kar9p, is dependent upon actin and proteins required for polarization. Miller RK, Matheos D, Rose MD. Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08544, USA. In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)- Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Delta, pea2Delta, bud6Delta, and bni1Delta, required for normal polarization and actin cytoskeleton functions and, of these, bni1Delta affected Kar9p localization most severely. Like kar9Delta, bni1Delta mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Delta, the bni1Delta mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Delta bni1Delta double mutants suggested that Kar9p retained some function in bni1Delta mitotic cells. Unlike the polarization mutants, kar9Delta shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Delta. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks. PMID: 10085294 [PubMed - indexed for MEDLINE] 782: J Cell Biol 1999 Mar 8;144(5):947-61 Control of mitotic spindle position by the Saccharomyces cerevisiae formin Bni1p. Lee L, Klee SK, Evangelista M, Boone C, Pellman D. Department of Pediatric Oncology, The Dana-Farber Cancer Institute and Department of Pediatric Hematology, The Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. Alignment of the mitotic spindle with the axis of cell division is an essential process in Saccharomyces cerevisiae that is mediated by interactions between cytoplasmic microtubules and the cell cortex. We found that a cortical protein, the yeast formin Bni1p, was required for spindle orientation. Two striking abnormalities were observed in bni1Delta cells. First, the initial movement of the spindle pole body (SPB) toward the emerging bud was defective. This phenotype is similar to that previously observed in cells lacking the kinesin Kip3p and, in fact, BNI1 and KIP3 were found to be in the same genetic pathway. Second, abnormal pulling interactions between microtubules and the cortex appeared to cause preanaphase spindles in bni1Delta cells to transit back and forth between the mother and the bud. We therefore propose that Bni1p may localize or alter the function of cortical microtubule-binding sites in the bud. Additionally, we present evidence that other bipolar bud site determinants together with cortical actin are also required for spindle orientation. PMID: 10085293 [PubMed - indexed for MEDLINE] 783: Mol Cell Biol 1999 Apr;19(4):2967-76 An activator binding module of yeast RNA polymerase II holoenzyme. Lee YC, Park JM, Min S, Han SJ, Kim YJ. Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University College of Medicine, Kangnam-ku, Seoul 135-230, Korea. The Mediator complex of Saccharomyces cerevisiae is required for both general and regulated transcription of RNA polymerase II (PolII) and is composed of two stable subcomplexes (Srb4 and Rgr1 subcomplexes). To decipher the function of each Mediator subcomplex and to delineate the functional relationship between the subcomplexes, we characterized the compositions and biochemical activities of PolII-Mediator complexes (holoenzymes) prepared from several Mediator mutant strains of S. cerevisiae. We found that holoenzymes devoid of a functional Gal11 module were defective for activated but not basal transcription in a reconstituted in vitro system. This activation-specific defect was correlated with a crippled physical interaction to transcriptional activator proteins, which could be bypassed by artificial recruitment of a mutant holoenzyme to a promoter. Consistent with this observation, a direct interaction between Gal11 and gene-specific transcriptional activator proteins was detected by far-Western analyses and column binding assays. In contrast, the srb5 deletion mutant holoenzyme was defective for both basal and activated transcription, despite its capacity for activator binding that is comparable to that of the wild-type holoenzyme. These results demonstrate that the Gal11 module of the Rgr1 subcomplex is required for the efficient recruitment of PolII holoenzyme to a promoter via activator-specific interactions, while the Srb4 subcomplex functions in the modulation of general polymerase activity. PMID: 10082564 [PubMed - indexed for MEDLINE] 784: Proc Natl Acad Sci U S A 1999 Mar 16;96(6):2656-61 The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks. Chuang RY, Kelly TJ. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21210, USA. The origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae as a protein that specifically binds to origins of DNA replication. Although ORC appears to play an essential role in the initiation of DNA replication in the cells of all eukaryotes, its interactions with DNA have not been defined in species other than budding yeast. We have characterized a Schizosaccharomyces pombe homologue of the ORC subunit, Orc4p. The homologue (Orp4p) consists of two distinct functional domains. The C-terminal domain shows strong sequence similarity to human, frog, and yeast Orc4 proteins, including conserved ATP-binding motifs. The N-terminal domain contains nine copies of the AT-hook motif found in a number of DNA-binding proteins, including the members of the HMG-I(Y) family of chromatin proteins. AT-hook motifs are known from biochemical and structural studies to mediate binding to the minor groove of AT-tracts in DNA. Orp4p is essential for viability of Sc. pombe and is expressed throughout the cell cycle. The Orp4 protein (and its isolated N-terminal domain) binds to the Sc. pombe replication origin, ars1. The DNA binding properties of Orp4p provide a plausible explanation for the characteristic features of Sc. pombe origins of replication, which differ significantly from those of Sa. cerevisiae. PMID: 10077566 [PubMed - indexed for MEDLINE] 785: EMBO J 1999 Mar 15;18(6):1621-9 A trans-acting peptide activates the yeast a1 repressor by raising its DNA-binding affinity. Stark MR, Escher D, Johnson AD. Department of Biochemistry and Biophysics, School of Medicine, University of California, San Francisco, CA 94143-0414, USA. The cooperative binding of gene regulatory proteins to DNA is a common feature of transcriptional control in both prokaryotes and eukaryotes. It is generally viewed as a simple energy coupling, through protein-protein interactions, of two or more DNA-binding proteins. In this paper, we show that the simple view does not account for the cooperative DNA binding of a1 and alpha2, two homeodomain proteins from budding yeast. Rather, we show through the use of chimeric proteins and synthetic peptides that, upon heterodimerization, alpha2 instructs a1 to bind DNA. This change is induced by contact with a peptide contributed by alpha2, and this contact converts a1 from a weak to a strong DNA-binding protein. This explains, in part, how high DNA-binding specificity is achieved only when the two gene regulatory proteins conjoin. We also provide evidence that features of the a1-alpha2 interaction can serve as a model for other examples of protein-protein interactions, including that between the herpes virus transcriptional activator VP16 and the mammalian homeodomain-containing protein Oct-l. PMID: 10075932 [PubMed - indexed for MEDLINE] 786: Biotechnol Appl Biochem 1999 Apr;29 ( Pt 2):165-84 Mutational analysis of sickle haemoglobin (Hb) gelation. Li X, Himanen JP, Martin de Llano JJ, Padovan JC, Chait BT, Manning JM. Department of Biology, Mugar 414, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA. The use of recombinant Hb has provided the advantage that any amino acid substitution can be made at sites not represented by natural mutants or that cannot be modified by chemical procedures. We have recently reported the expression of human sickle Hb (HbS) in the yeast Saccharomyces cerevisiae that carries a plasmid containing the human alpha- and beta-globin cDNA sequences; N-terminal nascent protein processing is correct and a soluble correctly folded Hb tetramer is produced. The yeast system produces a recombinant sickle Hb that is identical by about a dozen biochemical and physiological criteria with the natural sickle Hb purified from the red cells of sickle-cell anaemia patients. Most importantly, the gelling concentration of this recombinant sickle Hb is the same as that of the HbS purified from human sickle red cells. The misfolding of Hb reported for the Escherichia coli-expressed protein is not apparent for Hb expressed in yeast by any of the criteria that we have used for characterization. These findings indicate that this system is well suited to the production of HbS mutants to explore those areas of the HbS tetramer whose roles in the gelation process are not yet defined and to measure quantitatively the strength of such interactions at certain inter-tetrameric contact sites in the deoxy-HbS aggregate. This article reviews our studies on a number of sickle Hb mutants, including polymerization-enhancing HbS mutants and polymerization-inhibiting HbS mutants. PMID: 10075913 [PubMed - indexed for MEDLINE] 787: Methods 1999 Jan;17(1):28-37 Linking mRNA turnover and translation: assessing the polyribosomal association of mRNA decay factors and degradative intermediates. Mangus DA, Jacobson A. Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0122, USA. mRNA decay is a multistep process, often dependent on the active translation of an mRNA and on components of the translation apparatus. In Saccharomyces cerevisiae, several trans-acting factors required for mRNA decay associate with polyribosomes. We have explored the specificity of the interactions of these factors with polyribosomes, using sucrose gradient sedimentation analysis of the yeast UPF1 protein to test whether such interactions are altered when polyribosomes are disrupted by treatment with EDTA, digestion with micrococcal nuclease, or shifting of cells containing a temperature-sensitive eIF3 mutation to the nonpermissive temperature. These experiments, as well as others assaying the strength of factor association in high-salt sucrose gradients, lead us to conclude that Upf1p is tightly bound to the smallest polyribosomes, but not to the 40S or 60S ribosomal subunits. Similar experimental approaches were used to determine whether mRNA decay initiates prior to mRNA release from polyribosomes. Using sucrose gradient fractionation and Northern blotting, we can detect the polysomal association of a PGK1 mRNA decay intermediate and conclude that mRNA decay commences while an mRNA is still being translated. Copyright 1999 Academic Press. PMID: 10075880 [PubMed - indexed for MEDLINE] 788: J Virol 1999 Apr;73(4):2622-32 A brome mosaic virus intergenic RNA3 replication signal functions with viral replication protein 1a to dramatically stabilize RNA in vivo. Sullivan ML, Ahlquist P. Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA. Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins. The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain. Saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a BMV RNA3 template produced by in vivo transcription of a DNA copy of RNA3. Although insufficient for RNA3 replication, the expression of 1a protein alone results in a dramatic and specific stabilization of the RNA3 template in yeast. As one step toward understanding 1a-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replication, we have identified the cis-acting sequences required for this effect. We find that 1a-induced stabilization is mediated by a 150- to 190-base segment of the RNA3 intergenic region corresponding to a previously identified enhancer of RNA3 replication. Moreover, this segment is sufficient to confer 1a-induced stability on a heterologous beta-globin RNA. Within this intergenic segment, partial deletions that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in parallel decreases in the levels of negative- and positive-strand RNA3 replication products in yeast expressing 1a and 2a. In particular, a small deletion encompassing a motif corresponding to the box B element of RNA polymerase III promoters dramatically reduced the ability of RNAs to respond to 1a or 1a and 2a. These and other findings suggest that 1a-induced stabilization likely reflects an early template selection step in BMV RNA replication. PMID: 10074107 [PubMed - indexed for MEDLINE] 789: Genes Dev 1999 Mar 1;13(5):569-80 Interaction of the U1 snRNP with nonconserved intronic sequences affects 5' splice site selection. Puig O, Gottschalk A, Fabrizio P, Seraphin B. Gene Expression Program, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany. Intron definition and splice site selection occur at an early stage during assembly of the spliceosome, the complex mediating pre-mRNA splicing. Association of U1 snRNP with the pre-mRNA is required for these early steps. We report here that the yeast U1 snRNP-specific protein Nam8p is a component of the commitment complexes, the first stable complexes assembled on pre-mRNA. In vitro and in vivo, Nam8p becomes indispensable for efficient 5' splice site recognition when this process is impaired as a result of the presence of noncanonical 5' splice sites or the absence of a cap structure. Nam8p stabilizes commitment complexes in the latter conditions. Consistent with this, Nam8p interacts with the pre-mRNA downstream of the 5' splice site, in a region of nonconserved sequence. Substitutions in this region affect splicing efficiency and alternative splice site choice in a Nam8p-dependent manner. Therefore, Nam8p is involved in a novel mechanism by which a snRNP component can affect splice site choice and regulate intron removal through its interaction with a nonconserved sequence. This supports a model where early 5' splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA. PMID: 10072385 [PubMed - indexed for MEDLINE] 790: Genes Dev 1999 Mar 1;13(5):545-55 Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p. Sassoon I, Severin FF, Andrews PD, Taba MR, Kaplan KB, Ashford AJ, Stark MJ, Sorger PK, Hyman AA. Cell Biology Program, European Molecular Biology Laboratory, 69117 Heidelberg, Germany. We have investigated the role of protein phosphorylation in regulation of Saccharomyces cerevisiae kinetochores. By use of phosphatase inhibitors and a type 1 protein phosphatase mutant (glc7-10), we show that the microtubule binding activity, but not the centromeric DNA-binding activity, of the kinetochore complex is regulated by a balance between a protein kinase and the type 1 protein phosphatase (PP1) encoded by the GLC7 gene. glc7-10 mutant cells exhibit low kinetochore-microtubule binding activity in vitro and a high frequency of chromosome loss in vivo. Specifically, the Ndc10p component of the centromere DNA-binding CBF3 complex is altered by the glc7-10 mutation; Ndc10p is hyperphosphorylated in glc7-10 extracts. Furthermore, addition of recombinant Ndc10p reconstitutes the microtubule-binding activity of a glc7-10 extract to wild-type levels. Finally, the glc7-10-induced mitotic arrest is abolished in spindle checkpoint mutants, suggesting that defects in kinetochore-microtubule interactions caused by hyperphosphorylation of kinetochore proteins activate the spindle checkpoint. PMID: 10072383 [PubMed - indexed for MEDLINE] 791: Mol Gen Genet 1999 Feb;261(1):80-91 Functional implications of genetic interactions between genes encoding small GTPases involved in vesicular transport in yeast. Yoo JS, Grabowski R, Xing L, Trepte HH, Schmitt HD, Gallwitz D. Department of Molecular Genetics, Max Planck Institute for Biophysical Chemistry, Gottingen, Germany. Ras-related, guanine nucleotide-binding proteins of the Ypt/Rab family play a key role at defined steps in vesicular transport, both in yeast and in mammalian cells. In yeast, Ypt1p has an essential function late in endoplasmic reticulum (ER) to Golgi transport, and the redundant Ypt31/Ypt32 GTPases have been proposed to act in transport through and/or from the Golgi. Here we report that mutant alleles of YPT31 and YPT32, whose gene products have a reduced affinity for GTP, are able to suppress the dominant lethal phenotype of YPT1(N121I). Co-expression of YPT1(N121I) and the suppressor YPT31(N126I) allow essentially undisturbed secretory transport in the absence of the respective wild-type GTPases. Such mutant cells massively overaccumulate 60-100 nm vesicles and are heat sensitive. It appears likely that the mutant GTPases, which are defective in nucleotide binding, compete for the binding of common interacting protein(s). These and other genetic interactions between YPT1, YPT31/32, ARF1 and SEC4 described here strongly support the view that Ypt31p and Ypt32p have a central, Golgi-associated function in anterograde or retrograde transport. PMID: 10071213 [PubMed - indexed for MEDLINE] 792: Mol Biol Cell 1999 Mar;10(3):609-26 Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae. Brizzio V, Khalfan W, Huddler D, Beh CT, Andersen SS, Latterich M, Rose MD. Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA. During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles. PMID: 10069807 [PubMed - indexed for MEDLINE] 793: Microbiol Mol Biol Rev 1999 Mar;63(1):54-105 Cdc42: An essential Rho-type GTPase controlling eukaryotic cell polarity. Johnson DI. Department of Microbiology & Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA.dijohnso@zoo.uvm.edu Cdc42p is an essential GTPase that belongs to the Rho/Rac subfamily of Ras-like GTPases. These proteins act as molecular switches by responding to exogenous and/or endogenous signals and relaying those signals to activate downstream components of a biological pathway. The 11 current members of the Cdc42p family display between 75 and 100% amino acid identity and are functional as well as structural homologs. Cdc42p transduces signals to the actin cytoskeleton to initiate and maintain polarized gorwth and to mitogen-activated protein morphogenesis. In the budding yeast Saccharomyces cerevisiae, Cdc42p plays an important role in multiple actin-dependent morphogenetic events such as bud emergence, mating-projection formation, and pseudohyphal growth. In mammalian cells, Cdc42p regulates a variety of actin-dependent events and induces the JNK/SAPK protein kinase cascade, which leads to the activation of transcription factors within the nucleus. Cdc42p mediates these processes through interactions with a myriad of downstream effectors, whose number and regulation we are just starting to understand. In addition, Cdc42p has been implicated in a number of human diseases through interactions with its regulators and downstream effectors. While much is known about Cdc42p structure and functional interactions, little is known about the mechanism(s) by which it transduces signals within the cell. Future research should focus on this question as well as on the detailed analysis of the interactions of Cdc42p with its regulators and downstream effectors. Publication Types: Review Review, Tutorial PMID: 10066831 [PubMed - indexed for MEDLINE] 794: J Biol Chem 1999 Mar 12;274(11):7576-82 Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin-protein ligase interaction. Nuber U, Scheffner M. Deutsches Krebsforschungszentrum, Angewandte Tumorvirologie, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Members of the hect domain protein family are characterized by sequence similarity of their C-terminal regions to the C terminus of E6-AP, an E3 ubiquitin-protein ligase. An essential intermediate step in E6-AP-dependent ubiquitination is the formation of a thioester complex between E6-AP and ubiquitin in the presence of distinct E2 ubiquitin-conjugating enzymes including human UbcH5, a member of the UBC4/UBC5 subfamily of E2s. Similarly, several hect domain proteins, including Saccharomyces cerevisiae RSP5, form ubiquitin thioester complexes, indicating that hect domain proteins in general have E3 activity. We show here, by the use of chimeric E2s generated between UbcH5 and other E2s, that a region of UbcH5 encompassing the catalytic site cysteine residue is critical for its ability to interact with E6-AP and RSP5. Of particular importance is a phenylalanine residue at position 62 of UbcH5 that is conserved among the members of the UBC4/UBC5 subfamily but is not present in any of the other known E2s, whereas the N-terminal 60 amino acids do not contribute significantly to the specificity of these interactions. The conservation of this phenylalanine residue throughout evolution underlines the importance of the ability to interact with hect domain proteins for the cellular function of UBC4/UBC5 subfamily members. PMID: 10066826 [PubMed - indexed for MEDLINE] 795: Curr Opin Microbiol 1998 Apr;1(2):197-203 Protein chaperones and the heat shock response in Saccharomyces cerevisiae. Morano KA, Liu PC, Thiele DJ. Department of Biological Chemistry, University of Michigan Medical School, 1301 Catherine Road, Ann Arbor, MI 48109-0606, USA. kmorano@umich.edu Recent studies have shed new light on the complexities of the heat shock response in yeast. Multiple pathways for transcriptional induction of both classic and novel heat shock proteins are emerging together with a more detailed understanding of the interactions between protein chaperones and their physiological targets. New roles for heat shock proteins in defense and recovery from the impacts of thermal stress on critical cellular processes have expanded our understanding of these elaborate and ubiquitous proteins. Publication Types: Review Review, Tutorial PMID: 10066474 [PubMed - indexed for MEDLINE] 796: EMBO J 1999 Mar 1;18(5):1137-45 Physical interactions among circadian clock proteins KaiA, KaiB and KaiC in cyanobacteria. Iwasaki H, Taniguchi Y, Ishiura M, Kondo T. Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. The kai gene cluster, which is composed of three genes, kaiA, kaiB and kaiC, is essential for the generation of circadian rhythms in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. Here we demonstrate the direct association of KaiA, KaiB and KaiC in yeast cells using the two-hybrid system, in vitro and in cyanobacterial cells. KaiC enhanced KaiA-KaiB interaction in vitro and in yeast cells, suggesting that the three Kai proteins were able to form a heteromultimeric complex. We also found that a long period mutation kaiA1 dramatically enhanced KaiA-KaiB interaction in vitro. Thus, direct protein-protein association among the Kai proteins may be a critical process in the generation of circadian rhythms in cyanobacteria. PMID: 10064581 [PubMed - indexed for MEDLINE] 797: J Antimicrob Chemother 1998 Dec;42(6):747-53 Synergic effects of tactolimus and azole antifungal agents against azole-resistant Candida albican strains. Maesaki S, Marichal P, Hossain MA, Sanglard D, Vanden Bossche H, Kohno S. The Second Department of Internal Medicine, Nagasaki University School of Medicine, Japan. We investigated the effects of combining tacrolimus and azole antifungal agents in azole-resistant strains of Candida albicans by comparing the accumulation of [3H]itraconazole. The CDR1-expressing resistant strain C26 accumulated less itraconazole than the CaMDR-expressing resistant strain C40 or the azole-sensitive strain B2630. A CDR1-expressing Saccharomyces cerevisiae mutant, DSY415, showed a marked reduction in the accumulation of both fluconazole and itraconazole. A CaMDR-expressing S. cerevisiae mutant, DSY416, also showed lower accumulation of fluconazole, but not of itraconazole. The addition of sodium azide, an electron-transport chain inhibitor, increased the intracellular accumulation of itraconazole only in the C26 strain, and not in the C40 or B2630 strains. Addition of tacrolimus, an inhibitor of multidrug resistance proteins, resulted in the highest increase in itraconazole accumulation in the C26 strain. The combination of itraconazole and tacrolimus was synergic in azole-resistant C. albicans strains. In the C26 strain, the MIC of itraconazole decreased from >8 to 0.5 mg/L when combined with tacrolimus. Our results showed that two multidrug resistance phenotypes (encoded by the CDR1 and CaMDR genes) in C. albicans have different substrate specificity for azole antifungal agents and that a combination of tacrolimus and azole antifungal agents is effective against azole-resistant strains of C. albicans. PMID: 10052898 [PubMed - indexed for MEDLINE] 798: Nat Struct Biol 1999 Feb;6(2):157-65 X-ray structural analysis of the yeast cell cycle regulator Swi6 reveals variations of the ankyrin fold and has implications for Swi6 function. Foord R, Taylor IA, Sedgwick SG, Smerdon SJ. Division of Protein Structure, National Institute for Medical Research, London, UK. Swi6 is a 92,000 Mr protein common to two distinct transcriptional activation complexes (SBF and MBF) that coordinate gene expression at the G1-S boundary of the yeast cell cycle. The X-ray structure of a central 36,000 Mr fragment has been determined and refined at 2.1 A resolution. The structure reveals a basic framework of five ankyrin repeat modules that is elaborated through a series of helical insertions distinguishing it from structures of other ankyrin repeat proteins. A second domain contains an approximately 30-residue region of extended structure that interacts with the ankyrin repeat core over a substantial proportion of its surface. Conservation of residues buried by these interactions indicates that all members of the Swi6/Cdc10 family share a similar architecture. Several temperature-sensitive mutations within Swi6 and Cdc10 appear to disrupt these interdomain contacts rather than destabilize the ankyrin repeat core. The unusual domain arrangement may be crucial for the modulation of interactions with other co-regulatory molecules such as cyclin-CDK complexes, and has implications for the quaternary interactions within the multisubunit SBF and MBF transcription complexes. PMID: 10048928 [PubMed - indexed for MEDLINE] 799: Biochem Soc Trans 1998 Nov;26(4):601-6 Interactions of the human, plant and yeast ornithine decarboxylase subunits and human antizyme. Illingworth C, Michael AJ. Department of Genetics and Microbiology, Institute of Food Research, Norwich Research Park, Colney, U.K. PMID: 10047790 [PubMed - indexed for MEDLINE] 800: Exp Cell Res 1999 Feb 25;247(1):1-8 SNAREs and the secretory pathway-lessons from yeast. Pelham HR. MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, United Kingdom. hp@mrc-lmb.cam.ac.uk SNARE proteins lie at the heart of the membrane fusion events in the secretory and endocytic pathways. Physical interactions between them are thought not only to provide the driving force for bringing membranes together, but also to contribute to the specificity of vesicle targeting. Completion of the yeast genome sequence has allowed the full set of SNAREs to be identified. Characterization of these helps to define the number of distinct compartments and the nature of the transport steps between them, but also shows that SNAREs are by no means the sole determinants of fusion specificity. Evolutionary conservation of SNAREs suggests that despite the differences in scale and morphology, many features of membrane organization are similar in yeast and animal cells. This review summarizes current knowledge of the yeast SNAREs and the picture of the secretory pathway that emerges from such studies. Copyright 1999 Academic Press. Publication Types: Review Review, Tutorial PMID: 10047442 [PubMed - indexed for MEDLINE] 801: J Biol Chem 1999 Mar 5;274(10):6579-85 The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. Jungmann J, Rayner JC, Munro S. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, United Kingdom. In the yeast Saccharomyces cerevisiae many of the N-linked glycans on cell wall and periplasmic proteins are modified by the addition of mannan, a large mannose-containing polysaccharide. Mannan comprises a backbone of approximately 50 alpha-1,6-linked mannoses to which are attached many branches consisting of alpha-1,2-linked and alpha-1,3-linked mannoses. The initiation and subsequent elongation of the mannan backbone is performed by two complexes of proteins in the cis Golgi. In this study we show that the product of the MNN10/BED1 gene is a component of one of these complexes, that which elongates the backbone. Analysis of interactions between the proteins in this complex shows that Mnn10p, and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesis in vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p are responsible for the majority of the alpha-1, 6-polymerizing activity of the complex. PMID: 10037752 [PubMed - indexed for MEDLINE] 802: J Theor Biol 1999 Mar 7;197(1):63-76 Deviations from Chargaff's second parity rule correlate with direction of transcription. Bell SJ, Forsdyke DR. Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6. The distribution of deviations from Chargaff's second parity rule was examined for overlapping sequence windows of a length (1 kb) predicted to be suitable for detecting correlations with functional features of DNA. For long genomic segments from E. coli, Saccharomyces cerevisiae, and Vaccinia virus, Chargaff differences for the W bases and/or for the S bases correlate with transcription direction and gene location. For W-rich genomes, the mRNA-synonymous strand contains regions which, if extruded from negatively supercoiled DNA, would fold to generate stem-loop structures with A-rich loops. Similarly, for S-rich genomes the loops would be G-rich. We suggest that the disposition of genes in nucleic acid sequences arises from their having to adapt to a preexisting mosaic of genomic regions, each distinguished by its potential to extrude single-strand loops enriched for a particular base (or two non-Watson-Crick pairing bases). The mosaic would have facilitated the intrastrand and interstrand accounting required for correction of mutations, and would have evolved in the early RNA world before the emergence of protein-encoding capacity. The preexisting mosaic would have determined transcription direction since there is pressure for all mRNAs of a cell to have purine-rich loops, thus decreasing loop-loop interactions which might lead to formation of "self" sense-antisense RNA duplexes. Copyright 1999 Academic Press. PMID: 10036208 [PubMed - indexed for MEDLINE] 803: Genomics 1999 Feb 15;56(1):59-69 A gene upregulated in the acoustically damaged chick basilar papilla encodes a novel WD40 repeat protein. Adler HJ, Winnicki RS, Gong TW, Lomax MI. Department of Otolaryngology/Head-Neck Surgery, University of Michigan, Ann Arbor, Michigan, 48109, USA. The chick WDR1 gene is expressed at higher levels in the chick basilar papilla after acoustic overstimulation. The 3.3-kb WDR1 cDNA encodes a novel 67-kDa protein containing nine WD40 repeats, motifs that mediate protein-protein interactions. The predicted WDR1 protein has high sequence identity to WD40-repeat proteins in budding yeast (Saccharomyces cerevisiae), two slime molds (Dictyostelium discoideum and Physarum polycephalum), and the roundworm (Caenorhabditis elegans). The yeast and P. polycephalum proteins bind actin, suggesting that the novel chick protein may be an actin-binding protein. Sequence database comparisons identified mouse and human cDNAs with high sequence identity to the chick WDR1 cDNA. The mouse Wdr1 and human WDR1 proteins showed 95% sequence identity to each other and 86% identity to the chick WDR1 protein. Northern blot analysis of total RNA from the chick basilar papilla after noise trauma revealed increased levels of a 3.1-kb transcript in the lesioned area. The WDR1 gene was mapped to human chromosome 4, between 22 and 24 cM from the telomere of 4p. Copyright 1999 Academic Press. PMID: 10036186 [PubMed - indexed for MEDLINE] 804: Yeast 1999 Jan 15;15(1):35-41 Identification and characterization of the genes for two topoisomerase I-interacting proteins from Saccharomyces cerevisiae. Park H, Sternglanz R. Department of Biochemistry and Cell Biology, SUNY at Stony Brook 11794-5215, USA. The two-hybrid system was used to identify yeast genes encoding proteins that interact with DNA topoisomerase I. Two new genes were found and named TOF1 and TOF2. The Tof1 protein has 1238 amino acids and no obvious homologues in databases. The Tof2 protein has 771 amino acids; it is quite closely related to another yeast protein, the product of an uncharacterized ORF. Both tof1 and tof2 null mutants, and tof1-tof2 double mutants, grow normally and have normal levels of topoisomerase I activity. TOF2 shows various genetic interactions with TOP1 and HPR1. The implications of these interactions for TOF2 function are discussed. PMID: 10028183 [PubMed - indexed for MEDLINE] 805: Yeast 1999 Jan 15;15(1):23-33 Saccharomyces cerevisiae IRR1 protein is indirectly involved in colony formation. Kurlandzka A, Rytka J, Rozalska B, Wysocka M. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland. The ability of a microorganism to adhere to a solid support and to initiate a colony is often the first stage of microbial infections. To date, studies on S. cerevisiae cell-cell and cell-solid support interactions concerned only cell agglutination during mating and flocculation. Colony formation has not been studied before probably because this species is not pathogenic. However, S. cerevisiae can be a convenient model to study this process, thanks to well-developed genetics and the full knowledge of its nucleotide sequence. A preliminary characterization of the recently cloned essential IRR1 gene indicated that it may participate in cell-cell/substrate interactions. Here we show that lowering the level of expression of IRR1 (after fusion with a regulatory catalase A gene promoter) affects colony formation and disturbs zygote formation and spore germination. All these processes involve cell-cell or cell-solid support contacts. The IRR1 protein is localized in the cytosol as verified by immunofluorescence microscopy, and confirmed by cell fractionation and Western blotting. This indicates that Irr1p is not directly involved in the cell-solid support adhesion, but may be an element of a communication pathway between the cell and its surroundings. PMID: 10028182 [PubMed - indexed for MEDLINE] 806: Cancer Res 1999 Feb 15;59(4):816-22 hMSH5: a human MutS homologue that forms a novel heterodimer with hMSH4 and is expressed during spermatogenesis. Bocker T, Barusevicius A, Snowden T, Rasio D, Guerrette S, Robbins D, Schmidt C, Burczak J, Croce CM, Copeland T, Kovatich AJ, Fishel R. Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University and Medical College, Philadelphia, Pennsylvania 19107, USA. MutS homologues have been identified in nearly all organisms examined to date. They play essential roles in maintaining mitotic genetic fidelity and meiotic segregation fidelity. MutS homologues appear to function as a molecular switch that signals genomic manipulation events. Here we describe the identification of the human homologue of the Saccharomyces cerevisiae MSH5, which is known to participate in meiotic segregation fidelity and crossing-over. The human MSH5 (hMSH5) was localized to chromosome 6p22-21 and appears to play a role in meiosis because expression is induced during spermatogenesis between the late primary spermatocytes and the elongated spermatid phase. hMSH5 interacts specifically with hMSH4, confirming the generality of functional heterodimeric interactions in the eukaryotic MutS homologue, which also includes hMSH2-hMSH3 and hMSH2-hMSH6. PMID: 10029069 [PubMed - indexed for MEDLINE] 807: Sci Total Environ 1999 Jan 12;225(1-2):69-79 Monitoring of estrogen mimics by a recombinant yeast assay: synergy between natural and synthetic compounds? Graumann K, Breithofer A, Jungbauer A. Institute for Applied Microbiology, University of Agriculture, Forestry and Biotechnology, Vienna, Austria. Properties of mixtures of compounds exhibiting estrogenic potential have been questioned in the past. Synergistic effects of endocrine disrupters have been proposed, but could never be confirmed. In this study, the transactivational potential of xenoestrogens and phytoestrogens has been evaluated in a yeast test system. Pesticides such as endosulfan, dieldrin, atrazine, and the main metabolites, desethylatrazine and desisopropylatrazine, have been tested and their behavior as mixtures is compared to the behavior of the single compounds. Our results are in contrast to a report (Tran et al., 1996) on the inhibitive effects of xenoestrogens on 17 beta-estradiol-dependent transactivation. Phytoestrogens have been investigated in a similar manner. A synergistic effect could not be confirmed for both, xenoestrogens and phytoestrogens. These compounds are either weak estrogens or completely lack estrogenic potential. Their endocrine disrupting potential in more complex systems must be therefore attributed to other molecular mechanisms such as to metabolic modification or interference with steroidogenesis. This study shows that yeast systems are useful tools for monitoring pure estrogenic properties. PMID: 10028704 [PubMed - indexed for MEDLINE] 808: Mol Cell 1999 Jan;3(1):97-108 Erratum in: Mol Cell 1999 Apr;3(4):following 541 A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation. Gu W, Malik S, Ito M, Yuan CX, Fondell JD, Zhang X, Martinez E, Qin J, Roeder RG. Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021, USA. A novel human complex that can either repress activator-dependent transcription mediated by PC4, or, at limiting TFIIH, act synergistically with PC4 to enhance activator-dependent transcription has been purified. This complex contains homologs of a subset of yeast mediator/holoenzyme components (including SRB7, SRB10, SRB11, MED6, and RGR1), homologs of other yeast transcriptional regulatory factors (SOH1 and NUT2), and, significantly, some components (TRAP220, TRAP170/hRGR1, and TRAP100) of a human thyroid hormone receptor-associated coactivator complex. The complex shows direct activator interactions but, unlike yeast mediator, can act independently of the RNA polymerase II CTD. These findings demonstrate both positive and negative functional capabilities for the human complex, emphasize novel (CTD-independent) regulatory mechanisms, and link the complex to other human coactivator complexes. PMID: 10024883 [PubMed - indexed for MEDLINE] 809: Infect Immun 1999 Mar;67(3):1063-71 Non-serum-dependent chemotactic factors produced by Candida albicans stimulate chemotaxis by binding to the formyl peptide receptor on neutrophils and to an unknown receptor on macrophages. Edens HA, Parkos CA, Liang TW, Jesaitis AJ, Cutler JE, Miettinen HM. Department of Microbiology, Montana State University-Bozeman, Bozeman, Montana 59717, USA. hedens@tex2.oscs.montana.edu Serum-free culture filtrates of six Candida species and Saccharomyces cerevisiae were found to contain chemoattractants for human polymorphonuclear leukocytes (PMNs) and a mouse macrophage-like cell line, J774. The chemotactic factors differed for the PMN and J774 cells, however, in terms of heat stability, kinetics of liberation by the yeast cells, and divalent cation requirements for production. The chemoattractant in Candida albicans culture filtrates appeared to act through the formyl peptide receptor (FPR) of PMNs, since it was found to induce chemotaxis of Chinese hamster ovary (CHO) cells that were expressing the human FPR but did not induce chemotaxis of wild-type CHO cells. The C. albicans culture filtrates also induced migration of PMNs across confluent monolayers of a human gastrointestinal epithelial cell line, T84; migration occurred in the basolateral-to-apical direction but not the reverse direction, unless the epithelial tight junctions were disrupted. J774 cells did not migrate toward the formylated peptide (fMet-Leu-Phe; fMLF), and chemotaxis toward the C. albicans culture filtrate was not inhibited by an FPR antagonist (t-butoxycarbonyl-Met-Leu-Phe), suggesting that a different receptor mediated J774 cell chemotaxis. In conclusion, we have identified a receptor by which a non-serum-dependent chemotactic factor (NSCF) produced by C. albicans induced chemotaxis of PMNs. Additionally, we have shown that NSCF was active across epithelial monolayers. These findings suggest that NSCFs produced by C. albicans and other yeast species may influence host-pathogen interactions at the gastrointestinal tract mucosal surface by inducing phagocytic-cell infiltration. PMID: 10024544 [PubMed - indexed for MEDLINE] 810: Biochem J 1999 Mar 1;338 ( Pt 2):403-7 Characterization of the interaction domains of Ure2p, a prion-like protein of yeast. Fernandez-Bellot E, Guillemet E, Baudin-Baillieu A, Gaumer S, Komar AA, Cullin C. Centre de Genetique Moleculaire du C.N.R.S., Laboratoire Propre Associe a l'Universite Pierre-et-Marie-Curie, 91190 Gif-sur-Yvette, France. In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic element [URE3] behaves as a prion. A hypothesis has been put forward which states that [URE3] arises spontaneously from its cellular isoform Ure2p (the product of the URE2 gene), and propagates through interactions of the N-terminal domain of the protein, thus leading to its aggregation and loss of function. In the present study, various N- and C-terminal deletion mutants of Ure2p were constructed and their cross-interactions were tested in vitro and in vivo using affinity binding and a two-hybrid analysis. We show that the self-interaction of the protein is mediated by at least two domains, corresponding to the first third of the protein (the so-called prion-forming domain) and the C-terminal catalytic domain. PMID: 10024516 [PubMed - indexed for MEDLINE] 811: Biochem J 1999 Mar 1;338 ( Pt 2):375-86 Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin. Rostagno AA, Schwarzbauer JE, Gold LI. Department of Pathology, New York University Medical Center, 400 East 34th Street, New York, NY 10016, USA. Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction. PMID: 10024513 [PubMed - indexed for MEDLINE] 812: Protein Expr Purif 1999 Feb;15(1):127-45 A kinetic locking-on strategy for bioaffinity purification: further studies with alcohol dehydrogenase. O'flaherty M, McMahon M, Mulcahy P. Department of Applied Biology and Chemistry, Institute of Technology Carlow, Ireland. The kinetic locking-on strategy improves the selectivity of protein purification procedures based on immobilized cofactor derivatives through use of enzyme-specific substrate analogues in irrigants to promote biospecific adsorption. This paper describes the development and application of this strategy to the one-chromatographic step affinity purification of NAD(P)+-dependent alcohol dehydrogenases using 8'-azo-linked immobilized NAD(P)+, S6-linked and N6-linked immobilized NAD+, and N6-linked immobilized NADP+ derivatives. These studies were carried out using alcohol dehydrogenases from Saccharomyces cerevisiae (YADH, EC 1.1.1.1), equine liver (HLADH, EC 1.1.1.1), and Thermoanaerobium brockii (TBADH, EC 1.1.1.2). The results reveal that the factors which require careful consideration before development of a truly biospecific system based on the locking-on strategy include: (i) the stability of the immobilized cofactor derivative; (ii) the spacer-arm composition of the affinity derivative; (iii) the accessible immobilized cofactor concentration; (iv) the soluble locking-on ligand concentration; (v) the dissociation constant of locking-on ligand, and (vi) the identification and elimination of nonbiospecific interference. The S6-linked immobilized NAD+ derivative (synthesized with a hydrophilic spacer arm) proved to be the most suitable of the affinity adsorbents investigated in the present study for use with the locking-on strategy. This conclusion was based primarily on the observations that this affinity adsorbent was stable, retained cofactor activity with the "test" enzymes under study, and was not prone to nonbiospecific interactions. Using this immobilized derivative in conjunction with the locking-on strategy, alcohol dehydrogenase from Saccharomyces cerevisiae was purified to electrophoretic homogeneity in a single affinity chromatographic step. Copyright 1999 Academic Press. PMID: 10024480 [PubMed - indexed for MEDLINE] 813: J Cell Biochem 1999 Mar 1;72(3):356-67 180-kD bullous pemphigoid antigen/type XVII collagen: tissue-specific expression and molecular interactions with keratin 18. Aho S, Uitto J. Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA. sirpaaho@hotmail.com The 180-kD bullous pemphigoid antigen (BPAG2) is a hemidesmosomal transmembrane protein, also known as type XVII collagen. In this study, potential interactions of BPAG2 with other proteins expressed in epidermal keratinocytes were explored by yeast two-hybrid system using the amino-terminal intracellular domain of BPAG2 as a bait. Several independent interacting clones encoding keratin 18 (K18) were identified when the keratinocyte cDNA library, cloned into the yeast two-hybrid activation domain vector, was screened. The peptide sequence responsible for the interaction of BPAG2 was restricted to amino acids 15-25, and substitution of a valine residue in the middle of this sequence by a proline (V23P) by site-directed mutagenesis abolished the interaction. Further examination of the K18 sequences by restricted cDNA constructs in yeast two-hybrid system identified a carboxyl-terminal segment corresponding to helix 2B domain as critical for BPAG2 binding. The interaction of BPAG2/K18 was confirmed by an in vitro protein-protein interaction assay, which also confirmed that normal human keratinocytes express K18 in culture. The tissue specific expression of BPAG2 was first examined using a multi-tissue RNA blot. Human multiple tissue cDNA panels representing a variety of adult and fetal tissues as well as tumor cells were used as PCR-templates to study the expression patterns of both BPAG2 and K18. The results demonstrated significant level of expression of BPAG2, besides in epidermal keratinocytes, also in a variety of tissues with predominant epithelial component, such as mammary, salivary and thyroid glands, colon, prostate, testis, placenta, and adult and fetal thymus, as well as in colon, pancreatic and prostatic adenocarcinoma cell lines, and an ovarian carcinoma. As expected, K18 transcript is present in liver, pancreas, colon, placenta, and in fetal kidney. Collectively, the results suggest that BPAG2 has a relatively broad tissue distribution including specialized and simple epithelia, and that within the tissues such as colon and placenta, BPAG2 may have direct interactions with K18, a keratin characteristically expressed in a simple epithelia. PMID: 10022517 [PubMed - indexed for MEDLINE] 814: Mol Cell Biol 1999 Mar;19(3):2142-54 Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA. Massenet S, Motorin Y, Lafontaine DL, Hurt EC, Grosjean H, Branlant C. Laboratoire de Maturation des ARN et Enzymologie Moleculaire, UMR7567 CNRS-UHP, Faculte des Sciences, 54506 Vandoeuvre-les-Nancy Cedex, France. Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism. PMID: 10022901 [PubMed - indexed for MEDLINE] 815: Mol Cell Biol 1999 Mar;19(3):2000-7 The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3' exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae. Tran HT, Gordenin DA, Resnick MA. Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA. Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5'-->3' Exo1 and the 3'-->5' DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and highly sensitive mutation detection systems that are based on long and short homonucleotide runs. Genetic interactions were examined among DNA polymerase epsilon (pol2-4) and delta (pol3-01) mutants defective in 3'-->5' proofreading exonuclease, mutants defective in the 5'-->3' exonuclease Exo1, and mismatch repair mutants (msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01 mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they participate directly in postreplication mismatch repair as well as in other DNA metabolic processes. PMID: 10022887 [PubMed - indexed for MEDLINE] 816: Mol Cell Biol 1999 Mar;19(3):1627-39 Erratum in: Mol Cell Biol 1999 May;19(5):3929 Cooperative binding of heat shock factor to the yeast HSP82 promoter in vivo and in vitro. Erkine AM, Magrogan SF, Sekinger EA, Gross DS. Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 71130, USA. Previous work has shown that heat shock factor (HSF) plays a central role in remodeling the chromatin structure of the yeast HSP82 promoter via constitutive interactions with its high-affinity binding site, heat shock element 1 (HSE1). The HSF-HSE1 interaction is also critical for stimulating both basal (noninduced) and induced transcription. By contrast, the function of the adjacent, inducibly occupied HSE2 and -3 is unknown. In this study, we examined the consequences of mutations in HSE1, HSE2, and HSE3 on HSF binding and transactivation. We provide evidence that in vivo, HSF binds to these three sites cooperatively. This cooperativity is seen both before and after heat shock, is required for full inducibility, and can be recapitulated in vitro on both linear and supercoiled templates. Quantitative in vitro footprinting reveals that occupancy of HSE2 and -3 by Saccharomyces cerevisiae HSF (ScHSF) is enhanced approximately 100-fold through cooperative interactions with the HSF-HSE1 complex. HSE1 point mutants, whose basal transcription is virtually abolished, are functionally compensated by cooperative interactions with HSE2 and -3 following heat shock, resulting in robust inducibility. Using a competition binding assay, we show that the affinity of recombinant HSF for the full-length HSP82 promoter is reduced nearly an order of magnitude by a single-point mutation within HSE1, paralleling the effect of these mutations on noninduced transcript levels. We propose that the remodeled chromatin phenotype previously shown for HSE1 point mutants (and lost in HSE1 deletion mutants) stems from the retention of productive, cooperative interactions between HSF and its target binding sites. PMID: 10022851 [PubMed - indexed for MEDLINE] 817: EMBO J 1999 Feb 15;18(4):1071-80 The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. Guo W, Roth D, Walch-Solimena C, Novick P. Department of Cell Biology, Yale University School of Medicine, PO Box 208002, New Haven, CT 06520-8002, USA. Polarized secretion requires proper targeting of secretory vesicles to specific sites on the plasma membrane. Here we report that the exocyst complex plays a key role in vesicle targeting. Sec15p, an exocyst component, can associate with secretory vesicles and interact specifically with the rab GTPase, Sec4p, in its GTP-bound form. A chain of protein-protein interactions leads from Sec4p and Sec15p on the vesicle, through various subunits of the exocyst, to Sec3p, which marks the sites of exocytosis on the plasma membrane. Sec4p may control the assembly of the exocyst. The exocyst may therefore function as a rab effector system for targeted secretion. PMID: 10022848 [PubMed - indexed for MEDLINE] 818: Curr Opin Chem Biol 1999 Feb;3(1):64-70 Progress and variations in two-hybrid and three-hybrid technologies. Drees BL. Department of Genetics, Box 357360, University of Washington, Seattle WA,98195 USA. drees@u.washington.edu The original yeast two-hybrid system and its variants have proven to be effective tools for identification and analysis of protein-protein, protein-DNA and protein-RNA interactions. The two-hybrid assay is being applied to the entire complement of proteins of the yeast Saccharomyces cerevisiae to characterize the network of protein-protein interactions in the eukaryotic cell. The development of nontranscriptional cytosolic and membrane-associated two-hybrid methods has made it possible to detect and examine a number of protein-protein interactions in their normal cellular locations. Small-molecule hybrid systems have been developed which can be used to study protein-ligand interactions and to activate cellular processes by forcing protein associations. Publication Types: Review Review, Tutorial PMID: 10021404 [PubMed - indexed for MEDLINE] 819: Proc Natl Acad Sci U S A 1999 Feb 16;96(4):1738-43 A protein phosphatase 2C gene, LjNPP2C1, from Lotus japonicus induced during root nodule development. Kapranov P, Jensen TJ, Poulsen C, de Bruijn FJ, Szczyglowski K. Michigan State University-Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA. Symbiotic interactions between legumes and compatible strains of rhizobia result in root nodule formation. This new plant organ provides the unique physiological environment required for symbiotic nitrogen fixation by the bacterial endosymbiont and assimilation of this nitrogen by the plant partner. We have isolated two related genes (LjNPP2C1 and LjPP2C2) from the model legume Lotus japonicus that encode protein phosphatase type 2C (PP2C). Expression of the LjNPP2C1 gene was found to be enhanced specifically in L. japonicus nodules, whereas the LjPP2C2 gene was expressed at a similar level in nodules and roots. A glutathione S-transferase-LjNPP2C1 fusion protein was shown to have Mg2+- or Mn2+-dependent and okadaic acid-insensitive PP2C activity in vitro. A chimeric construct containing the full-length LjNPP2C1 cDNA, under the control of the Saccharomyces cerevisiae alcohol dehydrogenase promoter, was found to be able to complement a yeast PP2C-deficient mutant (pct1Delta). The transcript level of the LjNPP2C1 gene was found to increase significantly in mature nodules, and its highest expression level occurred after leghemoglobin (lb) gene induction, a molecular marker for late developmental events in nodule organogenesis. Expression of the LjNPP2C1 gene was found to be drastically altered in specific L. japonicus lines carrying monogenic-recessive mutations in symbiosis-related loci, suggesting that the product of the LjNPP2C1 gene may function at both early and late stages of nodule development. PMID: 9990094 [PubMed - indexed for MEDLINE] 820: Nat Genet 1999 Feb;21(2):204-8 Comment in: Nat Genet. 1999 Feb;21(2):151-2 Interaction between Set1p and checkpoint protein Mec3p in DNA repair and telomere functions. Corda Y, Schramke V, Longhese MP, Smokvina T, Paciotti V, Brevet V, Gilson E, Geli V. Laboratoire d'Ingenierie et de Dynamique des Systemes Macromoleculaires, CNRS, Marseille, France. The yeast protein Set1p, inactivation of which alleviates telomeric position effect (TPE), contains a conserved SET domain present in chromosomal proteins involved in epigenetic control of transcription. Mec3p is required for efficient DNA-damage-dependent checkpoints at G1/S, intra-S and G2/M (refs 3-7). We show here that the SET domain of Set1p interacts with Mec3p. Deletion of SET1 increases the viability of mec3delta mutants after DNA damage (in a process that is mostly independent of Rad53p kinase, which has a central role in checkpoint control) but does not significantly affect cell-cycle progression. Deletion of MEC3 enhances TPE and attenuates the Set1delta-induced silencing defect. Furthermore, restoration of TPE in a Set1delta mutant by overexpression of the isolated SET domain requires Mec3p. Finally, deletion of MEC3 results in telomere elongation, whereas cells with deletions of both SET1 and MEC3 do not have elongated telomeres. Our findings indicate that interactions between SET1 and MEC3 have a role in DNA repair and telomere function. PMID: 9988274 [PubMed - indexed for MEDLINE] 821: J Biol Chem 1999 Feb 19;274(8):5252-8 Topology and functional domains of the yeast pore membrane protein Pom152p. Tcheperegine SE, Marelli M, Wozniak RW. Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2A7, Canada. Integral membrane proteins associated with the nuclear pore complex (NPC) are likely to play an important role in the biogenesis of this structure. Here we have examined the functional roles of domains of the yeast pore membrane protein Pom152p in establishing its topology and its interactions with other NPC proteins. The topology of Pom152p was evaluated by alkaline extraction, protease protection, and endoglycosidase H sensitivity assays. The results of these experiments suggest that Pom152p contains a single transmembrane segment with its N terminus (amino acid residues 1-175) extending into the nuclear pore and its C terminus (amino acid residues 196-1337) positioned in the lumen of the nuclear envelope. The functional role of these different domains was investigated in mutants that are dependent on Pom152p for viability. The requirement for Pom152p in strains containing mutations allelic to the NPC protein genes NIC96 and NUP59 could be alleviated by Pom152p's N terminus, independent of its integration into the membrane. However, complementation of a mutation in NUP170 required both the N terminus and the transmembrane segment. Furthermore, mutations in NUP188 were rescued only by full-length Pom152p, suggesting that the lumenal structures play an important role in the function of pore-side NPC structures. PMID: 9988776 [PubMed - indexed for MEDLINE] 822: Mol Endocrinol 1999 Feb;13(2):286-96 Estrogen receptor domains E and F: role in dimerization and interaction with coactivator RIP-140. Peters GA, Khan SA. Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Ohio 45627, USA. We have used the yeast two-hybrid system to localize the ligand-dependent dimerization domain of the estrogen receptor-alpha (ER) to region E in vivo. In this system, the cDNAs corresponding to the A-D, E, E/F, A-E (deltaF), and full-length (wtER) domains of the human ER were each cloned into the yeast two-hybrid vectors GAL4 DB and GAL4 TA and expressed in different combinations in yeast harboring a GAL1-lacZ reporter. The reporter was used as a relative measure of the interaction between the ER domains, through reconstitution of GAL4 activity. We found that the interaction of E or E/F domains of the ER with full-length ER is estradiol dependent and estrogen responsive element independent, as measured by the reconstitution of GAL4 activity from GAL4-E domain-containing fusion protein interactions. In the presence of F domain, this activity is reduced 10-fold. The results suggest that sequences in the F domain are inhibitory to the dimerization signal that is present in the E region. We propose that the full-length ER contains intrinsic dimerization restraints contributed by regions outside domain E that are released upon binding hormone agonist. In addition, we have demonstrated that coactivator RIP140 is able to interact with the ER in vivo at the E domain of the receptor in the presence of estrogen. Yeast two-hybrid analysis shows that RIP140 does not homodimerize in the presence or absence of estrogens. We present evidence showing that the ER has the inherent ability to interact with RIP140 in the presence of antiestrogens, but sequences inherent in the ER itself that are present outside of the E domain compromise this ability. PMID: 9973258 [PubMed - indexed for MEDLINE] 823: Mol Biol Cell 1999 Feb;10(2):329-44 Detection of transient in vivo interactions between substrate and transporter during protein translocation into the endoplasmic reticulum. Dunnwald M, Varshavsky A, Johnsson N. Max-Delbruck-Laboratorium, D-50829 Koln, Germany. The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-alpha-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-alpha-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex. PMID: 9950680 [PubMed - indexed for MEDLINE] 824: Mol Biol Cell 1999 Feb;10(2):283-96 The multiple roles of Cyk1p in the assembly and function of the actomyosin ring in budding yeast. Shannon KB, Li R. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud junction during anaphase and has been shown to be required for the completion of cytokinesis. In this study, video microscopy analysis of cells expressing green fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a dynamic component of the contractile ring during cytokinesis. Furthermore, in the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p in actomyosin ring assembly and dynamics, we have investigated the role of the structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino terminal portion containing the calponin homology domain binds to actin filaments and is required for the assembly of actin filaments to the ring. This result supports the hypothesis that Cyk1p/Iqg1p plays a direct role in F-actin recruitment. Deletion of the domain harboring the eight IQ motifs abolishes the localization of Cyk1p/Iqg1p to the bud neck, suggesting that Cyk1p/Iqg1p may be localized through interactions with a calmodulin-like protein. Interestingly, deletion of the COOH-terminal GTPase-activating protein-related domain does not affect Cyk1p/Iqg1p localization or actin recruitment to the ring but prevents actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p binds to calmodulin, Cmd1p, in a calcium-dependent manner, and to Tem1p, a small GTP-binding protein previously found to be required for the completion of anaphase. These results demonstrate the critical function of Cyk1p/Iqg1p in regulating various steps of actomyosin ring assembly and cytokinesis. PMID: 9950677 [PubMed - indexed for MEDLINE] 825: J Biol Chem 1999 Feb 12;274(7):4027-35 Regulation of exocytosis by cyclin-dependent kinase 5 via phosphorylation of Munc18. Fletcher AI, Shuang R, Giovannucci DR, Zhang L, Bittner MA, Stuenkel EL. Departments of Physiology, University of Michigan, Ann Arbor, Michigan 48109, USA. Munc18a, a mammalian neuronal homologue of Saccharomyces cerevisiae Sec1p protein, is essential for secretion, likely as a result of its high affinity interaction with the target SNARE protein syntaxin 1a (where SNARE is derived from SNAP receptor (the soluble N-ethylmaleimide-sensitive fusion protein)). However, this interaction inhibits vesicle SNARE interactions with syntaxin that are required for secretory vesicles to achieve competency for membrane fusion. As such, regulation of the interaction between Munc18a and syntaxin 1a may provide an important mechanism controlling secretory responsiveness. Cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division kinases, co-purifies with Munc18a from rat brain, interacts directly with Munc18a in vitro, and utilizes Munc18a as a substrate for phosphorylation. We have now demonstrated that Cdk5 is capable of phosphorylating Munc18a in vitro within a preformed Munc18a.syntaxin 1a heterodimer complex and that this results in the disassembly of the complex. Using site-directed mutagenesis, the Cdk5 phosphorylation site on Munc18a was identified as Thr574. Stimulation of secretion from neuroendocrine cells produced a corresponding rapid translocation of cytosolic Cdk5 to a particulate fraction and an increase of Cdk5 kinase activity. Inhibition of Cdk5 with olomoucine decreased evoked norepinephrine secretion from chromaffin cells, an effect not observed with the inactive analogue iso-olomoucine. The effects of olomoucine were independent of calcium influx as evidenced by secretory inhibition in permeabilized chromaffin cells and in cells under whole-cell voltage clamp. Furthermore, transfection and expression in chromaffin cells of a neural specific Cdk5 activator, p25, led to a strong increase in nicotinic agonist-induced secretory responses. Our data suggest a model whereby Cdk5 acts to regulate Munc18a interaction with syntaxin 1a and thereby modulates the level of vesicle SNARE interaction with syntaxin 1a and secretory responsiveness. PMID: 9933594 [PubMed - indexed for MEDLINE] 826: Gene 1998 Dec 28;225(1-2):107-16 Isolation of hMRE11B: failure to complement yeast mre11 defects due to species-specific protein interactions. Chamankhah M, Wei YF, Xiao W. Department of Microbiology, University of Saskatchewan, 107 Wiggins Road, Saskatoon SK S7N 5E5, Canada. The Saccharomyces cerevisiae MRE11 gene plays an important role in meiotic recombination, mitotic DNA repair and telomere maintenance. We present the isolation of hMRE11B cDNA from a human HeLa cell cDNA library as an MRE11 homolog. Compared to the previously identified hMRE11, hMRE11B contains an additional 84bp sequence that results in a 28 amino-acid insertion close to the C-terminus. The expression pattern of hMRE11B in different tissues shows the presence of two mRNA species of approx. 2.6 and 7.5kb. Overexpression of hMRE11B does not complement the alkylation sensitivity of the mre11 null and temperature-sensitive mutant strains. In this study, we examine factors that may explain this lack of complementation. First, both Northern and Western analyses rule out the lack of hMRE11B transcription and/or translation in yeast. Second, we demonstrate that hMre11B, like the yeast Mre11 protein, dimerizes in vivo in a yeast two-hybrid system. This dimerization requires the C-terminal one-third of hMre11B protein, which includes the 28 amino acids absent in hMre11. However, hMre11B does not interact with Mre11, Rad50 and Xrs2. Hence, the lack of protein-protein interaction between hMre11B and the yeast Mre11, Rad50, and Xrs2 may explain the inability of hMRE11B to complement the yeast mre11 mutants. We rule out the hypothesis that the lack of interaction and, in turn of complementation, is due to the absence of sequence homology at the C-terminal domain of hMre11B compared to the yeast Mre11. Instead, we propose that the C-terminus of hMre11B participates in protein-protein interaction and functions in a species-specific manner. PMID: 9931460 [PubMed - indexed for MEDLINE] 827: Biochemistry 1999 Jan 26;38(4):1365-70 Nonspecific weak actomyosin interactions: relocation of charged residues in subdomain 1 of actin does not alter actomyosin function. Wong WW, Doyle TC, Reisler E. Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA. Yeast actin mutants with relocated charged residues within subdomain 1 were constructed so we could investigate the functional importance of individual clusters of acidic residues in mediating actomyosin weak-binding states in the cross-bridge cycle. Past studies have established a functional role for three distinct pairs of charged residues within this region of yeast actin (D2/E4, D24/D25, and E99/E100); the loss of any one of these pairs resulted in the same impairment in weak actomyosin interaction and in its function. However, the specificity of myosin interaction with these sites has not yet been addressed. To investigate this, we made and analyzed two new actin mutants, 4Ac/D24A/D25A and 4Ac/E99A/E100A. In these mutants, the acidic residues of the D24/D25 or E99/E100 sites were replaced with uncharged residues (alanines) and a pair of acidic residues was inserted at the N-terminus, maintaining the overall charge density of subdomain 1. Using the in vitro motility assays, we found that the sliding and force generation properties of these mutant actins were identical to those of wild-type actin. Similarly, actin-activated ATPase activities of the mutant and wild-type actins were also indistinguishable. Additionally, the binding of S1 to these mutant actins in the presence of ATP was similar to that of wild-type actin. These results show that relocation of charged residues in subdomain 1 of actin does not affect the weak actomyosin interactions and actomyosin function. PMID: 9930999 [PubMed - indexed for MEDLINE] 828: FEBS Lett 1999 Jan 22;443(1):41-7 C-terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction. Merkulova TI, Frolova LY, Lazar M, Camonis J, Kisselev LL. INSERM U248, Institut Curie, Paris, France. At the termination step of protein synthesis, hydrolysis of the peptidyl-tRNA is jointly catalysed at the ribosome by the termination codon and the polypeptide release factor (eRF1 in eukaryotes). eRF1 forms in vivo and in vitro a stable complex with release factor eRF3, an eRF1-dependent and ribosome-dependent GTPase. The role of the eRF1-eRF3 complex in translation remains unclear. We have undertaken a systematic analysis of the interactions between the human eRF1 and eRF3 employing a yeast two-hybrid assay. We show that the N-terminal parts of eRF1 (positions 1-280) and of eRF3 (positions 1477) are either not involved or non-essential for binding. Two regions in each factor are critical for mutual binding: positions 478-530 and 628-637 of eRF3 and positions 281-305 and 411-415 of eRF1. The GTP binding domain of eRF3 is not involved in complex formation with eRF1. The GILRY pentamer (positions 411-415) conserved in eukaryotes and archaebacteria is critical for eRF1's ability to stimulate eRF3 GTPase. The human eRF1 lacking 22 C-terminal amino acids remains active as a release factor and promotes an eRF3 GTPase activity whereas C-terminally truncated eRF3 is inactive as a GTPase. PMID: 9928949 [PubMed - indexed for MEDLINE] 829: EMBO J 1999 Feb 1;18(3):754-62 Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones. Prodromou C, Siligardi G, O'Brien R, Woolfson DN, Regan L, Panaretou B, Ladbury JE, Piper PW, Pearl LH. Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK. The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co-chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90. These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP-dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release. PMID: 9927435 [PubMed - indexed for MEDLINE] 830: EMBO J 1999 Feb 1;18(3):717-26 Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc. Salghetti SE, Kim SY, Tansey WP. Cold Spring Harbor Laboratory, 1 Bungtown Road, PO Box 100, Cold Spring Harbor, NY 11724, USA. The human proto-oncogene c-myc encodes a highly unstable transcription factor that promotes cell proliferation. Although the extreme instability of Myc plays an important role in preventing its accumulation in normal cells, little is known about how Myc is targeted for rapid destruction. Here, we have investigated mechanisms regulating the stability of Myc. We show that Myc is destroyed by ubiquitin-mediated proteolysis, and define two elements in Myc that oppositely regulate its stability: a transcriptional activation domain that promotes Myc destruction, and a region required for association with the POZ domain protein Miz-1 that stabilizes Myc. We also show that Myc is stabilized by cancer-associated and transforming mutations within its transcriptional activation domain. Our data reveal a complex network of interactions regulating Myc destruction, and imply that enhanced protein stability contributes to oncogenic transformation by mutant Myc proteins. PMID: 9927431 [PubMed - indexed for MEDLINE] 831: EMBO J 1999 Feb 1;18(3):632-43 Bcl-xL regulates apoptosis by heterodimerization-dependent and -independent mechanisms. Minn AJ, Kettlun CS, Liang H, Kelekar A, Vander Heiden MG, Chang BS, Fesik SW, Fill M, Thompson CB. Gwen Knapp Center for Lupus and Immunology Research, Chicago, IL, USA. A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3-containing death agonists. Mutants were constructed which did not bind to Bax but retained anti-apoptotic activity. Since Bcl-xL can form an ion channel in synthetic lipid membranes, the possibility that this property has a role in heterodimerization-independent cell survival was tested by replacing amino acids within the predicted channel-forming domain with the corresponding amino acids from Bax. The resulting chimera showed a reduced ability to adopt an open conductance state over a wide range of membrane potentials. Although this construct retained the ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation was introduced that rendered the chimera incapable of heterodimerization, the resulting protein failed to prevent both apoptosis in mammalian cells and Bax-mediated growth defect in yeast. Similar to mammalian cells undergoing apoptosis, yeast cells expressing Bax exhibited changes in mitochondrial properties that were inhibited by Bcl-xL through heterodimerization-dependent and -independent mechanisms. These data suggest that Bcl-xL regulates cell survival by at least two distinct mechanisms; one is associated with heterodimerization and the other with the ability to form a sustained ion channel. PMID: 9927423 [PubMed - indexed for MEDLINE] 832: EMBO J 1999 Feb 1;18(3):555-64 Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function. Poon PP, Cassel D, Spang A, Rotman M, Pick E, Singer RA, Johnston GC. Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. ARF proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the actions of GTPase-activating proteins (GAPs) for their function. The in vitro GTPase activity of the Saccharomyces cerevisiae ARF proteins Arf1 and Arf2 is stimulated by the yeast Gcs1 protein, and in vivo genetic interactions between arf and gcs1 mutations implicate Gcs1 in vesicular transport. However, the Gcs1 protein is dispensable, indicating that additional ARF GAP proteins exist. We show that the structurally related protein Glo3, which is also dispensable, also exhibits ARF GAP activity. Genetic and in vitro approaches reveal that Glo3 and Gcs1 have an overlapping essential function at the endoplasmic reticulum (ER)-Golgi stage of vesicular transport. Mutant cells deficient for both ARF GAPs cannot proliferate, undergo a dramatic accumulation of ER and are defective for protein transport between ER and Golgi. The glo3Delta and gcs1Delta single mutations each interact with a sec21 mutation that affects a component of COPI, which mediates vesicular transport within the ER-Golgi shuttle, while increased dosage of the BET1, BOS1 and SEC22 genes encoding members of a v-SNARE family that functions within the ER-Golgi alleviates the effects of a glo3Delta mutation. An in vitro assay indicates that efficient retrieval from the Golgi to the ER requires these two proteins. These findings suggest that Glo3 and Gcs1 ARF GAPs mediate retrograde vesicular transport from the Golgi to the ER. PMID: 9927415 [PubMed - indexed for MEDLINE] 833: Biochemistry 1998 Dec 22;37(51):17673-9 Dual function C-terminal domain of dynamin-1: modulation of self-assembly by interaction of the assembly site with SH3 domains. Scaife R, Venien-Bryan C, Margolis RL. Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS), Grenoble, France. Impairment of endocytosis by mutational targeting of dynamin-1 GTPases can result in paralysis and embryonic lethality. Dynamin-1 assembles at coated pits where it functions to cleave vesicles from donor membranes. Receptor endocytosis is modulated by SH3 (src homology 3) domain proteins, which directly bind to dynamin C-terminal proline motif sequences, affecting both the dynamin GTPase activity and its recruitment to coated pits. We have determined that dynamin-dynamin interactions, which are required for dynamin helix formation, involve these same SH3 domain-binding C-terminal proline motif sequences. Consequently, SH3 domain proteins induce the in vitro disassembly of dynamin helices. Our results therefore suggest the the dual function of the dynamin C-terminus (involving amino acids 800-840) permits direct regulation of dynamin assembly and function through interaction with SH3 domain proteins. Additionally, the N-terminal GTPase domain plays an important role in assembly. Finally, we show that the central PH (pleckstrin homology) domain exerts a strong inhibitory effect on the capacity for dynamin-1 self-assembly. PMID: 9922133 [PubMed - indexed for MEDLINE] 834: Biochemistry 1998 Dec 22;37(51):17637-41 Structure and function of the core histone N-termini: more than meets the eye. Hansen JC, Tse C, Wolffe AP. Department of Biochemistry, The University of Texas Health Science Center at San Antonio 78284-7760, USA. hansen@bioc02.uthscsa.edu For two decades, the core histone N-termini generally have been thought of as unstructured domains whose function is to bind to DNA and screen negative charge. New data indicates that both the molecular mechanisms of action and biological functions of the core histone N-termini in chromatin are considerably more complex. At the level of the chromatin fiber, multiple distinct functions of the N-termini are required to achieve higher order chromatin condensation, two of which apparently involve protein-protein rather than protein-DNA interactions. In addition, the N-termini have been documented to participate in specific interactions with many chromatin-associated regulatory proteins. Here, we discuss evidence supporting the new concepts that when functioning in their natural chromatin context, (1) the N-termini are engaged primarily in protein-protein interactions, (2) as a consequence of these interactions the N-termini adopt specific secondary structure, (3) posttranslational modifications such as acetylation disrupt the ability of the N-termini to form secondary structure, and (4) because the N-termini perform essential roles in both chromatin condensation and also bind specific chromatin-associated proteins, the global structure and function of any given region of the genome will be determined predominantly by the core histone N-termini and their specific interaction partners. Publication Types: Review Review, Tutorial PMID: 9922128 [PubMed - indexed for MEDLINE] 835: J Biol Chem 1999 Jan 29;274(5):2609-12 A built-in arginine finger triggers the self-stimulatory GTPase-activating activity of rho family GTPases. Zhang B, Zhang Y, Collins CC, Johnson DI, Zheng Y. Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163, USA. Signal transduction through the Rho family GTPases requires regulated cycling of the GTPases between the active GTP-bound state and the inactive GDP-bound state. Rho family members containing an arginine residue at position 186 in the C-terminal polybasic region were found to possess a self-stimulatory GTPase-activating protein (GAP) activity through homophilic interaction, resulting in significantly enhanced intrinsic GTPase activities. This arginine residue functions effectively as an "arginine finger" in the GTPase activating reaction to confer the catalytic GAP activity but is not essential for the homophilic binding interactions of Rho family proteins. The arginine 186-mediated negative regulation seems to be absent from Cdc42, a Rho family member important for cell-division cycle regulation, of lower eukaryotes, yet appears to be a part of the turn-off machinery of Cdc42 from higher eukaryotes. Introduction of the arginine 186 mutation into S. cerevisiae CDC42 led to phenotypes consistent with down-regulated CDC42 function. Thus, specific Rho family GTPases may utilize a built-in arginine finger, in addition to RhoGAPs, for negative regulation. PMID: 9915787 [PubMed - indexed for MEDLINE] 836: Eur J Biochem 1999 Jan;259(1-2):112-9 Protein interactions of Gts1p of Saccharomyces cerevisiae throughout a region similar to a cytoplasmic portion of some ATP-binding cassette transporters. Kawabata K, Mitsui K, Uno T, Tamura K, Tsurugi K. Department of Internal Medicine 2, Yamanaashi Medical University, Yamanashi, Japan. The GTS1 gene product, Gts1p, has pleiotropic effects on the timing of budding, cell size, heat tolerance, sporulation and the lifespan of the yeast Saccharomyces cerevisiae. In this study, we found (using the yeast two-hybrid system) that Gts1p forms homodimers throughout the 18-amino acid region 296-313 which has considerable similarity to a region downstream of the Walker nucleotide-binding motif A of some ATP-binding cassette (ABC) transporters. The region contains two aspartic acid residues at 301 and 310 preceded by hydrophobic amino acid residues, and Gts1p with an Asp310 to Ala substitution showed considerably reduced homodimerization, as shown by the two-hybrid assay. Overexpression of the point-mutated Gts1p did not efficiently induce the Gts1p-related phenotypes described above, suggesting that the homodimerization of Gts1p is required for it to function in vivo. The C-terminal cytoplasmic domain of the yeast ABC transporters Mdl1p (multidrug resistance-like transporter) and Ycf1p (yeast cadmium factor or glutathione S-conjugate pump) bound to Gts1p in the two-hybrid system, and the heterodimerization activity of the Gts1p with the Asp301 to Ala substitution was more affected than the Gts1p with the Asp310 to Ala substitution. Overexpression of GTS1 considerably reduced, and disruption of GTS1 slightly decreased, cellular resistance to cycloheximide, cadmium, cisplatin and 1-chloro-2,4-dinitrophenol, which (except for cycloheximide) are all substrates of Ycf1p. These results suggest that Gts1p interacts with some ABC transporters through the binding site overlapping that of homodimerization and modulates their activity. PMID: 9914482 [PubMed - indexed for MEDLINE] 837: Mol Gen Genet 1998 Dec;260(5):492-502 GRISEA, a copper-modulated transcription factor from Podospora anserina involved in senescence and morphogenesis, is an ortholog of MAC1 in Saccharomyces cerevisiae. Borghouts C, Osiewacz HD. Abteilung Molekulare Entwicklungsbiologie und Biotechnologie, Botanisches Institut Johann Wolfgang Goethe Universitat, Frankfurt am Main, Germany. The initial characterization of Grisea suggested that this gene codes for a transcription factor involved in the genetic control of cellular copper homeostasis in Podospora anserina. Here we demonstrate that GRISEA activates in vivo gene expression in Saccharomyces cerevisiae and is characterized by a modular organization. The DNA-binding domain was mapped to the first 168 N-terminal amino acids and the transactivation domain to the C-terminal half of the protein. Increased levels of copper in the growth medium lead to repression of the transactivation function possibly via intramolecular interactions between parts of the DNA-binding domain and the transactivation domain. The wild-type copy of Grisea was found to complement the phenotype of the mac1-1 mutant of S. cerevisiae. GRISEA is able to bind to the promoter of CTR1, a MAC1 target gene that encodes a high-affinity copper transporter. Taken together, the data reported here and in earlier investigations indicate that GRISEA is an ortholog of the yeast transcription factor MAC1 and suggest at least a partial conservation of the molecular machinery involved in the control of cellular copper homeostasis in eukaryotes. Remarkably, in P. anserina, the spectrum of phenotypes affected by this regulatory protein is much broader than that known in yeast and includes morphogenetic traits as well as lifespan and senescence. PMID: 9894921 [PubMed - indexed for MEDLINE] 838: Nature 1999 Jan 7;397(6714):69-72 GABA(A)-receptor-associated protein links GABA(A) receptors and the cytoskeleton. Wang H, Bedford FK, Brandon NJ, Moss SJ, Olsen RW. Molecular Biology Institute, University of California, Los Angeles 90095, USA. Type-A receptors for the neurotransmitter GABA (gamma-aminobutyric acid) are ligand-gated chloride channels that mediate inhibitory neurotransmission. Each subunit of the pentameric receptor protein has ligand-binding sites in the amino-terminal extracellular domain and four membrane-spanning regions, one of which forms a wall of the ion channel. Each subunit also has a large intracellular loop that may be a target for protein kinases and be required for subcellular targeting and membrane clustering of the receptor, perhaps by anchoring the receptor to the cytoskeleton. Neurotransmitter receptors need to be positioned in high density in the cell membrane at sites postsynaptic to nerve terminals releasing that neurotransmitter. Other members of the superfamily of ligand-gated ion-channel receptors associate in postsynaptic-membrane clusters by binding to the proteins rapsyn or gephyrin. Here we identify a new cellular protein, GABA(A)-receptor-associated protein (GABARAP), which can interact with the gamma2 subunit of GABA(A) receptors. GABARAP binds to GABA(A) receptors both in vitro and in vivo, and co-localizes with the punctate staining of GABA(A) receptors on cultured cortical neurons. Sequence analysis shows similarity between GABARAP and light chain-3 of microtubule-associated proteins 1A and 1B. Moreover, the N terminus of GABARAP is highly positively charged and features a putative tubulin-binding motif. The interactions among GABA(A) receptors, GABARAP and tubulin suggest a mechanism for the targeting and clustering of GABA(A) receptors. PMID: 9892355 [PubMed - indexed for MEDLINE] 839: Mol Cell Biol 1999 Feb;19(2):1547-57 Interactions between a nuclear transporter and a subset of nuclear pore complex proteins depend on Ran GTPase. Seedorf M, Damelin M, Kahana J, Taura T, Silver PA. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and The Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. Proteins to be transported into the nucleus are recognized by members of the importin-karyopherin nuclear transport receptor family. After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous pore channel. Once cargo is released, the importin then moves back through the channel for new rounds of transport. Thus, importin and exportin, another member of this family involved in export, are thought to continuously shuttle between the nuclear interior and the cytoplasm. In order to understand how nuclear transporters traverse the NPC, we constructed functional protein fusions between several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent protein (GFP). Complexes containing nuclear transporters were isolated by using highly specific anti-GFP antibodies. Pse1-GFP was studied in the most detail. Pse1-GFP is in a complex with importin-alpha and -beta (Srp1p and Kap95p in yeast cells) that is sensitive to the nucleotide-bound state of the Ran GTPase. In addition, Pse1p associates with the nucleoporins Nsp1p, Nup159p, and Nup116p, while Sxm1p, Xpo1p, and Kap95p show different patterns of interaction with nucleoporins. Association of Pse1p with nucleoporins also depends on the nucleotide-bound state of Ran; when Ran is in the GTP-bound state, the nucleoporin association is lost. A mutant form of Pse1p that does not bind Ran also fails to interact with nucleoporins. These data indicate that transport receptors such as Pse1p interact in a Ran-dependent manner with certain nucleoporins. These nucleoporins may represent major docking sites for Pse1p as it moves in or out of the nucleus via the NPC. PMID: 9891088 [PubMed - indexed for MEDLINE] 840: Mol Cell Biol 1999 Feb;19(2):1325-33 Antagonistic interactions between yeast chaperones Hsp104 and Hsp70 in prion curing. Newnam GP, Wegrzyn RD, Lindquist SL, Chernoff YO. School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230, USA. The maintenance of [PSI], a prion-like form of the yeast release factor Sup35, requires a specific concentration of the chaperone protein Hsp104: either deletion or overexpression of Hsp104 will cure cells of [PSI]. A major puzzle of these studies was that overexpression of Hsp104 alone, from a heterologous promoter, cures cells of [PSI] very efficiently, yet the natural induction of Hsp104 with heat shock, stationary-phase growth, or sporulation does not. These observations pointed to a mechanism for protecting the genetic information carried by the [PSI] element from vicissitudes of the environment. Here, we show that simultaneous overexpression of Ssa1, a protein of the Hsp70 family, protects [PSI] from curing by overexpression of Hsp104. Ssa1 protein belongs to the Ssa subfamily, members of which are normally induced with Hsp104 during heat shock, stationary-phase growth, and sporulation. At the molecular level, excess Ssa1 prevents a shift of Sup35 protein from the insoluble (prion) to the soluble (cellular) state in the presence of excess Hsp104. Overexpression of Ssa1 also increases nonsense suppression by [PSI] when Hsp104 is expressed at its normal level. In contrast, hsp104 deletion strains lose [PSI] even in the presence of overproduced Ssa1. Overproduction of the unrelated chaperone protein Hsp82 (Hsp90) neither cured [PSI] nor antagonized the [PSI]-curing effect of overproduced Hsp104. Our results suggest it is the interplay between Hsp104 and Hsp70 that allows the maintenance of [PSI] under natural growth conditions. PMID: 9891066 [PubMed - indexed for MEDLINE] 841: Mol Cell Biol 1999 Feb;19(2):1056-67 A complex containing RNA polymerase II, Paf1p, Cdc73p, Hpr1p, and Ccr4p plays a role in protein kinase C signaling. Chang M, French-Cornay D, Fan HY, Klein H, Denis CL, Jaehning JA. Department of Biochemistry and Molecular Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. Yeast contains at least two complex forms of RNA polymerase II (Pol II), one including the Srbps and a second biochemically distinct form defined by the presence of Paf1p and Cdc73p (X. Shi et al., Mol. Cell. Biol. 17:1160-1169, 1997). In this work we demonstrate that Ccr4p and Hpr1p are components of the Paf1p-Cdc73p-Pol II complex. We have found many synthetic genetic interactions between factors within the Paf1p-Cdc73p complex, including the lethality of paf1Delta ccr4Delta, paf1Delta hpr1Delta, ccr4Delta hpr1Delta, and ccr4Delta gal11Delta double mutants. In addition, paf1Delta and ccr4Delta are lethal in combination with srb5Delta, indicating that the factors within and between the two RNA polymerase II complexes have overlapping essential functions. We have used differential display to identify several genes whose expression is affected by mutations in components of the Paf1p-Cdc73p-Pol II complex. Additionally, as previously observed for hpr1Delta, deleting PAF1 or CDC73 leads to elevated recombination between direct repeats. The paf1Delta and ccr4Delta mutations, as well as gal11Delta, demonstrate sensitivity to cell wall-damaging agents, rescue of the temperature-sensitive phenotype by sorbitol, and reduced expression of genes involved in cell wall biosynthesis. This unusual combination of effects on recombination and cell wall integrity has also been observed for mutations in genes in the Pkc1p-Mpk1p kinase cascade. Consistent with a role for this novel form of RNA polymerase II in the Pkc1p-Mpk1p signaling pathway, we find that paf1Delta mpk1Delta and paf1Delta pkc1Delta double mutants do not demonstrate an enhanced phenotype relative to the single mutants. Our observation that the Mpk1p kinase is fully active in a paf1Delta strain indicates that the Paf1p-Cdc73p complex may function downstream of the Pkc1p-Mpk1p cascade to regulate the expression of a subset of yeast genes. PMID: 9891041 [PubMed - indexed for MEDLINE] 842: Mol Cell Biol 1999 Feb;19(2):1049-55 Selective interaction of vitamin D receptor with transcriptional coactivators by a vitamin D analog. Takeyama K, Masuhiro Y, Fuse H, Endoh H, Murayama A, Kitanaka S, Suzawa M, Yanagisawa J, Kato S. Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113, Japan. The nuclear vitamin D receptor (VDR) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor. A family of cotranscriptional activators (SRC-1, TIF2, and AIB-1) interacts with and activates the transactivation function of nuclear receptors in a ligand-dependent way. We examined interaction of VDR with these coactivators that was induced by several vitamin D analogs, since they exert differential subsets of the biological action of vitamin D through unknown mechanisms. Unlike other vitamin D analogs tested, OCT (22-oxa-1alpha,25-dihydroxyvitamin D3) induced interaction of VDR with TIF2 but not with SRC-1 or AIB-1. Consistent with these interactions, only TIF2 was able to potentiate the transactivation function of VDR bound to OCT. Thus, the present findings suggest that the structure of VDR is altered in a vitamin D analog-specific way, resulting in selective interactions of VDR with coactivators. Such selective interaction of coactivators with VDR may specify the array of biological actions of a vitamin D analog like OCT, possibly through activating a particular set of target gene promoters. PMID: 9891040 [PubMed - indexed for MEDLINE] 843: J Biol Chem 1999 Jan 22;274(4):1928-33 Heterochromatin organization of a natural yeast telomere. Recruitment of Sir3p through interaction with histone H4 N terminus is required for the establishment of repressive structures. Venditti S, Vega-Palas MA, Di Mauro E. Fondazione "Istituto Pasteur-Fondazione Cenci-Bolognetti", c/o Dipartimento di Genetica e Biologia Molecolare Universita "La Sapienza", P. le A. Moro 5, 00185-Roma Italy. The chromatin organization of eukaryotic telomeres is essential for telomeric function and is currently receiving great attention. In yeast, the structural organization of telomeres involves a complex interplay of telomeric proteins that results in the formation of heterochromatin. This telomeric heterochromatin involves homotypic and heterotypic protein interactions that have been summarized in a general model. Recent analyses have focused on the study of the structural complexity at yeast telomeres to the level of specific nucleosomes and of the distribution of protein complexes in a natural telomeric region (LIII). In this report, we further analyze the structural complexity of LIII and the implication of this structure on telomeric silencing. It is shown that the establishment of repressive heterochromatin structures at LIII requires the recruitment of Sir3p through interaction with the N terminus of histone H4. The establishment of such structures does not require acetylation of any of four lysines located in the H4 N terminus (lysines 5, 8, 12, and 16). PMID: 9890947 [PubMed - indexed for MEDLINE] 844: Nat Struct Biol 1999 Jan;6(1):22-7 Structure of HAP1-18-DNA implicates direct allosteric effect of protein-DNA interactions on transcriptional activation. King DA, Zhang L, Guarente L, Marmorstein R. The Wistar Institute and The Department of Chemistry, University of Pennsylvania, Philadelphia 19104, USA. HAP1 is a yeast transcriptional activator that binds with equal affinity to the dissimilar upstream activation sequences UAS1 and UAS(CYC7), but activates transcription differentially when bound to each site. HAP1-18 harbors an amino acid change in the DNA binding domain. While binding UAS1 poorly, HAP1-18 binds UAS(CYC7) with wild-type properties and activates transcription at elevated levels relative to HAP1. We have determined the structure of HAP1-18-UAS(CYC7) and have compared it to HAP1-UAS(CYC7). Unexpectedly, the single amino acid substitution in HAP1-18 nucleates a significantly altered hydrogen bond interface between the protein and DNA resulting in DNA conformational changes and an ordering of one N-terminal arm of the protein dimer along the DNA minor groove. These observations, together with a large subset of transcriptionally defective mutations in the HAP1 DNA-binding domain that map to the HAP1-DNA interface, suggest that protein-DNA interactions may have direct allosteric effects on transcriptional activation. PMID: 9886287 [PubMed - indexed for MEDLINE] 845: Toxicol Appl Pharmacol 1999 Jan 1;154(1):76-83 New screening methods for chemicals with hormonal activities using interaction of nuclear hormone receptor with coactivator. Nishikawa J, Saito K, Goto J, Dakeyama F, Matsuo M, Nishihara T. Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka, 565-0871, Japan. nishihara@phs.osaka-u.ac.jp The endocrine system exerts important functions in a multitude of physiological processes including embryogenesis, differentiation, and homeostasis. Xenobiotics may modify natural endocrine function and so affect human health and wildlife. It is necessary, therefore, to understand the degree to which xenobiotics can disrupt endocrine systems. The key targets of endocrine disruptors are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. We have developed relevant assay systems based on the ligand-dependent interaction between nuclear hormone receptor and coactivator. The coactivators used in this study contained CBP, p300, RIP140, SRC1, TIF1, and TIF2. By two hybrid assay in yeast, the interactions of estrogen receptor with RIP140, SRC1, TIF1, and TIF2 were detected and they were completely dependent on the presence of estrogen. Specificity of this assay was assessed by determining the effect of steroids, known estrogen receptor agonists, and phytoestrogens. The pattern of response to chemicals were consistent with estrogenic activity measured by other assay systems, indicating that this assay system is reliable for measuring estrogenic activity. In addition, we carried out in vitro binding studies: GST pull-down assay and surface plasmon resonance analysis. The estrogen receptor also bound to coactivator in response to chemicals depending on their estrogenic activity in vitro. These data demonstrate that the measurement of interaction between steroid hormone receptor and coactivator serves as a useful tool for identifying chemicals that interact with steroid receptors. Copyright 1999 Academic Press. PMID: 9882594 [PubMed - indexed for MEDLINE] 846: J Biochem (Tokyo) 1999 Jan;125(1):130-7 Identification of SEC12, SED4, truncated SEC16, and EKS1/HRD3 as multicopy suppressors of ts mutants of Sar1 GTPase. Saito Y, Yamanushi T, Oka T, Nakano A. Molecular Membrane Biology Laboratory, RIKEN, Wako, Saitama, 351-0198, Japan. The yeast SAR1 gene encodes a low-molecular-weight GTPase which is essential for the formation of transport vesicles from the endoplasmic reticulum (ER). To understand how the Sar1p function is regulated in its GTPase cycle, we searched for multicopy suppressors of sar1 temperature-sensitive mutants and identified SEC12, SED4, truncated SEC16, and EKS1. EKS1 turns out to be identical to HRD3, which was independently isolated as a gene implicated in the degradation of an HMG-CoA reductase isozyme, Hmg2p. In this paper, we show that the product of EKS1/HRD3 is a type-I transmembrane glycoprotein and resides in the ER. The eks1/hrd3 disrupted cells are normal in growth and transport of cargo proteins, but missecrete BiP (Kar2p). The overexpression of EKS1/HRD3, which stabilizes Hmg2p, did not affect the stability of wild-type or mutant Sar1p or any early Sec proteins we examined. These results suggest that the role of Eks1p/Hrd3p is not involved in the ER protein degradation in general but rather required for the maintenance of the ER membrane functions. The novel genetic interactions unveiled between SAR1, SEC12, SEC16, and SED4 will provide useful information as to how the complex machinery of vesicle budding operates. PMID: 9880808 [PubMed - indexed for MEDLINE] 847: Biochim Biophys Acta 1999 Jan 6;1426(2):323-34 The KTR and MNN1 mannosyltransferase families of Saccharomyces cerevisiae. Lussier M, Sdicu AM, Bussey H. Department of Biology, McGill University, 1205 Dr. Penfield Avenue, Montreal, Que. H3A 1B1, Canada. Glycosylation constitutes one of the most important of all the post-translational modifications and may have numerous effects on the function, structure, physical properties and targeting of particular proteins. Eukaryotic glycan structures are progressively elaborated in the secretory pathway. Following the addition of a core N-linked carbohydrate in the endoplasmic reticulum, glycoproteins move to the Golgi complex where the elongation of O-linked sugar chains and processing of complex N-linked oligosaccharide structures take place. In order to better define how such post-translational modifications occur, we have been studying the yeast KTR and MNN1 mannosyltransferase gene families. The KTR family contains nine members: KRE2, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6 and KTR7. The MNN1 family contains six members: MNN1, TTP1, YGL257c, YNR059w, YIL014w and YJL86w. In this review, we address protein structure, sequence similarities and enzymatic activity in the context of each gene family. In addition, a description of the known function of many family members in O- and N-linked glycosylation is included. Finally, the genetic interactions and functional redundancies within a gene family are also discussed. Publication Types: Review Review, Tutorial PMID: 9878809 [PubMed - indexed for MEDLINE] 848: Biochim Biophys Acta 1999 Jan 6;1426(2):309-22 Asparagine-linked glycosylation in the yeast Golgi. Dean N. Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, NY 11794-5215, USA. ndean@mcbsgi.bio.sunnysb.edu The Golgi complex is the site where the terminal carbohydrate modification of proteins and lipids occurs. These carbohydrates play a variety of biological roles, ranging from the stabilization of glycoprotein structure to the provision of ligands for cell-cell interactions to the regulation of cell surface properties. Progress in our understanding of the biosynthesis and regulation of glycoconjugates has been accelerating at a rapid pace. Recent advances in the field of yeast glycobiology have been particularly impressive. This review focuses on glycosylation of proteins in the Golgi of the yeast Saccharomyces cerevisiae, with emphasis on the candidate mannosyltransferases that participate in the synthesis of N-linked oligosaccharides. Current views on how these enzymes may be regulated and how glycosylation relates on other cellular processes are also discussed. Publication Types: Review Review, Tutorial PMID: 9878803 [PubMed - indexed for MEDLINE] 849: J Mol Biol 1998 Dec 18;284(5):1341-51 Conserved core structure in the internal transcribed spacer 1 of the Schizosaccharomyces pombe precursor ribosomal RNA. Lalev AI, Nazar RN. Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, N1G 2W1, Canada. The structure of the internal transcribed spacer 1 (ITS1) in Schizosaccharomyces pombe was examined with respect to phylogenetically conserved features in yeasts as well as the binding of transacting factors that potentially play a role in ribosomal maturation. Computer analyses and probes for nuclease protection indicate a compact, more highly organized structure than previously proposed in Saccharomyces cerevisiae, with distinct structural features which can be recognized in S. cerevisiae. These include a central extended hairpin structure as well as smaller hairpins immediately adjacent to the maturing termini. Comparisons with ITS sequences in more diverse organisms indicate that the same features also can be recognized. This is especially clear in organisms which contain very short sequences in which the putative structures are much less ambiguous. Again nuclease protection analyses in one of these, Verticillium albo-atrum, confirm a central hairpin with additional hairpins linked to the maturing termini. Protein binding and gel retardation studies with the S. pombe ITS1 further indicate that, as observed in the 3' external transcripted spacer (ETS) region, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing, but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data provide evidence that the spacer regions act not only to organize the maturing terminal sequences but also may serve to organize specific soluble factors, possibly acting in a manner which is analogous with that of the free small nucleolar ribonucleo protein particles (snoRNPs). Copyright 1998 Academic Press PMID: 9878354 [PubMed - indexed for MEDLINE] 850: EMBO J 1999 Jan 4;18(1):58-64 Epistatic interactions of deletion mutants in the genes encoding the F1-ATPase in yeast Saccharomyces cerevisiae. Lai-Zhang J, Xiao Y, Mueller DM. Department of Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, IL 60064, USA. The F1-ATPase is a multimeric enzyme (alpha3 beta3 gamma delta epsilon) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding alpha-, beta-, gamma- or delta-subunits were unable to grow, while the strain with a null mutation in epsilon was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in gamma or delta became 100% rho0/rho- and the strain with the null mutation in gamma grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: Delta alpha Delta gamma, Delta beta Delta gamma, Deltaatp11 Delta gamma, Delta alpha Delta delta, Delta beta Delta delta or Deltaatp11 Delta delta. These results indicate that epsilon is not an essential component of the ATP synthase and that mutations in the genes encoding the alpha- and beta-subunits and in ATP11 are epistatic to null mutations in the genes encoding the gamma- and delta-subunits. These data suggest that the propensity to form rho0/rho- mutations in the gamma and delta null deletion mutant stains and the slow growing phenotypes of the null gamma mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells. PMID: 9878050 [PubMed - indexed for MEDLINE] 851: Genetics 1999 Jan;151(1):31-44 A region of the Sir1 protein dedicated to recognition of a silencer and required for interaction with the Orc1 protein in saccharomyces cerevisiae. Gardner KA, Rine J, Fox CA. Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA. Silencing of the cryptic mating-type loci HMR and HML requires the recognition of DNA sequence elements called silencers by the Sir1p, one of four proteins dedicated to the assembly of silenced chromatin in Saccharomyces cerevisiae. The Sir1p is thought to recognize silencers indirectly through interactions with proteins that bind the silencer DNA directly, such as the origin recognition complex (ORC). Eight recessive alleles of SIR1 were discovered that encode mutant Sir1 proteins specifically defective in their ability to recognize the HMR-E silencer. The eight missense mutations all map within a 17-amino-acid segment of Sir1p, and this segment was also required for Sir1p's interaction with Orc1p. The mutant Sir1 proteins could function in silencing if tethered to a silencer directly through a heterologous DNA-binding domain. Thus the amino acids identified are required for Sir1 protein's recognition of the HMR-E silencer and interaction with Orc1p, but not for its ability to function in silencing per se. The approach used to find these mutations may be applicable to defining interaction surfaces on proteins involved in other processes that require the assembly of macromolecular complexes. PMID: 9872946 [PubMed - indexed for MEDLINE] 852: Genes Dev 1998 Dec 15;12(24):3843-56 Maintenance of sister-chromatid cohesion at the centromere by the Drosophila MEI-S332 protein. Tang TT, Bickel SE, Young LM, Orr-Weaver TL. Whitehead Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts USA 02142, USA. Sister-chromatid cohesion is essential for the faithful segregation of chromosomes during cell division. Recently biochemical analysis with Xenopus extracts suggests that cohesion is established during S phase by a cohesion complex but that other proteins must maintain it in mitosis. The Drosophila melanogaster MEI-S332 protein is present on centromeres in mitosis and meiosis and is essential for cohesion at the centromeres in meiosis II. Here, we analyze the timing of MEI-S332 assembly onto centromeres and the functional domains of the MEI-S332 protein. We find that MEI-S332 is first detectable on chromosomes during prometaphase, and this localization is independent of microtubules. MEI-S332 contains two separable functional domains, as mutations within these domains show intragenic complementation. The carboxy-terminal basic region is required for chromosomal localization. The amino-terminal coiled-coil domain may facilitate protein-protein interactions between MEI-S332 and male meiotic proteins. MEI-S332 interacts with itself in the yeast two-hybrid assay and in immunoprecipitates from Drosophila oocyte and embryo extracts. Thus it appears that MEI-S332 assembles into a multimeric protein complex that localizes to centromeric regions during prometaphase and is required for the maintenance of sister-chromatid cohesion until anaphase, rather than its establishment in S phase. PMID: 9869638 [PubMed - indexed for MEDLINE] 853: Nature 1998 Dec 10;396(6711):587-90 Decoupling of nucleotide- and microtubule-binding sites in a kinesin mutant. Song H, Endow SA. Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA. Molecular motors require ATP to move along microtubules or actin filaments. To understand how molecular motors function, it is crucial to know how binding of the motor to its filamentous track stimulates the hydrolysis of ATP by the motor, enabling it to move along the filament. A mechanism for the enhanced ATP hydrolysis has not been elucidated, but it is generally accepted that conformational changes in the motor proteins occur when they bind to microtubules or actin filaments, facilitating the release of ADP. Here we report that a mutation in the motor domain of the microtubule motor proteins Kar3 and Ncd uncouples nucleotide- and microtubule-binding by the proteins, preventing activation of the motor ATPase by microtubules. Unlike the wild-type motors, the mutants bind tightly to both ADP and microtubules, indicating that interactions between the nucleotide- and microtubule-binding sites are blocked. The region of the motor that includes the mutated amino acid could transmit or undergo a conformational change required to convert the motor ATPase into a microtubule-stimulated state. PMID: 9859995 [PubMed - indexed for MEDLINE] 854: Mol Cell Biol 1999 Jan;19(1):826-34 Ribosomal protein S14 of Saccharomyces cerevisiae regulates its expression by binding to RPS14B pre-mRNA and to 18S rRNA. Fewell SW, Woolford JL Jr. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA. Production of ribosomal protein S14 in Saccharomyces cerevisiae is coordinated with the rate of ribosome assembly by a feedback mechanism that represses expression of RPS14B. Three-hybrid assays in vivo and filter binding assays in vitro demonstrate that rpS14 directly binds to an RNA stem-loop structure in RPS14B pre-mRNA that is necessary for RPS14B regulation. Moreover, rpS14 binds to a conserved helix in 18S rRNA with approximately five- to sixfold-greater affinity. These results support the model that RPS14B regulation is mediated by direct binding of rpS14 either to its pre-mRNA or to rRNA. Investigation of these interactions with the three-hybrid system reveals two regions of rpS14 that are involved in RNA recognition. D52G and E55G mutations in rpS14 alter the specificity of rpS14 for RNA, as indicated by increased affinity for RPS14B RNA but reduced affinity for the rRNA target. Deletion of the C terminus of rpS14, where multiple antibiotic resistance mutations map, prevents binding of rpS14 to RNA and production of functional 40S subunits. The emetine-resistant protein, rpS14-EmRR, which contains two mutations near the C terminus of rpS14, does not bind either RNA target in the three-hybrid or in vitro assays. This is the first direct demonstration that an antibiotic resistance mutation alters binding of an r protein to rRNA and is consistent with the hypothesis that antibiotic resistance mutations can result from local alterations in rRNA structure. PMID: 9858605 [PubMed - indexed for MEDLINE] 855: Mol Cell Biol 1999 Jan;19(1):602-11 Genetic evidence for Pak1 autoinhibition and its release by Cdc42. Tu H, Wigler M. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA. Pak1 protein kinase of Schizosaccharomyces pombe, a member of the p21-GTPase-activated protein kinase (PAK) family, participates in signaling pathways including sexual differentiation and morphogenesis. The regulatory domain of PAK proteins is thought to inhibit the kinase catalytic domain, as truncation of this region renders kinases more active. Here we report the detection in the two-hybrid system of the interaction between Pak1 regulatory domain and the kinase catalytic domain. Pak1 catalytic domain binds to the same highly conserved region on the regulatory domain that binds Cdc42, a GTPase protein capable of activating Pak1. Two-hybrid, mutant, and genetic analyses indicated that this intramolecular interaction rendered the kinase in a closed and inactive configuration. We show that Cdc42 can induce an open configuration of Pak1. We propose that Cdc42 interaction disrupts the intramolecular interactions of Pak1, thereby releasing the kinase from autoinhibition. PMID: 9858584 [PubMed - indexed for MEDLINE] 856: Mol Cells 1998 Oct 31;8(5):606-13 Isoform-specific interaction of the cytoplasmic domains of Na,K-ATPase. Yoon T, Lee K. College of Pharmacy, Ewha Woman's University, Seoul, Korea. The Na,K-ATPase is a heterodimer consisting of an alpha and a beta subunit, which exchanges intracellular Na+ for extracellular K+ using the energy of ATP hydrolysis. Several studies have demonstrated that the enzyme exists as an (alphabeta)2 heterotetramer, an oligomer of alphabeta dimers within the cell membrane, at least during some portion of the transport cycle although its functional significance is unknown. In our study, we employed the yeast two-hybrid system to identify the cytoplasmic domains of the Na,K-ATPase which might be involved in intersubunit and/or intrasubunit interactions to form higher order oligmers. Our data demonstrate that the N-terminus and the cytoplasmic loop 1 of the alpha2 subunit interact with each other, while those of the alpha1 subunit do not, suggesting that the interaction is isoform-specific. Therefore, the N-terminal and the cytoplasmic loop 1 might be the regions where the alpha2 subunit, which are involved in alpha alpha interactions, stabilize Na,K-ATPase as alphabeta protomer, diprotomer, or higher order oligomer because the interaction can be intrasubunit as well as intersubunit interactions. Our study suggests that there may be an isoform-specific difference in the alpha-alpha interaction and that the isoform-specific interaction may contribute significantly to the differences of the physiological function and regulation among the alpha isoforms. PMID: 9856349 [PubMed - indexed for MEDLINE] 857: Biochem J 1999 Jan 1;337 ( Pt 1):89-95 The role of the C-terminal region in phosphoglycerate mutase. Walter RA, Nairn J, Duncan D, Price NC, Kelly SM, Rigden DJ, Fothergill-Gilmore LA. Department of Biochemistry, University of Edinburgh, George Square, Edinburgh EH8 9XD, Scotland, U.K. Removal of the C-terminal seven residues from phosphoglycerate mutase from Saccharomyces cerevisiae by limited proteolysis is associated with loss of mutase activity, but no change in phosphatase activity. The presence of the cofactor 2, 3-bisphosphoglycerate, or of the cofactor and substrate 3-phosphoglycerate together, confers protection against proteolysis. The substrate alone offers no protection. Replacement of either or both of the two lysines at the C-terminus by glycines has only limited effects on the kinetic properties of phosphoglycerate mutase, indicating that these residues are unlikely to be involved in crucial electrostatic interactions with the substrate, intermediate or product in the reaction. However, the double-mutant form of the enzyme is more sensitive to proteolysis and is no longer protected against proteolysis by the presence of cofactor. The proteolysed wild-type and two of the mutated forms of the enzyme show a reduced response to 2-phosphoglycollate, which enhances the instability of the phospho form of the native enzyme. The phosphoglycerate mutase from Schizosaccharomyces pombe, which lacks the analogous C-terminal tail, has an inherently lower mutase activity and is also less responsive to stimulation by 2-phosphoglycollate. It is proposed that the C-terminal region of phosphoglycerate mutase helps to maintain the enzyme in its active phosphorylated form and assists in the retention of the bisphosphoglycerate intermediate at the active site. However, its role seems not to be to contribute directly to ligand binding, but rather to exert indirect effects on the transfer of the phospho group between substrate, enzyme, intermediate and product. PMID: 9854029 [PubMed - indexed for MEDLINE] 858: Biol Pharm Bull 1998 Nov;21(11):1215-7 Evaluation of fluorescence polarization method for binding study in carbohydrate-lectin interaction. Oda Y, Kinoshita M, Nakayama K, Kakehi K. Faculty of Pharmaceutical Sciences, Kinki University, Higashi-osaka, Japan. The fluorescence polarization (FP) technique was evaluated to determine molecular interaction between plant lectins and polysaccharides, yeast cells and glycopeptide after labeling the lectins with fluorescein isothiocyanate. Use of Lycoris radiata agglutinin allowed determination of the molecular interactions with large biomolecules containing mannose oligomers and polymers. Another example using a fluorescein-labeled glycopeptide also indicated that use of the FP method would allow easy observation of the molecular interactions on the quantitative base. The present technique is highly sensitive and facile because it does not require any washing procedures before measurement. PMID: 9853416 [PubMed - indexed for MEDLINE] 859: J Biol Chem 1998 Dec 18;273(51):34653-60 Promoter structure and transcriptional activation with chromatin templates assembled in vitro. A single Gal4-VP16 dimer binds to chromatin or to DNA with comparable affinity. Pazin MJ, Hermann JW, Kadonaga JT. Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0347, USA. To gain a better understanding of the role of chromatin in the regulation of transcription by RNA polymerase II, we examined the relation between promoter structure and the ability of Gal4-VP16 to function with chromatin templates assembled in vitro. First, to investigate whether there are synergistic interactions among multiple bound factors, we studied promoter constructions containing one or five Gal4 sites and found that a single recognition site is sufficient for Gal4-VP16 to bind to chromatin, to induce nucleosome rearrangement, and to activate transcription. Notably, we observed that Gal4-VP16 binds to a single site in chromatin with affinity comparable with that which it binds to naked DNA, even in the absence of ATP-dependent nucleosome remodeling activity. Second, to explore the relation between translational nucleosome positioning and transcriptional activation, we analyzed a series of promoter constructions in which nucleosomes were positioned by Gal4-VP16 at different locations relative to the RNA start site. These experiments revealed that the positioning of a nucleosome over the RNA start site is not an absolute barrier to transcriptional activation. Third, to determine the contribution of core promoter elements to transcriptional activation with chromatin templates, we tested the ability of Gal4-VP16 to activate transcription with TATA box- versus DPE-driven core promoters and found that the TATA box is not required to achieve transcriptional activation by Gal4-VP16 with chromatin templates. These results suggest that a single protomer of a strong activator is able to bind to chromatin, to induce nucleosome remodeling, and to activate transcription in conjunction with a broad range of chromatin structures and core promoter elements. PMID: 9852139 [PubMed - indexed for MEDLINE] 860: J Biol Chem 1998 Dec 18;273(51):34328-34 Evidence for a salt bridge between transmembrane segments 5 and 6 of the yeast plasma-membrane H+-ATPase. Gupta SS, DeWitt ND, Allen KE, Slayman CW. Departments of Genetics and Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. The plasma-membrane H+-ATPase of Saccharomyces cerevisiae, which belongs to the P2 subgroup of cation-transporting ATPases, is encoded by the PMA1 gene and functions physiologically to pump protons out of the cell. This study has focused on hydrophobic transmembrane segments M5 and M6 of the H+-ATPase. In particular, a conserved aspartate residue near the middle of M6 has been found to play a critical role in the structure and biogenesis of the ATPase. Site-directed mutants in which Asp-730 was replaced by an uncharged residue (Asn or Val) were abnormally sensitive to trypsin, consistent with the idea that the proteins were poorly folded, and immunofluorescence confocal microscopy showed them to be arrested in the endoplasmic reticulum. Similar defects are known to occur when either Arg-695 or His-701 in M5 is replaced by a neutral residue (Dutra, M. B., Ambesi, A., and Slayman, C. W. (1998) J. Biol. Chem. 273, 17411-17417). To search for possible charge-charge interactions between Asp-730 and Arg-695 or His-701, double mutants were constructed in which positively and negatively charged residues were swapped or eliminated. Strikingly, two of the double mutants (R695D/D730R and R695A/D730A) regained the capacity for normal biogenesis and displayed near-normal rates of ATP hydrolysis and ATP-dependent H+ pumping. These results demonstrate that neither Arg-695 nor Asp-730 is required for enzymatic activity or proton transport, but suggest that there is a salt bridge between the two residues, linking M5 and M6 of the 100-kDa polypeptide. PMID: 9852098 [PubMed - indexed for MEDLINE] 861: RNA 1998 Dec;4(12):1675-86 Corrected and republished from: RNA 1998 Oct; 4(10):1239-50 Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae. Dix I, Russell CS, O'Keefe RT, Newman AJ, Beggs JD. Institute of Cell and Molecular Biology, University of Edinburgh, United Kingdom. We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome. Publication Types: Corrected and Republished Article PMID: 9848662 [PubMed - indexed for MEDLINE] 862: Proc Natl Acad Sci U S A 1998 Dec 8;95(25):14897-902 Identification of a proline-binding motif regulating CD2-triggered T lymphocyte activation. Nishizawa K, Freund C, Li J, Wagner G, Reinherz EL. Laboratory of Immunobiology, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, MA 02115, USA. An intracellular protein termed CD2 binding protein 2 (CD2BP2), which binds to a site containing two PPPGHR segments within the cytoplasmic region of CD2, was identified. Mutagenesis and NMR analysis demonstrated that the CD2 binding region of CD2BP2 includes a 17-aa motif (GPY[orF]xxxxM[orV]xxWxxx GYF), also found in several yeast and Caenorhabditis elegans proteins of unknown function. In Jurkat T cells, over-expression of the isolated CD2BP2 domain binding to CD2 enhances the production of interleukin 2 on crosslinking of CD2 but not the T cell receptor. Hence, a proline-binding module distinct from SH3 and WW domains regulates protein-protein interactions. PMID: 9843987 [PubMed - indexed for MEDLINE] 863: Proc Natl Acad Sci U S A 1998 Dec 8;95(25):14799-804 The terminal tail region of a yeast myosin-V mediates its attachment to vacuole membranes and sites of polarized growth. Catlett NL, Weisman LS. Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA. The Saccharomyces cerevisiae myosin-V, Myo2p, has been implicated in the polarized movement of several organelles and is essential for yeast viability. We have shown previously that Myo2p is required for the movement of a portion of the lysosome (vacuole) into the bud and consequently for proper inheritance of this organelle during cell division. Class V myosins have a globular carboxyl terminal tail domain that is proposed to mediate localization of the myosin, possibly through interaction with organelle-specific receptors. Here we describe a myo2 allele whose phenotypes support this hypothesis. vac15-1/myo2-2 has a single mutation in this globular tail domain, causing defects in vacuole movement and inheritance. Although a portion of wild-type Myo2p fractionates with the vacuole, the myo2-2 gene product does not. In addition, the mutant protein does not concentrate at sites of active growth, the predominant location of wild-type Myo2p. Although deletion of the tail domain is lethal, the myo2-2 gene product retains the essential functions of Myo2p. Moreover, myo2-2 does not cause the growth defects and lethal genetic interactions seen in myo2-66, a mutant defective in the actin-binding domain. These observations suggest that the myo2-2 mutation specifically disrupts interactions with selected myosin receptors, namely those on the vacuole membrane and those at sites of polarized growth. PMID: 9843969 [PubMed - indexed for MEDLINE] 864: Mol Biol Cell 1998 Dec;9(12):3533-45 Specific molecular chaperone interactions and an ATP-dependent conformational change are required during posttranslational protein translocation into the yeast ER. McClellan AJ, Endres JB, Vogel JP, Palazzi D, Rose MD, Brodsky JL. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA. The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST-63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST-63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST-63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners. PMID: 9843586 [PubMed - indexed for MEDLINE] 865: Mol Biol Cell 1998 Dec;9(12):3475-92 Functional characterization of a Nup159p-containing nuclear pore subcomplex. Belgareh N, Snay-Hodge C, Pasteau F, Dagher S, Cole CN, Doye V. Centre National de la Recherche Scientifique, UMR144, Institut Curie, 75 248 Paris cedex 05, France. Nup159p/Rat7p is an essential FG repeat-containing nucleoporin localized at the cytoplasmic face of the nuclear pore complex (NPC) and involved in poly(A)+ RNA export and NPC distribution. A detailed structural-functional analysis of this nucleoporin previously demonstrated that Nup159p is anchored within the NPC through its essential carboxyl-terminal domain. In this study, we demonstrate that Nup159p specifically interacts through this domain with both Nsp1p and Nup82p. Further analysis of the interactions within the Nup159p/Nsp1p/Nup82p subcomplex using the nup82Delta108 mutant strain revealed that a deletion within the carboxyl-terminal domain of Nup82p prevents its interaction with Nsp1p but does not affect the interaction between Nup159p and Nsp1p. Moreover, immunofluorescence analysis demonstrated that Nup159p is delocalized from the NPC in nup82Delta108 cells grown at 37 degrees C, a temperature at which the Nup82Delta108p mutant protein becomes degraded. This suggests that Nup82p may act as a docking site for a core complex composed of the repeat-containing nucleoporins Nup159p and Nsp1p. In vivo transport assays further revealed that nup82Delta108 and nup159-1/rat7-1 mutant strains have little if any defect in nuclear protein import and protein export. Together our data suggest that the poly(A)+ RNA export defect previously observed in nup82 mutant cells might be due to the loss from the NPCs of the repeat-containing nucleoporin Nup159p. PMID: 9843582 [PubMed - indexed for MEDLINE] 866: EMBO J 1998 Dec 1;17(23):7105-17 A map of the binding site for catalytic domain 5 in the core of a group II intron ribozyme. Konforti BB, Liu Q, Pyle AM. Department of Biochemistry and Molecular Biophysics, Columbia University, 701 W. 168th Street, Room 616, Hammer Health Sciences Center, New York, NY 10032, USA. Group II introns are ribozymes with a complex tertiary architecture that is of great interest as a model for RNA folding. Domain 5 (D5) is a highly conserved region of the intron that is considered one of the most critical structures in the catalytic core. Despite its central importance, the means by which D5 interacts with other core elements is unclear. To obtain a map of potential interaction sites, dimethyl sulfate was used to footprint regions of the intron that are involved in D5 binding. These studies were complemented by measurements of D5 binding to a series of truncated intron derivatives. In this way, the minimal region of the intron required for strong D5 association was defined and the sites most likely to represent thermodynamically significant positions of tertiary contact were identified. These studies show that ground-state D5 binding is mediated by tertiary contacts to specific regions of D1, including a tetraloop receptor and an adjacent three-way junction. In contrast, D2 and D3 are not found to stabilize D5 association. These data highlight the significance of D1-D5 interactions and will facilitate the identification of specific tertiary contacts between them. PMID: 9843514 [PubMed - indexed for MEDLINE] 867: Biochemistry 1998 Nov 10;37(45):15842-9 DNA sequence-specific recognition by the Saccharomyces cerevisiae "TATA" binding protein: promoter-dependent differences in the thermodynamics and kinetics of binding. Petri V, Hsieh M, Jamison E, Brenowitz M. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA. The equilibrium binding and association kinetics of the Saccharomyces cerevisiae TATA Binding Protein (TBP) to the E4 and Major Late promoters of adenovirus (TATATATA and TATAAAAG, respectively), have been directly compared by quantitative DNase I titration and quench-flow "footprinting". The equilibrium binding of TBP to both promoters is described by the equilibrium TBP + DNA"TATA" left and right arrow TBP-DNA"TATA". The salt dependence of TBP binding to both promoters is identical within experimental error while the temperature dependence differs significantly. The observed rate of association follows simple second-order kinetics over the TBP concentration ranges investigated. The salt and temperature dependencies of the second-order association rate constants for TBP binding the two promoters reflect the dependencies determined by equilibrium binding. The TBP-E4 promoter interaction is entropically driven at low temperature and enthalpically driven at high temperature while the TBP-Major Late promoter reaction is entropically driven over virtually the entire temperature range investigated. These data suggest that the reaction mechanisms of TBP-promoter interactions are TATA sequence-specific and provide for differential regulation of promoters as a function of environmental variables. PMID: 9843390 [PubMed - indexed for MEDLINE] 868: Biochemistry 1998 Nov 10;37(45):15726-36 SH3 binding domains in the dopamine D4 receptor. Oldenhof J, Vickery R, Anafi M, Oak J, Ray A, Schoots O, Pawson T, von Zastrow M, Van Tol HH. Department of Pharmacology, Institute of Medical Science, University of Toronto, Ontario, Canada. The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization. PMID: 9843378 [PubMed - indexed for MEDLINE] 869: Microbiol Mol Biol Rev 1998 Dec;62(4):1492-553 Posttranscriptional control of gene expression in yeast. McCarthy JE. Posttranscriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), Manchester M60 1QD, United Kingdom. J.McCarthy@umist.ac.uk Studies of the budding yeast Saccharomyces cerevisiae have greatly advanced our understanding of the posttranscriptional steps of eukaryotic gene expression. Given the wide range of experimental tools applicable to S. cerevisiae and the recent determination of its complete genomic sequence, many of the key challenges of the posttranscriptional control field can be tackled particularly effectively by using this organism. This article reviews the current knowledge of the cellular components and mechanisms related to translation and mRNA decay, with the emphasis on the molecular basis for rate control and gene regulation. Recent progress in characterizing translation factors and their protein-protein and RNA-protein interactions has been rapid. Against the background of a growing body of structural information, the review discusses the thermodynamic and kinetic principles that govern the translation process. As in prokaryotic systems, translational initiation is a key point of control. Modulation of the activities of translational initiation factors imposes global regulation in the cell, while structural features of particular 5' untranslated regions, such as upstream open reading frames and effector binding sites, allow for gene-specific regulation. Recent data have revealed many new details of the molecular mechanisms involved while providing insight into the functional overlaps and molecular networking that are apparently a key feature of evolving cellular systems. An overall picture of the mechanisms governing mRNA decay has only very recently begun to develop. The latest work has revealed new information about the mRNA decay pathways, the components of the mRNA degradation machinery, and the way in which these might relate to the translation apparatus. Overall, major challenges still to be addressed include the task of relating principles of posttranscriptional control to cellular compartmentalization and polysome structure and the role of molecular channelling in these highly complex expression systems. Publication Types: Review Review, Academic PMID: 9841679 [PubMed - indexed for MEDLINE] 870: J Immunol Methods 1998 Nov 1;220(1-2):179-88 A yeast surface display system for the discovery of ligands that trigger cell activation. Cho BK, Kieke MC, Boder ET, Wittrup KD, Kranz DM. Department of Biochemistry, University of Illinois, Urbana 61801, USA. Opposing cells often communicate signalling events using multivalent interactions between receptors present on their cell surface. For example, T cells are typically activated when the T cell receptor (TCR) and its associated costimulatory molecules are multivalently engaged by the appropriate ligands present on an antigen presenting cell. In this report, yeast expressing high cell-surface levels of a TCR ligand (a recombinant antibody to the TCR Vbeta domain) were shown to act as 'pseudo' antigen presenting cells and induce T cell activation as monitored by increased levels of CD25 and CD69 and by downregulation of cell surface TCR. Similar levels of T cell activation could occur even when a 30-fold excess of irrelevant yeast was present, suggesting that such a yeast display system, by virtue of its ability to present ligands multivalently, may be used in highly sensitive procedures to identify novel polypeptides that interact multivalently with cell surface receptors and thereby trigger specific cellular responses. PMID: 9839939 [PubMed - indexed for MEDLINE] 871: Nucleic Acids Res 1998 Dec 15;26(24):5707-18 Crystal structure of the MATa1/MATalpha2 homeodomain heterodimer in complex with DNA containing an A-tract. Li T, Jin Y, Vershon AK, Wolberger C. Department of Biophysics and Biophysical Chemistry and The Howard Hughes Medical Institute,Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA. The crystal structure of the heterodimer formed by the DNA binding domains of the yeast mating type transcription factors, MATa1 and MATalpha2, bound to a 21 bp DNA fragment has been determined at 2.5 A resolution. The DNA fragment in the present study differs at four central base pairs from the DNA sequence used in the previously studied ternary complex. These base pair changes give rise to a (dA5).(dT5) tract without changing the overall base composition of the DNA. The resulting A-tract occurs near the center of the overall 60 degrees bend in the DNA. Comparison of the two structures shows that the structural details of the DNA bend are maintained despite the DNA sequence changes. Analysis of the A5-tract DNA subfragment shows that it contains a bend toward the minor groove centered at one end of the A-tract. The observed bend is larger than that observed in the crystal structures of A-tracts embedded in uncomplexed DNA, which are straight and have been presumed to be quite rigid. Variation of the central DNA base sequence reverses the two AT base pairs contacted in the minor groove by Arg7 of the alpha2 N-terminal arm without significantly altering the DNA binding affinity of the a1/alpha2 heterodimer. The Arg7 side chain accommodates the sequence change by forming alternate H bond interactions, in agreement with the proposal that minor groove base pair recognition is insensitive to base pair reversal. Furthermore, the minor groove spine of hydration, which stabilizes the narrowed minor groove caused by DNA bending, is conserved in both structures. We also find that many of the water-mediated hydrogen bonds between the a1 and alpha2 homeodomains and the DNA are highly conserved, indicating an important role for water in stabilization of the a1/alpha2-DNA complex. PMID: 9838003 [PubMed - indexed for MEDLINE] 872: Nucleic Acids Res 1998 Dec 15;26(24):5562-7 In vitro assembly of an archaeal D-L-N RNA polymerase subunit complex reveals a eukaryote-like structural arrangement. Eloranta JJ, Kato A, Teng MS, Weinzierl RO. Department of Biochemistry, Imperial College of Science, Technology and Medicine, Exhibition Road, London SW7 2AY, UK. Archaeal RNA polymerases (RNAPs) resemble the eukaryotic nuclear RNAPs in complexity, and many of their subunits display a high degree of sequence similarity to their eukaryotic counterparts. Here we describe specific protein-protein contacts present between individual recombinant RNAP subunits from the archaeon Methanococcus jannaschii. Subunits D and L interact specifically with each other in two-hybrid assays. D also interacts under the same conditions with the RPB11 and AC19 subunits from the yeast Saccharomyces cerevisiae, suggesting that essential elements of the binding surface between these proteins have been conserved across the archaeal/eukaryotic evolutionary domain boundary. Interactions between L and RPB3 or AC40 were, however, not detectable. Recombinant D and L subunits associate under in vitro conditions and copurify with each other during size-exclusion chromatography. Addition of an another recombinant subunit (N) to the D-L complex results in the formation of a triple complex. This D-L-N complex resembles the RPB3-RPB11-RPB10 or AC40-AC19-RPB10 complexes in eukaryotic RNAPIIand RNAPI/RNAPIII, respectively. Our data provide evidence for a close similarity in the quaternary arrangement of a subset of archaeal and eukaryotic RNA polymerase subunits and the conservation of the protein-protein contacts formed between them. PMID: 9837983 [PubMed - indexed for MEDLINE] 873: Genetics 1998 Dec;150(4):1407-17 Mapping of a yeast G protein betagamma signaling interaction. Dowell SJ, Bishop AL, Dyos SL, Brown AJ, Whiteway MS. Glaxo Wellcome Research and Development, Stevenage, SG1 2NY, United Kingdom. sd14041@glaxowellcome.co.uk The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein betagamma subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gbetagamma coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gbetagamma coiled-coil in Ste5p binding may set a precedent for Gbetagamma-effector interactions in more complex organisms. PMID: 9832519 [PubMed - indexed for MEDLINE] 874: Genes Dev 1998 Nov 15;12(22):3482-7 Allosteric interactions between capping enzyme subunits and the RNA polymerase II carboxy-terminal domain. Cho EJ, Rodriguez CR, Takagi T, Buratowski S. Harvard Medical School, Boston, Massachusetts 02115 USA. mRNA capping is a cotranscriptional event mediated by the association of capping enzyme with the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Here we map interactions between Ceg1, Cet1, and the CTD. Although the guanylyltransferase subunit can bind alone to the CTD, it cannot be guanylylated unless the triphosphatase subunit is also present. Therefore, the yeast mRNA guanylyltransferase is regulated by allosteric interactions with both the triphosphatase and CTD. PMID: 9832501 [PubMed - indexed for MEDLINE] 875: Proc Natl Acad Sci U S A 1998 Nov 24;95(24):14278-83 Physical interaction between components of DNA mismatch repair and nucleotide excision repair. Bertrand P, Tishkoff DX, Filosi N, Dasgupta R, Kolodner RD. Charles A. Dana Division of Human Cancer Genetics, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA. Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as "bait," and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes. PMID: 9826691 [PubMed - indexed for MEDLINE] 876: Proc Natl Acad Sci U S A 1998 Nov 24;95(24):14272-7 An artificial cell-cycle inhibitor isolated from a combinatorial library. Cohen BA, Colas P, Brent R. Department of Molecular Biology, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114, USA. Understanding the genetic networks that operate inside cells will require the dissection of interactions among network members. Here we describe a peptide aptamer isolated from a combinatorial library that distinguishes among such interactions. This aptamer binds to cyclin-dependent kinase 2 (Cdk2) and inhibits its kinase activity. In contrast to naturally occurring inhibitors, such as p21(Cip1), which inhibit the activity of Cdk2 on all its substrates, inhibition by pep8 has distinct substrate specificity. We show that the aptamer binds to Cdk2 at or near its active site and that its mode of inhibition is competitive. Expression of pep8 in human cells retards their progression through the G1 phase of the cell cycle. Our results suggest that the aptamer inhibits cell-cycle progression by blocking the activity of Cdk2 on substrates needed for the G1-to-S transition. This work demonstrates the feasibility of selection of artificial proteins to perform functions not developed during evolution. The ability to select proteins that block interactions between a gene product and some partners but not others should make sophisticated genetic manipulations possible in human cells and other currently intractable systems. PMID: 9826690 [PubMed - indexed for MEDLINE] 877: J Mol Biol 1998 Dec 4;284(3):673-87 Functional and structural characterization of the prp3 binding domain of the yeast prp4 splicing factor. Ayadi L, Callebaut I, Saguez C, Villa T, Mornon JP, Banroques J. Centre de Genetique Moleculaire du CNRS, Laboratoire Propre Associe a l'Universite P. & M. Curie, Gif-sur-Yvette, 91198, France. Nuclear pre-mRNA splicing occurs in a large RNA-protein complex containing four small nuclear ribonucleoprotein particles (snRNPs) and additional protein factors. The yeast Prp4 (yPrp4) protein is a specific component of the U4/U6 and U4/U6-U5 snRNPs, which associates transiently with the spliceosome before the first step of splicing. In this work, we used the in vivo yeast two-hybrid system and in vitro immunoprecipitation assays to show that yPrp4 interacts with yPrp3, another U4/U6 snRNP protein. To investigate the domain of yPrp4 that directly contacts yPrp3, we introduced deletions in the N-terminal half of yPrp4 and point mutations in the C-terminal half of the molecule, and we tested the resulting prp4 mutants for cell viability and for their ability to interact with yPrp3. We could not define any particular sequence in the first 161 amino acid residues that are specifically required for protein-protein interactions. However, deletion of a small basic-rich region of 30 amino acid residues is lethal to the cells. Analysis of the C terminus prp4 mutants obtained clearly shows that this region of yPrp4 represents the primary domain of interaction with yPrp3. Interestingly, yPrp4 shows significant similarity in its C-terminal half to the beta-subunits of G proteins. We have generated a three-dimensional computer model of this domain, consisting of a seven-bladed beta-propeller based on the crystalline structure of beta-transducin. Several lines of evidence suggested that yPrp4 is contacting yPrp3 through a large flat surface formed by the long variable loops linking the beta-strands of the propeller. This surface could be used as a scaffold for generating an RNA-protein complex. Copyright 1998 Academic Press PMID: 9826507 [PubMed - indexed for MEDLINE] 878: J Biol Chem 1998 Nov 27;273(48):32360-8 Interactions of p62(dok) with p210(bcr-abl) and Bcr-Abl-associated proteins. Bhat A, Johnson KJ, Oda T, Corbin AS, Druker BJ. Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon 97201, USA. A 62-kDa Ras GTPase-activating protein (RasGAP)-associated protein is tyrosine-phosphorylated under a variety of circumstances including growth factor stimulation and in cells transformed by activated tyrosine kinases. A cDNA for p62(dok), reported to be the RasGAP-associated 62-kDa protein, was recently cloned from Abl-transformed cells. In this study, the interactions of p62(dok) with Bcr-Abl and associated proteins were examined. In 32D myeloid cells and Rat-1 fibroblasts transformed by p210(bcr-abl), p62(dok) is tyrosine-phosphorylated and co-immunoprecipitates with Bcr-Abl, RasGAP, and CrkL, a Src homology 2 (SH2) and SH3 domain-containing adaptor protein. Tyrosine-phosphorylated p62(dok) from cells expressing p210(bcr-abl) bound directly to the SH2 domains of Abl and CrkL in a gel overlay assay. Previous work has shown that an SH2 domain deletion mutant of Bcr-Abl is defective in transforming fibroblasts but remains capable of inducing myeloid growth factor independence. In both fibroblasts and myeloid cells expressing this mutant, p62(dok) is underphosphorylated as compared with cells expressing full-length p210(bcr-abl) but remains capable of associating with Bcr-Abl. However, in a gel overlay assay, p62(dok) from cells expressing the SH2 domain deletion was incapable of associating directly with SH2 domains of Abl and CrkL. Interestingly, no direct binding between Bcr-Abl and p62(dok) could be demonstrated in a yeast two-hybrid assay. These data suggest that indirect interactions mediate the interaction between Bcr-Abl and p62(dok) and that the SH2 domain of Bcr-Abl is required for hyperphosphorylation of p62(dok). Further, hyperphosphorylation of p62(dok) correlates with the ability of Bcr-Abl to transform fibroblasts but not with the induction of growth factor independence in myeloid cells. PMID: 9822717 [PubMed - indexed for MEDLINE] 879: J Biol Chem 1998 Nov 27;273(48):32254-64 Identification of highly conserved amino-terminal segments of dTAFII230 and yTAFII145 that are functionally interchangeable for inhibiting TBP-DNA interactions in vitro and in promoting yeast cell growth in vivo. Kotani T, Miyake T, Tsukihashi Y, Hinnebusch AG, Nakatani Y, Kawaichi M, Kokubo T. Division of Gene Function in Animals, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. TFIID is a multiprotein complex composed of TBP and several TAFIIs. Small amino-terminal segments (TAF N-terminal domain (TAND)) of Drosophila TAFII230 (dTAFII230) and yeast TAFII145 (yTAFII145) bind strongly to TBP and inhibit TBP-DNA interactions. yTAFII145 TAND (yTAND) was divided into two subdomains, yTANDI10-37 and yTANDII46-71, that function cooperatively. Here, we identify dTANDII within the amino terminus of dTAFII230 at 118-143 amino acids in addition to dTANDI18-77, reported previously. dTANDII exhibits pronounced sequence similarity to yTANDII, and the two were shown to be functionally equivalent in binding to TBP and inhibiting TBP-DNA interactions in vitro. Alanine scanning mutation analysis demonstrated that Phe-57 (yTANDII) and Tyr-129 (dTANDII) are critically required for the interaction with TBP. Yeast strains containing mutant yTAFII145 lacking yTANDI or yTANDII showed a temperature-sensitive growth phenotype. The conserved core of dTANDII could substitute for the yTANDII core, and Phe-57 or Tyr-129 described above was critically required for the function of this segment in promoting normal cell growth at 37 degreesC. In these respects, the impact of yTANDII mutations on cell growth paralleled their effects on TBP binding in vitro, strongly suggesting that the yTAFII145-TBP interaction and its negative effects on TFIID binding to core promoters are physiologically important. PMID: 9822704 [PubMed - indexed for MEDLINE] 880: Mol Cell Biol 1998 Dec;18(12):7466-77 The 2 micrometer plasmid stability system: analyses of the interactions among plasmid- and host-encoded components. Velmurugan S, Ahn YT, Yang XM, Wu XL, Jayaram M. Department of Microbiology and Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA. The stable inheritance of the 2 micrometer plasmid in a growing population of Saccharomyces cerevisiae is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In this study we demonstrate that short carboxy-terminal deletions of Rep1p and Rep2p severely diminish their normal capacity to localize to the yeast nucleus. The nuclear targeting, as well as their functional role in plasmid partitioning, can be restored by the addition of a nuclear localization sequence to the amino or the carboxy terminus of the shortened Rep proteins. Analyses of deletion derivatives of the Rep proteins by using the in vivo dihybrid genetic test in yeast, as well as by glutathione S-transferase fusion trapping assays in vitro demonstrate that the amino-terminal portion of Rep1p (ca. 150 amino acids long) is responsible for its interactions with Rep2p. In a monohybrid in vivo assay, we have identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of interacting with the STB locus. The Shf1 protein expressed in Escherichia coli can bind with high specificity to the STB sequence in vitro. In a yeast strain deleted for the SHF1 locus, a 2 micrometer circle-derived plasmid shows relatively poor stability. PMID: 9819432 [PubMed - indexed for MEDLINE] 881: Mol Cell Biol 1998 Dec;18(12):7344-52 CNS1 encodes an essential p60/Sti1 homolog in Saccharomyces cerevisiae that suppresses cyclophilin 40 mutations and interacts with Hsp90. Dolinski KJ, Cardenas ME, Heitman J. Departments of Genetics, Pharmacology and Cancer Biology, and Medicine, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA. Cyclophilins are cis-trans-peptidyl-prolyl isomerases that bind to and are inhibited by the immunosuppressant cyclosporin A (CsA). The toxic effects of CsA are mediated by the 18-kDa cyclophilin A protein. A larger cyclophilin of 40 kDa, cyclophilin 40, is a component of Hsp90-steroid receptor complexes and contains two domains, an amino-terminal prolyl isomerase domain and a carboxy-terminal tetratricopeptide repeat (TPR) domain. There are two cyclophilin 40 homologs in the yeast Saccharomyces cerevisiae, encoded by the CPR6 and CPR7 genes. Yeast strains lacking the Cpr7 enzyme are viable but exhibit a slow-growth phenotype. In addition, we show here that cpr7 mutant strains are hypersensitive to the Hsp90 inhibitor geldanamycin. When overexpressed, the TPR domain of Cpr7 alone complements both cpr7 mutant phenotypes, while overexpression of the cyclophilin domain of Cpr7, full-length Cpr6, or human cyclophilin 40 does not. The open reading frame YBR155w, which has moderate identity to the yeast p60 homolog STI1, was isolated as a high-copy-number suppressor of the cpr7 slow-growth phenotype. We show that this Sti1 homolog Cns1 (cyclophilin seven suppressor) is constitutively expressed, essential, and found in protein complexes with both yeast Hsp90 and Cpr7 but not with Cpr6. Cyclosporin A inhibited Cpr7 interactions with Cns1 but not with Hsp90. In summary, our findings identify a novel component of the Hsp90 chaperone complex that shares function with cyclophilin 40 and provide evidence that there are functional differences between two conserved sets of Hsp90 binding proteins in yeast. PMID: 9819421 [PubMed - indexed for MEDLINE] 882: Mol Cell Biol 1998 Dec;18(12):7304-16 Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain. Romano PR, Zhang F, Tan SL, Garcia-Barrio MT, Katze MG, Dever TE, Hinnebusch AG. Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA. The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR. PMID: 9819417 [PubMed - indexed for MEDLINE] 883: Mol Cell Biol 1998 Dec;18(12):7205-15 Identification of a polar region in transmembrane domain 6 that regulates the function of the G protein-coupled alpha-factor receptor. Dube P, Konopka JB. Program in Molecular and Cellular Biology, State University of New York, Stony Brook, New York 11794-5222, USA. The alpha-factor pheromone receptor (Ste2p) of the yeast Saccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs). Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the alpha-factor receptor by mutating these residues to nonpolar leucine. Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity. The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the alpha-factor induced maximum signal. Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity. Comparison of nine different mutations affecting Ser254 showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing. Thus, these studies indicate that Gln253 and Ser254 are likely to be involved in intramolecular interactions with other TMDs. Furthermore, Gln253 and Ser254 fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated. These results suggest that Gln253 and Ser254 face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor. Consistent with this, we identified two residues in TMD7 (Ser288 and Ser292) that are potential contact residues for Gln253 because mutations affecting these residues also cause constitutive activity. Altogether, these results identify a new domain of the alpha-factor receptor that regulates its ability to enter the activated conformation. PMID: 9819407 [PubMed - indexed for MEDLINE] 884: Curr Opin Chem Biol 1998 Oct;2(5):597-603 Technological advances in high-throughput screening. Fernandes PB. Small Molecule Therapeutics Inc., Monmouth Junction, NJ 08852, USA. fernandes@smtherapeutics.com A variety of assay technologies continue to be developed for high-throughput screening. These include cell-based assays, surrogate systems using microbial cells such as yeast and bacterial two-hybrid and three-hybrid systems, and systems to measure nucleic acid-protein and receptor-ligand interactions. Modifications have been developed for cell-free, homogeneous assay systems, such as time-resolved fluorescence, fluorescence polarization and the scintillation proximity assay. Innovations in engineering and chemistry have led to delivery systems for nanoliter volumes and sensitive biosensors for ultra-high-throughout screening conducted in nanoliter and picoliter volumes. Spectroscopic methods have been extended to read single molecule fluorescence. Technologies are being developed to identify new targets from genomic information in order to design the next generation of screens. Publication Types: Review Review, Tutorial PMID: 9818185 [PubMed - indexed for MEDLINE] 885: J Cell Biol 1998 Nov 16;143(4):887-99 Nuclear import and the evolution of a multifunctional RNA-binding protein. Rosenblum JS, Pemberton LF, Bonifaci N, Blobel G. Laboratory of Cell Biology, Howard Hughes Medical Institute and Rockefeller University, New York, New York 10021, USA. La (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p. Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La. Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p. We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin alpha2, karyopherin beta1, Ran, and p10. As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins. Our results are consistent with an intermingling of the nuclear import and evolution of La. PMID: 9817748 [PubMed - indexed for MEDLINE] 886: RNA 1998 Nov;4(11):1357-72 Synthetic lethal interactions with conditional poly(A) polymerase alleles identify LCP5, a gene involved in 18S rRNA maturation. Wiederkehr T, Pretot RF, Minvielle-Sebastia L. Department of Cell Biology, Biozentrum, University of Basel, Switzerland. To identify new genes involved in 3'-end formation of mRNAs in Saccharomyces cerevisiae, we carried out a screen for synthetic lethal mutants with the conditional poly(A) polymerase allele, pap1-7. Five independent temperature-sensitive mutations called Icp1 to Icp5 (for lethal with conditional pap1 allele) were isolated. Here, we describe the characterization of the essential gene LCP5 which codes for a protein with a calculated molecular mass of 40.8 kD. Unexpectedly, we found that mutations in LCP5 caused defects in pre-ribosomal RNA (pre-rRNA) processing, whereas mRNA 3'-end formation in vitro was comparable to wild-type. Early cleavage steps (denoted A0 to A2) that lead to the production of mature 18S rRNA were impaired. In vivo depletion of Lcp5p also inhibited pre-rRNA processing. As a consequence, mutant and depleted cells showed decreased levels of polysomes compared to wild-type cells. Indirect immunofluorescence indicated a predominant localization of Lcp5p in the nucleolus. In addition, antibodies directed against Lcp5p specifically immunoprecipitated the yeast U3 snoRNA snR17, suggesting that the protein is directly involved in pre-rRNA processing. PMID: 9814757 [PubMed - indexed for MEDLINE] 887: Proc Natl Acad Sci U S A 1998 Nov 10;95(23):13543-8 A transcriptional activating region with two contrasting modes of protein interaction. Ansari AZ, Reece RJ, Ptashne M. Program in Molecular Biology, Memorial Sloan Kettering Cancer Center, Box 595, 1275 York Avenue, New York, NY 10021, USA. A C-terminal segment of the yeast activator Gal4 manifests two functions: When tethered to DNA, it elicits gene activation, and it binds the inhibitor Gal80. Here we examine the effects on these two functions of cysteine and proline substitutions. We find that, although certain cysteine substitutions diminish interaction with Gal80, those substitutions have little effect on the activating function in vivo and interaction with TATA box-binding protein (TBP) in vitro. Proline substitutions introduced near residues critical for Gal80 binding abolish that interaction but once again have no effect on the activating function. Crosslinking experiments show that a defined position in the activating peptide is in close proximity to TBP and Gal80 in the two separate reactions and show that binding of the inhibitor blocks binding to TBP. Thus, the same stretch of amino acids are involved in two quite different protein-protein interactions: binding to Gal80, which depends on a precise sequence and the formation of a defined secondary structure, or interactions with the transcriptional machinery in vivo, which are not impaired by perturbations of either sequence or structure. PMID: 9811836 [PubMed - indexed for MEDLINE] 888: Mol Cell Endocrinol 1998 Aug 25;143(1-2):133-42 Studies of dehydroepiandrosterone (DHEA) with the human estrogen receptor in yeast. Nephew KP, Sheeler CQ, Dudley MD, Gordon S, Nayfield SG, Khan SA. Medical Sciences Program, Indiana University School of Medicine, Bloomington 47405-4401, USA. knephew@indiana.edu Dehydroepiandrosterone (DHEA) is a C19 adrenal steroid synthesized in the human adrenal cortex and serving as a biosynthetic precursor to testosterone and 17beta-estradiol. Despite the fact that it is one of the most abundant steroid hormones in circulation, the physiological role of DHEA in humans remains unclear. The action of DHEA itself, such as its interactions with receptors and nuclear transcription factors, is not well understood, and a specific DHEA receptor has yet to be identified. Although the activity of DHEA can be due to its metabolism into androgens and estrogens, DHEA has been shown to interact with the androgen receptor and the estrogen receptor (ER) in vitro. We demonstrate in this study that DHEA (3beta-Hydroxy-5alpha-androstan-17-one) inhibits 17beta-estradiol (E2) binding to its receptor in vivo in yeast. DHEA stimulates human ER dimerization in yeast, as determined by ER fusion protein interactions, GAL4 reconstitution and subsequent measurement of increased beta-galactosidase activity. DHEA causes an increase in estrogen response element-dependent beta-galactosidase activity, demonstrating that the ER dimer induced by DHEA is transcriptionally active, but at a concentration of DHEA about 1000 times greater than E2. Inclusion of the nuclear receptor co-activator RIP140 in the yeast enhances ER transactivation by DHEA or E2 in a ligand-dependent manner; moreover, only in the presence of RIP140 is DHEA able to stimulate beta-galactosidase activity to levels similar to those achieved by E2. Ligand-receptor interaction for other C19-steroids was also examined. While 5-androstene-3beta, 17beta-diol (ADIOL) displayed estrogenic activity in this system, 4-androstene-17-dione (androstenedione) and 4-androstene-17beta-ol,3-one (testosterone) did not. We have investigated whether DHEA can interact with the human ER in vivo. Our findings demonstrate a mechanism by which DHEA interacts directly with estrogen signaling systems; however, because DHEA is several orders of magnitude less potent than E2 in this system, we conclude that it essentially is not an estrogen agonist. PMID: 9806358 [PubMed - indexed for MEDLINE] 889: J Biol Chem 1998 Nov 13;273(46):30279-86 Lipid products of phosphoinositide 3-kinase interact with Rac1 GTPase and stimulate GDP dissociation. Missy K, Van Poucke V, Raynal P, Viala C, Mauco G, Plantavid M, Chap H, Payrastre B. Institut Federatif de Recherche en Immunologie Cellulaire et Moleculaire, Universite Paul Sabatier and Centre Hospitalo-Universitaire de Toulouse, INSERM Unite 326, France. A number of reports suggest that under different conditions leading to cytoskeleton reorganization the GTPase Rac1 and possibly RhoA are downstream targets of phosphoinositide 3-kinase (PI 3-kinase). In order to gain more insight into this particular signaling pathway, we have addressed the question of a possible direct interaction of PI 3-kinase products with the Rho family GTPases RhoA, Rac1, and Cdc42. Using recombinant proteins, we found that Rac1 and, to a lesser extent, RhoA but not Cdc42 were capable to selectively bind to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in a mixture of crude brain phosphoinositides. Nucleotide-depleted Rac1 was the most efficient, but the GDP- and GTP-bound forms retained significant PtdIns(3,4,5)P3 binding activity. This protein-lipid association involved electrostatic as well as hydrophobic interactions, since both phosphate groups located at specific positions of the inositol ring and fatty-acyl chains were absolutely required. Based on the sequence of Rac1, two potential binding sites were identified, one at the C terminus and one in the extra alpha-helical domain. Deletion of these two domains resulted in a complete loss of binding to PI 3-kinase products. Finally, PtdIns(3, 4,5)P3 strongly stimulated GDP dissociation from Rac1 in a dose-dependent manner. In agreement, data obtained in intact cells suggest that PtdIns(3,4,5)P3 might target Rac1 to peculiar membrane domains, allowing formation of specific clusters containing not only small GTPases but other partners bearing pleckstrin homology domains such as specific exchange factors required for Rac1 and RhoA activation. PMID: 9804788 [PubMed - indexed for MEDLINE] 890: FEBS Lett 1998 Oct 16;437(1-2):56-60 Identification of a possible MAP kinase cascade in Arabidopsis thaliana based on pairwise yeast two-hybrid analysis and functional complementation tests of yeast mutants. Mizoguchi T, Ichimura K, Irie K, Morris P, Giraudat J, Matsumoto K, Shinozaki K. Laboratory of Plant Molecular Biology, Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center, Ibaraki, Japan. A possible MAP kinase (MAPK) cascade of Arabidopsis thaliana was identified on the basis of both yeast 2-hybrid analysis and complementation analysis of yeast mutants. Specific protein-protein interactions between ATMPK4 (a MAPK) and MEK1 (a MAPKK) and interactions between MEK1 and ATMEKK1 (a MAPKKK) were detected by using the 2-hybrid system. A growth defect of the yeast mpk1delta mutant was reversed by coexpression of ATMPK4 and MEK1. Coexpression of the N-terminal deletion form of ATMEKK1 increased the ability of MEK1 to suppress a growth defect of the yeast pbs2delta mutant. These results suggest that ATMPK4, MEK1, and ATMEKK1 may interact with each other and constitute a specific MAPK cascade in Arabidopsis. This is the first demonstration of a possible MAPK cascade in plants. PMID: 9804171 [PubMed - indexed for MEDLINE] 891: Mol Biol Cell 1998 Nov;9(11):3071-83 Hsp90 is required for pheromone signaling in yeast. Louvion JF, Abbas-Terki T, Picard D. Departement de Biologie Cellulaire, Universite de Geneve Sciences III, CH-1211 Geneve 4, Switzerland. The heat-shock protein 90 (Hsp90) is a cytosolic molecular chaperone that is highly abundant even at normal temperature. Specific functions for Hsp90 have been proposed based on the characterization of its interactions with certain transcription factors and kinases including Raf in vertebrates and flies. We therefore decided to address the role of Hsp90 for MAP kinase pathways in the budding yeast, an organism amenable to both genetic and biochemical analyses. We found that both basal and induced activities of the pheromone-signaling pathway depend on Hsp90. Signaling is defective in strains expressing low levels or point mutants of yeast Hsp90 (Hsp82), or human Hsp90beta instead of the wild-type protein. Ste11, a yeast equivalent of Raf, forms complexes with wild-type Hsp90 and depends on Hsp90 function for accumulation. For budding yeast, Ste11 represents the first identified endogenous "substrate" of Hsp90. Moreover, Hsp90 functions in steroid receptor and pheromone signaling can be genetically separated as the Hsp82 point mutant T525I and the human Hsp90beta are specifically defective for the former and the latter, respectively. These findings further corroborate the view that molecular chaperones must also be considered as transient or stable components of signal transduction pathways. PMID: 9802897 [PubMed - indexed for MEDLINE] 892: Dev Genes Evol 1998 Oct;208(8):440-6 Specific interactions between vestigial and scalloped are required to promote wing tissue proliferation in Drosophila melanogaster. Paumard-Rigal S, Zider A, Vaudin P, Silber J. Institut Jacques Monod, L.G.Q.M., 2, Place Jussieu, Tour 43, F-75251 Paris cedex 05, France. The two genes vestigial (vg) and scalloped (sd) are required for wing development in Drosophila melanogaster. They present similar patterns of expression in second and third instar wing discs and similar wing mutant phenotypes. vg encodes a nuclear protein without any recognized nucleic acid-binding motif. Sd is a transcription factor homologous to the human TEF-1 factor whose promoter activity depends on cell-specific cofactors. We postulate that Vg could be a cofactor of Sd in the wing morphogenetic process and that, together, they could constitute a functional transcription complex. We investigated genetic interactions between the two genes. We show here that vg and sd co-operate in vivo in a manner dependent on the structure of the Vg protein. We ectopically expressed vg in the patch (ptc) domains. We show evidence that wing-like outgrowths induced by ectopic expression of vg are severely reduced in vg or sd mutant backgrounds. Accordingly, we demonstrate that ptc-GAL4-driven expression of vg induces both expressions of the endogenous vg and sd genes and that the two Vg and Sd proteins have to be produced together to promote wing proliferation. Furthermore, we show an interaction between the two proteins by double hybrid experiments in yeast. Our results therefore support the hypothesis that Sd and Vg directly interact in vivo to form a complex regulating the proliferation of wing tissue. PMID: 9799424 [PubMed - indexed for MEDLINE] 893: Genetics 1998 Nov;150(3):987-1005 Sth1p, a Saccharomyces cerevisiae Snf2p/Swi2p homolog, is an essential ATPase in RSC and differs from Snf/Swi in its interactions with histones and chromatin-associated proteins. Du J, Nasir I, Benton BK, Kladde MP, Laurent BC. Department of Microbiology and Immunology and Morse Institute for Molecular Genetics, State University of New York, Brooklyn, New York 11203, USA. The essential Sth1p is the protein most closely related to the conserved Snf2p/Swi2p in Saccharomyces cerevisiae. Sth1p purified from yeast has a DNA-stimulated ATPase activity required for its function in vivo. The finding that Sth1p is a component of a multiprotein complex capable of ATP-dependent remodeling of the structure of chromatin (RSC) in vitro, suggests that it provides RSC with ATP hydrolysis activity. Three sth1 temperature-sensitive mutations map to the highly conserved ATPase/helicase domain and have cell cycle and non-cell cycle phenotypes, suggesting multiple essential roles for Sth1p. The Sth1p bromodomain is required for wild-type function; deletion mutants lacking portions of this region are thermosensitive and arrest with highly elongated buds and 2C DNA content, indicating perturbation of a unique function. The pleiotropic growth defects of sth1-ts mutants imply a requirement for Sth1p in a general cellular process that affects several metabolic pathways. Significantly, an sth1-ts allele is synthetically sick or lethal with previously identified mutations in histones and chromatin assembly genes that suppress snf/swi, suggesting that RSC interacts differently with chromatin than Snf/Swi. These results provide a framework for understanding the ATP-dependent RSC function in modeling chromatin and its connection to the cell cycle. PMID: 9799253 [PubMed - indexed for MEDLINE] 894: Virology 1998 Oct 25;250(2):302-15 The vaccinia virus E3L gene product interacts with both the regulatory and the substrate binding regions of PKR: implications for PKR autoregulation. Sharp TV, Moonan F, Romashko A, Joshi B, Barber GN, Jagus R. Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland, 21202, USA. The vaccinia virus E3L gene product, pE3, is a dsRNA binding protein that prevents activation of the interferon-induced, dsRNA-activated protein kinase, PKR. Activation of PKR, which results in phosphorylation of the translation initiation factor, eIF2alpha, leads to the inhibition of protein synthesis, a process involved in defense against virus infection. The E3L gene product has a conserved dsRNA binding domain (DRBD) in its carboxyl-terminal region and has been shown to function in vitro by sequestration of dsRNA. We have utilized in vitro binding assays and the yeast two-hybrid system to demonstrate direct interactions of pE3 with PKR. By these methods, we demonstrate that pE3 interacts with two distinct regions in PKR, the amino-terminal (amino acids 1-99) located in the regulatory domain and the carboxyl-terminal (amino acids 367-523) located in the catalytic domain. The amino-terminal region of PKR that interacts with pE3 contains a conserved DRBD, suggesting that PKR can form nonfunctional heterodimers with pE3, analogous to those seen with other dsRNA binding proteins. Interaction of pE3 with the amino-terminal region of PKR is enhanced by dsRNA. In contrast, dsRNA reduces the interaction of pE3 with the carboxyl-terminal region of PKR. Competition experiments demonstrate that the carboxyl-terminal region of PKR, to which pE3 binds, overlaps the region with which eIF2alpha and the pseudosubstrate pK3 interact, suggesting that pE3 may also prevent PKR activation by masking the substrate binding domain. Like pE3, the amino-terminal region of PKR also interacts with the carboxyl-terminal domain of PKR. These interactions increase our understanding of the mechanisms by which pE3 downregulates PKR. In addition, the PKR-PKR interactions observed leads us to suggest a novel autoregulatory mechanism for activation of PKR in which dsRNA binding to the DRBD(s) induces a conformational change that results in release of the amino terminal region from the substrate binding domain, allowing access to eIF2alpha and its subsequent phosphorylation. Copyright 1998 Academic Press PMID: 9792841 [PubMed - indexed for MEDLINE] 895: Biochem Biophys Res Commun 1998 Oct 29;251(3):903-6 The use of the yeast two hybrid system to evaluate ErbB-3 interactions with SH2 domain containing proteins. Yoo JY, Hamburger AW. Molecular and Cellular Biology Program, University of Maryland, Baltimore, Maryland, 21201, USA. Several mutations in the tyrosine kinase domain of ErbB-3 have been postulated to render this enzyme catalytically inactive. To test which amino acid mutations in ErbB-3 might be critical for kinase inactivation, we used a yeast two hybrid assay of protein-protein interaction. We monitored restoration of ErbB-3 kinase activity by investigating the ability of wild type or mutant ErbB-3 to associate with the SH2 containing proteins Syp and Phosphatidyl-inositol-3-kinase (PI3K). Our results demonstrate that changing individual amino acids to tyrosine kinase consensus sequences did not increase the interaction of ErbB-3 with Syp or PI3K. Mutation of the consensus Asp832 of rat ErbB-3 to Asn observed in human and bovine ErbB-3 significantly increased the interaction of ErbB-3 and Syp and PI3K 11 or 26 fold respectively. A double mutant (Asp832Asn, Asp757 His) exhibited a 96 or 350 fold increase in the ability to bind PI3K and Syp. Copyright 1998 Academic Press. PMID: 9791008 [PubMed - indexed for MEDLINE] 896: Biochem Biophys Res Commun 1998 Oct 29;251(3):732-6 Shs1p: a novel member of septin that interacts with spa2p, involved in polarized growth in saccharomyces cerevisiae. Mino A, Tanaka K, Kamei T, Umikawa M, Fujiwara T, Takai Y. Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, 565-0871, Japan. The Rho family small G proteins regulate various cell functions including cytokinesis. We have shown that Bni1p, a potential target of Rho1p, interacts with Spa2p and that Spa2p is required for the localization of Bni1p at the growth sites in Saccharomyces cerevisiae. We isolated here a novel member of the septin family, implicated in cytokinesis, as a Spa2p-binding protein by the yeast two-hybrid method. We named this gene SHS1 (Seventh Homolog of Septin). The shs1 mutant cells showed cytokinesis deficiency and Shs1p was localized at the bud neck in budded cells. The Spa2p-Shs1p interactions may play an important role in cytokinesis. Copyright 1998 Academic Press. PMID: 9790978 [PubMed - indexed for MEDLINE] 897: Nat Biotechnol 1998 Oct;16(10):946-50 Erratum in: Nat Biotechnol 1998 Nov;16(11):1074 Comment in: Nat Biotechnol. 1998 Oct;16(10):906. Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen. Young K, Lin S, Sun L, Lee E, Modi M, Hellings S, Husbands M, Ozenberger B, Franco R. Wyeth-Ayerst Research, CNS Disorders, Princeton, NJ 08543, USA. The interaction of the N-type calcium channel beta3 subunit with the alpha1B subunit alters the activation/inactivation kinetics and the maximal conductance of the channel. The defined protein-protein interaction of the human alpha1B and beta3 subunits provides a target for small-molecule modulation of N-type channel activity. We describe a high throughput screen based on a counterselection yeast two-hybrid assay, which was used to identify small molecules that disrupt alpha1B-beta3 subunit interactions and inhibit N-type calcium channel activity. These small molecules may be a new class of calcium channel antagonists with therapeutic potential. PMID: 9788351 [PubMed - indexed for MEDLINE] 898: Nucleic Acids Res 1998 Nov 1;26(21):4965-74 A specific RNA-protein interaction at yeast polyadenylation efficiency elements. Chen S, Hyman LE. Department of Biochemistry, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans,LA 70112-2699, USA. The specific RNA-protein interactions responsible for the production of mature 3' ends of eukaryotic mRNAs are not well understood. Sequence elements at the 3' ends of yeast genes have been identified that specify the position of the poly(A) site and the efficiency of polyadenylation. To provide additional insights into the interaction between important sequences that direct 3'-end formation in vivo and nuclear proteins, we utilized gel mobility shift assays and UV-crosslinking studies. The data indicate that a protein, with an apparent molecular weight of 80 kDa, interacts specifically with pre-mRNA at the (UA)3efficiency element. Although the interaction is specific, it can be competed by RNA sequences that do not contain the same type of efficiency element; that is, a sequence lacking a (UA)3repeat. This result implies that the protein binding site is flexible. Using immunoprecipitation techniques, the protein has been identified as Hrp1, a heteronuclear RNA binding protein. The role of Hrp1p in 3'-end formation including RNA processing and transcription termination is addressed. PMID: 9776761 [PubMed - indexed for MEDLINE] 899: Mol Cell Biol 1998 Nov;18(11):6365-73 Erratum in: Mol Cell Biol 2000 Mar;20(5):1898 Cak1 is required for Kin28 phosphorylation and activation in vivo. Espinoza FH, Farrell A, Nourse JL, Chamberlin HM, Gileadi O, Morgan DO. Departments of Physiology and Biochemistry & Biophysics, University of California, San Francisco, California 94143-0444, USA. Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1 mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28. PMID: 9774652 [PubMed - indexed for MEDLINE] 900: J Biol Chem 1998 Oct 23;273(43):28341-5 Interaction of Bnr1p with a novel Src homology 3 domain-containing Hof1p. Implication in cytokinesis in Saccharomyces cerevisiae. Kamei T, Tanaka K, Hihara T, Umikawa M, Imamura H, Kikyo M, Ozaki K, Takai Y. Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565-0871, Osaka, Japan. Proteins containing the formin homology (FH) domains FH1 and FH2 are involved in cytokinesis or establishment of cell polarity in a variety of organisms. We have shown that the FH proteins Bni1p and Bnr1p are potential targets of the Rho family small GTP-binding proteins and bind to an actin-binding protein, profilin, at their proline-rich FH1 domains to regulate reorganization of the actin cytoskeleton in the yeast Saccharomyces cerevisiae. We found here that a novel Src homology 3 (SH3) domain-containing protein, encoded by YMR032w, interacted with Bnr1p in a GTP-Rho4p-dependent manner through the FH1 domain of Bnr1p and the SH3 domain of Ymr032wp. Ymr032wp weakly bound to Bni1p. Ymr032wp was homologous to cdc15p, which is involved in cytokinesis in Schizosaccharomyces pombe, and we named this gene HOF1 (homolog of cdc 15). Both Bnr1p and Hof1p were localized at the bud neck, and both the bnr1 and hof1 mutations showed synthetic lethal interactions with the bni1 mutation. The hof1 mutant cells showed phenotypes similar to those of the septin mutants, indicating that HOF1 is involved in cytokinesis. These results indicate that Bnr1p directly interacts with Hof1p as well as with profilin to regulate cytoskeletal functions in S. cerevisiae. PMID: 9774458 [PubMed - indexed for MEDLINE] 901: J Biol Chem 1998 Oct 23;273(43):28085-90 Negative charges in the C-terminal domain stabilize the alphaB-crystallin complex. Boelens WC, Croes Y, de Ruwe M, de Reu L, de Jong WW. Department of Biochemistry, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands. w.boelens@bioch.kun.nl alphaB-Crystallin is one of the six known mammalian small heat-shock proteins (sHsps). These are characterized by the presence of a conserved sequence of 80-100 residues, which constitutes the putative C-terminal domain. Like other sHsps, alphaB-crystallin forms multimeric globular complexes, often in combination with related sHsps. Here we show that in a yeast two-hybrid system, alphaB-crystallin can specifically interact with itself as well as with alphaA-crystallin and Hsp27. Analyses of the separate domains show that the conserved C-terminal domain (CalphaB) is essential for this interaction between subunits. To try and detect residues that are important in subunit interaction, the CalphaB domain was used in a two-hybrid screen as bait to select randomly mutated CalphaB mutants. In this way we obtained nine mutants that were still able to interact with wild-type CalphaB despite the presence of up to 15 replacements. Similarly, we obtained 16 mutants that were unable to bind, because of the presence of just three to nine replacements. In binding CalphaB mutants, lysine residues were most often replaced by glutamic acid residues, and in non-binding CalphaB mutants, acidic residues were often found to be replaced by non-charged residues. This indicates that negative charges are important for subunit interaction and we propose a model to explain this role of acidic residues. Furthermore, we observed that two homologs of alphaB-crystallin, alphaA-crystallin and Hsp27, generally interact similarly with the binding and non-binding CalphaB mutants as does alphaB-crystallin. This suggests that interactions involved in the complex formation of these three sHsps are largely comparable. PMID: 9774426 [PubMed - indexed for MEDLINE] 902: J Biol Chem 1998 Oct 23;273(43):28073-7 Inhibition of the interaction between tyrosine-based motifs and the medium chain subunit of the AP-2 adaptor complex by specific tyrphostins. Crump CM, Williams JL, Stephens DJ, Banting G. Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom. Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs found within the cytosolic domains of certain integral membrane proteins. Many of these tyrosine motifs conform to the consensus YXXPhi (where Phi represents a bulky hydrophobic residue). This YXXPhi motif has been shown to interact with the medium chain subunits of adaptor complexes that generally link relevant integral membrane protein cytosolic domains to the clathrin coat involved in vesicle formation. The motif YXXPhi is also very similar to motifs that are targets for phosphorylation by tyrosine kinases. Tyrosine kinase inhibitors known as tyrphostins are structural analogues of tyrosine, and so it is possible that tyrphostins could also inhibit interactions between medium chains and YXXPhi motifs. TGN38 is a type I integral membrane protein containing a tyrosine motif, YQRL, within the cytosolic domain. We have previously shown that this motif interacts directly with the medium chain subunit of the plasma membrane localized AP-2 adaptor complex (mu2). We have investigated a range of tyrphostins and demonstrated a specific inhibition of the interaction between mu2 and the TGN38 cytosolic domain by tyrphostin A23 through in vitro analysis and the yeast two-hybrid system. These data raise the exciting possibility that different membrane traffic events could be inhibited by specific tyrphostins. PMID: 9774424 [PubMed - indexed for MEDLINE] 903: J Biol Chem 1998 Oct 23;273(43):27761-4 Oncoprotein TLS interacts with serine-arginine proteins involved in RNA splicing. Yang L, Embree LJ, Tsai S, Hickstein DD. Medical Research Service, Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108, USA. The gene encoding the human TLS protein, also termed FUS, is located at the site of chromosomal translocations in human leukemias and sarcomas where it forms a chimeric fusion gene with one of several different genes. To identify interacting partners of TLS, we screened a yeast two-hybrid cDNA library constructed from mouse hematopoietic cells using the C-terminal region of TLS in the bait plasmid. Two cDNAs encoding members of the serine-arginine (SR) family of proteins were isolated. The first SR protein is the mouse homolog of human splicing factor SC35, and the second SR member is a novel 183-amino acid protein that we term TASR (TLS-associated serine-arginine protein). cDNA cloning of human TASR indicated that mouse and human TASR have identical amino acid sequences. The interactions between TLS and these two SR proteins were confirmed by co-transfection and immunoprecipitation studies. In vivo splicing assays indicated that SC35 and TASR influence splice site selection of adenovirus E1A pre-mRNA. TLS may recruit SR splicing factors to specific target genes through interaction with its C-terminal region, and chromosomal translocations that truncate the C-terminal region of TLS may prevent this interaction. Thus TLS translocations may alter RNA processing and play a role in malignant transformation. PMID: 9774382 [PubMed - indexed for MEDLINE] 904: EMBO J 1998 Oct 15;17(20):6028-38 Nucleosome structure of the yeast CHA1 promoter: analysis of activation-dependent chromatin remodeling of an RNA-polymerase-II-transcribed gene in TBP and RNA pol II mutants defective in vivo in response to acidic activators. Moreira JM, Holmberg S. Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Oster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark. The Saccharomyces cerevisiae CHA1 gene encodes the catabolic L-serine (L-threonine) dehydratase. We have previously shown that the transcriptional activator protein Cha4p mediates serine/threonine induction of CHA1 expression. We used accessibility to micrococcal nuclease and DNase I to determine the in vivo chromatin structure of the CHA1 chromosomal locus, both in the non-induced state and upon induction. Upon activation, a precisely positioned nucleosome (nuc-1) occluding the TATA box and the transcription start site is removed. A strain devoid of Cha4p showed no chromatin alteration under inducing conditions. Five yeast TBP mutants defective in different steps in activated transcription abolished CHA1 expression, but failed to affect induction-dependent chromatin rearrangement of the promoter region. Progressive truncations of the RNA polymerase II C-terminal domain caused a progressive reduction in CHA1 transcription, but no difference in chromatin remodeling. Analysis of swi1, swi3, snf5 and snf6, as well as gcn5, ada2 and ada3 mutants, suggested that neither the SWI/SNF complex nor the ADA/GCN5 complex is involved in efficient activation and/or remodeling of the CHA1 promoter. Interestingly, in a sir4 deletion strain, repression of CHA1 is partly lost and activator-independent remodeling of nuc-1 is observed. We propose a model for CHA1 activation based on promoter remodeling through interactions of Cha4p with chromatin components other than basal factors and associated proteins. PMID: 9774346 [PubMed - indexed for MEDLINE] 905: Proc Natl Acad Sci U S A 1998 Oct 13;95(21):12486-91 Rap1 protein regulates telomere turnover in yeast. Krauskopf A, Blackburn EH. Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0414, USA. Telomere length is maintained through a dynamic balance between addition and loss of the terminal telomeric DNA. Normal telomere length regulation requires telomerase as well as a telomeric protein-DNA complex. Previous work has provided evidence that in the budding yeasts Kluyveromyces lactis and Saccharomyces cerevisiae, the telomeric double-stranded DNA binding protein Rap1p negatively regulates telomere length, in part by nucleating, by its C-terminal tail, a higher-order DNA binding protein complex that presumably limits access of telomerase to the chromosome end. Here we show that in K. lactis, truncating the Rap1p C-terminal tail (Rap1p-DeltaC mutant) accelerates telomeric repeat turnover in the distal region of the telomere. In addition, combining the rap1-DeltaC mutation with a telomerase template mutation (ter1-kpn), which directs the addition of mutated telomeric DNA repeats to telomeres, synergistically caused an immediate loss of telomere length regulation. Capping of the unregulated telomeres of these double mutants with functionally wild-type repeats restored telomere length control. We propose that the rate of terminal telomere turnover is controlled by Rap1p specifically through its interactions with the most distal telomeric repeats. PMID: 9770512 [PubMed - indexed for MEDLINE] 906: RNA 1998 Oct;4(10):1239-50 Corrected and republished in: RNA 1998 Dec;4(12):1675-86 Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae. Dix I, Russell CS, O'Keefe RT, Newman AJ, Beggs JD. Institute of Cell and Molecular Biology, University of Edinburgh, United Kingdom. We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome. PMID: 9769098 [PubMed - indexed for MEDLINE] 907: J Virol 1998 Nov;72(11):9318-22 A map of interactions between the proteins of a retrotransposon. Steele SJ, Levin HL. Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. The yeast two-hybrid system and in vitro binding assays were used to characterize 54 potential interactions between the proteins of Tf1, an LTR-retrotransposon found in Schizosaccharomyces pombe. The Tf1 integrase (IN) protein was found to interact strongly with itself and not with other control proteins. In addition, the IN core domain interacted strongly with itself and full-length IN. Interestingly, the two-hybrid analysis detected an interaction between the RNase H domain of reverse transcriptase and IN. The biological implications of these interactions are discussed. PMID: 9765482 [PubMed - indexed for MEDLINE] 908: J Virol 1998 Nov;72(11):9192-200 Mutations in the N terminus of the brome mosaic virus polymerase affect genetic RNA-RNA recombination. Figlerowicz M, Nagy PD, Tang N, Kao CC, Bujarski JJ. Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland. Previously, we have observed that mutations in proteins 1a and 2a, the two virally encoded components of the brome mosaic virus (BMV) replicase, can affect the frequency of recombination and the locations of RNA recombination sites (P. D. Nagy, A. Dzianott, P. Ahlquist, and J. J. Bujarski, J. Virol. 69:2547-2556, 1995; M. Figlerowicz, P. D. Nagy, and J. J. Bujarski, Proc. Natl. Acad. Sci. USA 94:2073-2078, 1997). Also, it was found before that the N-terminal domain of 2a, the putative RNA polymerase protein, participates in the interactions between 1a and 2a (C. C. Kao, R. Quadt, R. P. Hershberger, and P. Ahlquist, J. Virol. 66:6322-6329, 1992; E. O'Reilly, J. Paul, and C. C. Kao, J. Virol. 71:7526-7532, 1997). In this work, we examine how mutations within the N terminus of 2a influence RNA recombination in BMV. Because of the likely electrostatic character of 1a-2a interactions, five 2a mutants, MF1 to MF5, were generated by replacing clusters of acidic amino acids with their neutral counterparts. MF2 and MF5 retained nearly wild-type levels of 1a-2a interaction and were infectious in Chenopodium quinoa. However, compared to that in wild-type virus, the frequency of nonhomologous recombination in both MF2 and MF5 was markedly decreased. Only in MF2 was the frequency of homologous recombination reduced and the occurrence of imprecise homologous recombination increased. In MF5 there was also a 3' shift in the positions of homologous crossovers. The observed effects of MF2 and MF5 reveal that the 2a N-terminal domain participates in different ways in homologous and in nonhomologous BMV RNA recombination. This work maps specific locations within the N terminus involved in 1a-2a interaction and in recombination and further suggests that the mechanisms of the two types of crossovers in BMV are different. PMID: 9765466 [PubMed - indexed for MEDLINE] 909: Mol Biol Cell 1998 Oct;9(10):2803-17 A late mitotic regulatory network controlling cyclin destruction in Saccharomyces cerevisiae. Jaspersen SL, Charles JF, Tinker-Kulberg RL, Morgan DO. Department of Physiology, University of California, San Francisco, California 94143-0444, USA. Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase-cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15, cdc5, cdc14, dbf2, and tem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis. We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, and SIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1. PMID: 9763445 [PubMed - indexed for MEDLINE] 910: Mol Biol Cell 1998 Oct;9(10):2729-38 The role of glucosidase I (Cwh41p) in the biosynthesis of cell wall beta-1,6-glucan is indirect. Abeijon C, Chen LY. Department of Molecular and Cell Biology, Boston University-Goldman School of Dental Medicine, Boston, Massachusetts 02118, USA. cabeijon@bu.edu CWH41, a gene involved in the assembly of cell wall beta-1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal alpha-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of beta-1,6-glucan, we constructed a double mutant, alg5Delta (lacking dolichol-P-glucose synthase) cwh41Delta, and found that it has the same phenotype as the alg5Delta single mutant. It contains wild-type levels of cell wall beta-1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41Delta single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the beta-1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5Delta). The triple mutant alg5Deltacwh41Deltakre6Delta is viable, whereas the double mutant cwh41Deltakre6Delta in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall beta-1,6-glucan, characteristic of the cwh41Deltakre1Delta double mutant, are not observed in the triple mutant alg5Deltacwh41Deltakre1Delta. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41Delta strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall beta-1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway. PMID: 9763440 [PubMed - indexed for MEDLINE] 911: J Cell Biol 1998 Oct 5;143(1):49-63 Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1. Chan GK, Schaar BT, Yen TJ. Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. We have identified a 350-amino acid domain in the kinetochore motor CENP-E that specifies kinetochore binding in mitosis but not during interphase. The kinetochore binding domain was used in a yeast two-hybrid screen to isolate interacting proteins that included the kinetochore proteins CENP-E, CENP-F, and hBUBR1, a BUB1-related kinase that was found to be mutated in some colorectal carcinomas (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Wilson, S.D. Markowitz, K.W. Kinzler, and B. Vogelstein. 1998. Nature. 392:300-303). CENP-F, hBUBR1, and CENP-E assembled onto kinetochores in sequential order during late stages of the cell cycle. These proteins therefore define discrete steps along the kinetochore assembly pathway. Kinetochores of unaligned chromosome exhibited stronger hBUBR1 and CENP-E staining than those of aligned chromosomes. CENP-E and hBUBR1 remain colocalized at kinetochores until mid-anaphase when hBUBR1 localized to portions of the spindle midzone that did not overlap with CENP-E. As CENP-E and hBUBR1 can coimmunoprecipitate with each other from HeLa cells, they may function as a motor-kinase complex at kinetochores. However, the complex distribution pattern of hBUBR1 suggests that it may regulate multiple functions that include the kinetochore and the spindle midzone. PMID: 9763420 [PubMed - indexed for MEDLINE] 912: Biochemistry 1998 Oct 6;37(40):14257-66 Kinetics of dimerization and interactions of p13suc1 with cyclin-dependent kinases. Morris MC, Heitz F, Divita G. Centre de Recherches de Biochimie Macromoleculaire, CNRS, Montpellier, France. The impact of p13suc1 on the conformation and regulation of cyclin-dependent kinases (cdks) and cyclins was investigated by spectroscopic and rapid kinetic approaches. In the absence of phosphorylation on cdks, p13suc1 formed stable complexes, mainly stabilized by hydrophobic interactions, specifically with cdk2 and cdc2. The presence of cyclin A, associated with cdk2 or cdc2, increased the stability of the interaction between cdk2 and p13suc1 by a factor of 2. However, cyclin A did not modify the association rate of p13suc1 to cdk2, but the dissociation rate, which was decreased 3-fold. Moreover, binding of p13suc1 to cdk2 resulted in a 2-fold decrease in the release of nucleotide from cdk2, indicating that p13suc1 induces a marked change in the structure of the nucleotide binding site of cdks. On the basis of the structure of cdk2/CksHs1 complex and on our kinetic results, we propose that the binding of Cks proteins to C-lobe of cdk2 is stabilized by the presence of cyclin A and that it may modify the orientation of the loop carrying residues 14 and 15 and their consequent access for dephosphorylation by cdc25 phosphatases. Finally, we have shown that dimerization of p13suc1 in the presence of zinc abolishes its interaction with cdks, which suggests that the binding of p13suc1 to cdk2 or cdk2/cyclin A may be regulated by dimerization of p13suc1 in vivo. PMID: 9760264 [PubMed - indexed for MEDLINE] 913: EMBO J 1998 Oct 1;17(19):5796-804 Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs. Sanchirico ME, Fox TD, Mason TL. Department of Biochemistry and Molecular Biology and The Graduate Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003-4505, USA. The essential products of the yeast mitochondrial translation system are seven hydrophobic membrane proteins and Var1p, a hydrophilic protein in the small ribosomal subunit. Translation of the membrane proteins depends on nuclearly encoded, mRNA-specific translational activators that recognize the 5'-untranslated leaders of their target mRNAs. These translational activators are themselves membrane associated and could therefore tether translation to the inner membrane. In this study, we tested whether chimeric mRNAs with the untranslated sequences normally present on the mRNA encoding soluble Var1p, can direct functional expression of coding sequences specifying the integral membrane proteins Cox2p and Cox3p. DNA sequences specifying these chimeric mRNAs were inserted into mtDNA at the VAR1 locus and expressed in strains containing a nuclearly localized plasmid that supplies a functional form of Var1p, imported from the cytoplasm. Although cells expressing these chimeric mRNAs actively synthesized both membrane proteins, they were severely deficient in cytochrome c oxidase activity and in the accumulation of Cox2p and Cox3p, respectively. These data strongly support the physiological importance of interactions between membrane-bound mRNA-specific translational activators and the native 5'-untranslated leaders of the COX2 and COX3 mRNAs for localizing productive synthesis of Cox2p and Cox3p to the inner membrane. PMID: 9755179 [PubMed - indexed for MEDLINE] 914: EMBO J 1998 Oct 1;17(19):5679-88 The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage. Vialard JE, Gilbert CS, Green CM, Lowndes NF. Imperial Cancer Research Fund, Clare Hall Laboratories, CDC Laboratory, South Mimms, Hertfordshire EN6 3LD, UK. The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53. PMID: 9755168 [PubMed - indexed for MEDLINE] 915: EMBO J 1998 Oct 1;17(19):5525-8 DNA damage checkpoint in budding yeast. Longhese MP, Foiani M, Muzi-Falconi M, Lucchini G, Plevani P. Dipartimento di Genetica e Biologia dei Microrganismi, Via Celoria 26, 20133 Milano, Italy. Eukaryotic cells have evolved a network of control mechanisms, known as checkpoints, which coordinate cell-cycle progression in response to internal and external cues. The yeast Saccharomyces cerevisiae has been invaluable in dissecting genetically the DNA damage checkpoint pathway. Recent results on posttranslational modifications and protein-protein interactions of some key factors provide new insights into the architecture of checkpoint protein complexes and their order of function. Publication Types: Review Review, Tutorial PMID: 9755152 [PubMed - indexed for MEDLINE] 916: Nucleic Acids Res 1998 Oct 15;26(20):4771-7 Interaction of myocyte enhancer factor 2 (MEF2) with a mitogen-activated protein kinase, ERK5/BMK1. Yang CC, Ornatsky OI, McDermott JC, Cruz TF, Prody CA. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. Myocyte enhancer factor 2 (MEF2) has been implicated in the complex hierarchical regulation of muscle-specific gene expression and differentiation. While the MyoD family members are able to initiate the skeletal muscle differentiation program, whether MEF2 is sufficient in directing skeletal muscle differentiation is still controversial. Furthermore, how MEF2 transactivates its target genes is not fully understood. It has been suggested that the interactions of MEF2 with other factors modify its transcriptional activity. Therefore, the identification of MEF2-interacting factors may be important in understanding the mechanism by which MEF2 activates its target genes. In this study, a mitogen-activated protein kinase (MAP kinase), ERK5/BMK1 was found to interact with MEF2 in a yeast two hybrid screen. The interaction was confirmed by a glutathione S -transferase-pull down assay and a co-immunoprecipitation study indicating that endogenous ERK5 and MEF2 interact with each other in vivo . The interacting domain of MEF2 was mapped to the N-terminus which contains the highly conserved MADS and MEF2 domains. Functionally, ERK5/BMK1 was able to phosphorylate MEF2 in vitro . Furthermore, when cotransfected with ERK5/BMK1, the transactivation capacity of MEF2 was enhanced. These results suggest that the functions of MEF2 could be regulated through ERK5/BMK1. PMID: 9753748 [PubMed - indexed for MEDLINE] 917: Biochem Biophys Res Commun 1998 Sep 18;250(2):212-6 Protein-protein interactions of the yeast Golgi t-SNARE Sed5 protein distinct from its neural plasma membrane cognate syntaxin 1. Kosodo Y, Noda Y, Yoda K. Department of Biotechnology, University of Tokyo, Japan. Targeting of vesicles to the acceptor membrane in protein transport depends on membrane proteins called SNAREs. Saccharomyces cerevisiae Golgi t-SNARE Sed5 protein and its neural cognate syntaxin 1 have similar three alpha-helices which are predicted to form coiled coils. We dissected the helices of Sed5 and found several characteristics unexpectedly distinct from those of syntaxin 1. Most importantly, only the N-terminal helix is responsible for the binding of Sly1 protein while almost the entire molecule of syntaxin is necessary for the binding of the cognate, Munc-18. The N-terminal region of Sed5 protein also binds to the C-terminal helix and Sly1 protein interfered this binding. PMID: 9753609 [PubMed - indexed for MEDLINE] 918: Proc Natl Acad Sci U S A 1998 Sep 29;95(20):11590-5 Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors. Levanon D, Goldstein RE, Bernstein Y, Tang H, Goldenberg D, Stifani S, Paroush Z, Groner Y. Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel. The mammalian AML/CBFalpha runt domain (RD) transcription factors regulate hematopoiesis and osteoblast differentiation. Like their Drosophila counterparts, most mammalian RD proteins terminate in a common pentapeptide, VWRPY, which serves to recruit the corepressor Groucho (Gro). Using a yeast two-hybrid assay, in vitro association and pull-down experiments, we demonstrate that Gro and its mammalian homolog TLE1 specifically interact with AML1 and AML2. In addition to the VWRPY motif, other C-terminal sequences are required for these interactions with Gro/TLE1. TLE1 inhibits AML1-dependent transactivation of the T cell receptor (TCR) enhancers alpha and beta, which contain functional AML binding sites, in transfected Jurkat T cells. LEF-1 is an additional transcription factor that mediates transactivation of TCR enhancers. LEF-1 and its Drosophila homolog Pangolin (Pan) are involved in the Wnt/Wg signaling pathway through interactions with the coactivator beta-catenin and its highly conserved fly homolog Armadillo (Arm). We show that TLE/Gro interacts with LEF-1 and Pan, and inhibits LEF-1:beta-catenin-dependent transcription. These data indicate that, in addition to their activity as transcriptional activators, AML1 and LEF-1 can act, through recruitment of the corepressor TLE1, as transcriptional repressors in TCR regulation and Wnt/Wg signaling. PMID: 9751710 [PubMed - indexed for MEDLINE] 919: Biochim Biophys Acta 1998 Oct 1;1400(1-3):3-18 Structure of DNA topoisomerases. Berger JM. Division of Biochemistry and Molecular Biology, Department of Molecular and Cellular Biology, 229 Stanley Hall, University of California, Berkeley, Berkeley, CA 94720, USA. Over the last several years topoisomerases have finally begun to yield to high-resolution structural studies. These models have greatly aided our understanding of the mechanisms of topoisomerase catalysis and drug interactions. This review will cover advances in the structural biology of topoisomerases and discuss their implications for topoisomerase function. Publication Types: Review Review, Tutorial PMID: 9748476 [PubMed - indexed for MEDLINE] 920: Mol Gen Genet 1998 Aug;259(2):142-9 The chromatin structure of the GAL1 promoter forms independently of Reb1p in Saccharomyces cerevisiae. Reagan MS, Majors JE. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA. mreagan@csbsju.edu Positive and negative regulation of the GAL1 promoter of the yeast Saccharomyces cerevisiae results from a network of interactions between transcription factors and chromatin. In this study we used footprinting procedures to characterize these interactions in vivo. DNase I analysis of the GAL1 upstream activating sequence (UAS(GAL1/10)) showed expected Gal4 activator protein binding during growth in galactose, and also revealed binding of the Reb1 protein (Reb1p) during growth in glucose. In addition, we mapped to nucleotide resolution a positioned nucleosome that, in the inactive promoter, packages DNA between the UAS(GAL1/10) and the GAL1 TATA sequence, leaving both of these elements nucleosome free. The nucleosome footprint was lost when the promoter was activated. Surprisingly, mutation of the Reb1p binding site had no effect on nucleosome positioning or on the kinetics or extent of activation or repression of either the GAL1 or GAL10 promoters under any of the conditions assayed. PMID: 9747705 [PubMed - indexed for MEDLINE] 921: Curr Biol 1998 Aug 27;8(17):959-62 The WASp homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast. Naqvi SN, Zahn R, Mitchell DA, Stevenson BJ, Munn AL. Institute of Molecular Agrobiology, National University of Singapore, Republic of Singapore. Several end mutations that block the internalisation step of endocytosis in Saccharomyces cerevisiae also affect the cortical actin cytoskeleton [1]. END5 encodes a proline-rich protein (End5p or verprolin) required for a polarised cortical actin cytoskeleton and endocytosis [2,3]. End5p interacts with actin [4], but its exact function is not yet known. To help elucidate End5p function, we sought other End5p-interacting proteins and identified the LAS17/BEE1 gene (encoding the yeast homologue of the human Wiskott-Aldrich Syndrome protein, WASp) as a high-copy-number suppressor of the temperature-sensitive growth and endocytic defects of end5-1 cells (carrying a frameshift mutation affecting the last 213 residues of End5p). LAS17 is unable to suppress a full deletion of END5 (end5 delta), however, suggesting that the defective End5-1p in end5-1 mutants may be stabilised by Las17p. The amino terminus of Las17p interacts with the carboxyl terminus of End5p in the yeast two-hybrid system and similar interactions have been shown between WASp and a mammalian End5p homologue, WASp-interacting protein (WIP) [5]. As las17 delta deletion mutants are blocked in endocytosis, we conclude that Las17p and End5p interact and are essential for endocytosis. PMID: 9742397 [PubMed - indexed for MEDLINE] 922: Mol Cell Biol 1998 Oct;18(10):6110-20 Functional characterization of the N terminus of Sir3p. Gotta M, Palladino F, Gasser SM. Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges/Lausanne, Switzerland. Silent information regulator 3 is an essential component of the Saccharomyces cerevisiae silencing complex that functions at telomeres and the silent mating-type loci, HMR and HML. We show that expression of the N- and C-terminal-encoding halves of SIR3 in trans partially complements the mating defect of the sir3 null allele, suggesting that the two domains have distinct functions. We present here a functional characterization of these domains. The N-terminal domain (Sir3N) increases both the frequency and extent of telomere-proximal silencing when expressed ectopically in SIR+ yeast strains, although we are unable to detect interaction between this domain and any known components of the silencing machinery. In contrast to its effect at telomeres, Sir3N overexpression derepresses transcription of reporter genes inserted in the ribosomal DNA (rDNA) array. Immunolocalization of Sir3N-GFP and Sir2p suggests that Sir3N directly antagonizes nucleolar Sir2p, releasing an rDNA-bound population of Sir2p so that it can enhance repression at telomeres. Overexpression of the C-terminal domain of either Sir3p or Sir4p has a dominant-negative effect on telomeric silencing. In strains overexpressing the C-terminal domain of Sir4p, elevated expression of either full-length Sir3p or Sir3N restores repression and the punctate pattern of Sir3p and Rap1p immunostaining. The similarity of Sir3N and Sir3p overexpression phenotypes suggests that Sir3N acts as an allosteric effector of Sir3p, either enhancing its interactions with other silencing components or liberating the full-length protein from nonfunctional complexes. PMID: 9742128 [PubMed - indexed for MEDLINE] 923: Mol Cell Biol 1998 Oct;18(10):5942-51 Processivity of the Saccharomyces cerevisiae poly(A) polymerase requires interactions at the carboxyl-terminal RNA binding domain. Zhelkovsky A, Helmling S, Moore C. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111-1800, USA. The interaction of the Fip1 subunit of polyadenylation factor I with the Saccharomyces cerevisiae poly(A) polymerase (PAP) was assayed in vivo by two-hybrid analysis and was found to involve two separate regions on PAP, located at opposite ends of the protein sequence. In vitro, Fip1 blocks access of the RNA primer to an RNA binding site (RBS) that overlaps the Fip1 carboxy-terminal interaction region and, in doing so, shifts PAP to a distributive mode of action. Partial truncation of this RBS has the same effect, indicating that this site is required for processivity. A comparison of the utilization of ribo- and deoxyribonucleotides as substrates indicates the existence on PAP of a second RBS which recognizes the last three nucleotides at the 3' end of the primer. This site discriminates against deoxyribonucleotides at the 3' end, and interactions at this site are not affected by Fip1. Further analysis revealed that the specificity of PAP for adenosine is not simply a function of the ATP binding site but also reflects interactions with bases at the 3' end of the primer and at another contact site 14 nucleotides upstream of the 3' end. These results suggest that the unique specificity of PAP for ribose and base, and thus the extent and type of activity with different substrates, depends on interactions at multiple nucleotide binding sites. PMID: 9742111 [PubMed - indexed for MEDLINE] 924: J Biol Chem 1998 Sep 25;273(39):25041-4 Ribosomal P-protein stalk function is targeted by sordarin antifungals. Gomez-Lorenzo MG, Garcia-Bustos JF. Research Department, Glaxo Wellcome, S. A., Severo Ochoa 2, 28760 Tres Cantos, Spain. Sordarin derivatives are remarkably selective inhibitors of fungal protein synthesis. Available evidence points to a binding site for these inhibitors on elongation factor 2, but high affinity binding requires the presence of ribosomes. The gene mutated in one of the two isolated complementation groups of Saccharomyces cerevisiae mutants resistant to the sordarin derivative GM193663 has now been identified. It is RPP0, encoding the essential protein of the large ribosomal subunit stalk rpP0. Resistant mutants are found to retain most of the binding capacity for the drug, indicating that mutations in rpP0 endow the ribosome with the capacity to perform translation elongation in the presence of the inhibitor. Other proteins of the ribosomal stalk influence the expression of resistance, pointing to a wealth of interactions between stalk components and elongation factors. The involvement of multiple elements of the translation machinery in the mode of action of sordarin antifungals may explain the large selectivity of these compounds, even though the individual target components are highly conserved proteins. PMID: 9737960 [PubMed - indexed for MEDLINE] 925: J Mol Biol 1998 Sep 25;282(3):525-41 Mutant alleles of the MRS2 gene of yeast nuclear DNA suppress mutations in the catalytic core of a mitochondrial group II intron. Schmidt U, Maue I, Lehmann K, Belcher SM, Stahl U, Perlman PS. Department of Microbiology and Genetics, University of Technology, Berlin, D-13355, Germany. schm1534@mailszrz.zrz.tu-berlin.de Previous studies show that some yeast strains carrying point mutations of domain 5 that block splicing of a mitochondrial group II intron yield spontaneous revertants in which splicing is partially restored by dominant mutations of nuclear genes. Here we cloned and sequenced the suppressor allele of one such gene, and found it to be a missense mutation of the MRS2 gene (MRS2-L232F). The MRS2 gene was first implicated in group II intron splicing by the finding that overexpression of the wild-type gene weakly suppresses the splicing defect of a mutation of another intron. Tetrad analysis showed that independently isolated suppressors of two other domain 5 mutations are also allelles of the MRS2 gene and DNA sequencing identified a new missense mutation in each strain (MRS2-T230I and MRS2-L213M). All three suppressor mutations cause a temperature-sensitive respiration defect that is dominant negative in heterozygous diploids, but those strains splice the mutant intron at the elevated temperature. The three mutations are in a domain of the protein that is likely to be a helix-turn-helix region, so that effects of the mutations on protein-protein interactions may contribute to these phenotypes. These mutations suppress the splicing defect of many, but not all, of the available splicing defective mutations of aI5gamma, including mutations of several intron domains. Protein and RNA blot experiments show that the level of the protein encoded by the MRS2 gene, but not the mRNA, is elevated by these mutations. Interestingly, overexpression of the wild-type protein restores much lower levels of splicing than were obtained with similar elevated levels of the mutated Mrs2 proteins. The splicing phenotypes of these strains suggest a direct role for Mrs2 protein on group II intron splicing, but an indirect effect is not yet ruled out. Copyright 1998 Academic Press. PMID: 9737920 [PubMed - indexed for MEDLINE] 926: EMBO J 1998 Sep 15;17(18):5438-48 L-arginine recognition by yeast arginyl-tRNA synthetase. Cavarelli J, Delagoutte B, Eriani G, Gangloff J, Moras D. UPR 9004 Biologie Structurale, Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch Cedex, France. The crystal structure of arginyl-tRNA synthetase (ArgRS) from Saccharomyces cerevisiae, a class I aminoacyl-tRNA synthetase (aaRS), with L-arginine bound to the active site has been solved at 2.75 A resolution and refined to a crystallographic R-factor of 19.7%. ArgRS is composed predominantly of alpha-helices and can be divided into five domains, including the class I-specific active site. The N-terminal domain shows striking similarity to some completely unrelated proteins and defines a module which should participate in specific tRNA recognition. The C-terminal domain, which is the putative anticodon-binding module, displays an all-alpha-helix fold highly similar to that of Escherichia coli methionyl-tRNA synthetase. While ArgRS requires tRNAArg for the first step of the aminoacylation reaction, the results show that its presence is not a prerequisite for L-arginine binding. All H-bond-forming capability of L-arginine is used by the protein for the specific recognition. The guanidinium group forms two salt bridge interactions with two acidic residues, and one H-bond with a tyrosine residue; these three residues are strictly conserved in all ArgRS sequences. This tyrosine is also conserved in other class I aaRS active sites but plays several functional roles. The ArgRS structure allows the definition of a new framework for sequence alignments and subclass definition in class I aaRSs. PMID: 9736621 [PubMed - indexed for MEDLINE] 927: Methods 1998 Jul;15(3):207-23 Using genetic means to dissect homologous and heterologous protein-protein interactions of PKR, the interferon-induced protein kinase. Tan SL, Katze MG. School of Medicine, University of Washington, Seattle, Washington, 98195, USA. The interferon-induced protein kinase, PKR, is a pivotal component of interferon (IFN)-induced cellular antiviral and antiproliferative response. The identification and characterization of proteins, of both viral and cellular origins, that interact with PKR have proven to be a valuable probe for unraveling the cellular regulation and function of PKR. Several studies have demonstrated that PKR forms dimers and that dimerization is likely to be required for activation and/or catalytic function. It is therefore important to elucidate the mechanism of PKR dimer formation and the role of PKR effectors in modulating kinase dimerization. Herein we describe the use of the two genetic approaches, the lambda repressor fusion and the yeast two-hybrid systems, to detect and analyze homo- and heterotypic interactions with PKR. We also describe several biochemical methodologies commonly used in our laboratory to validate the genetic results. Although the examples in this article focus on PKR, the techniques can easily be adapted to investigate protein-protein associations in a variety of experimental systems. Finally, given the important role of PKR as a mediator of IFN-induced antiviral and antiproliferative effects, these studies may provide clues to the development of reagents that target PKR to enhance the therapeutic use of IFN in the treatment of disease. Copyright 1998 Academic Press. PMID: 9735306 [PubMed - indexed for MEDLINE] 928: Genes Cells 1998 Jun;3(6):357-69 Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase delta and replication factor C. Oku T, Ikeda S, Sasaki H, Fukuda K, Morioka H, Ohtsuka E, Yoshikawa H, Tsurimoto T. Faculty of Biological Science, Nara Institute of Science and Technology, Takayama, Ikoma, Japan. BACKGROUND: PCNA, an eukaryotic DNA sliding clamp interacts with replication factors and the cell cycle protein, p21(Cip1/Waf1) and functions as a molecular switch for DNA elongation. To understand how DNA replication is regulated through PCNA, elucidation of the precise mechanisms of these protein interactions is necessary. RESULTS: Loop-region mutants in which human PCNA sequences were substituted with the corresponding Saccharomyces cerevisiae PCNA regions were prepared. Analysis of their functions, along with previously prepared alanine scanning mutants, demonstrated that some loops interact with DNA polymerase delta (pol delta) and replication factor C (RFC). The p21 binding sites of PCNA, mapped by affinity measurement of the mutant forms, found to be located within a distinct structure of the PCNA monomer, overlap with RFC- and pol delta-interaction sites. Competition between p21 and pol delta or RFC for binding to PCNA results in efficient inhibition of its stimulation of pol delta DNA synthesis and RFC ATPase but not of PCNA loading on DNA by RFC. CONCLUSIONS: Semi-saturated amounts of p21 selectively block formation of the active pol delta complex but not the RFC-PCNA complex at 3'-ends of DNA primers. This differential effect may explain the specific inhibition of DNA replication by p21. PMID: 9734782 [PubMed - indexed for MEDLINE] 929: Genes Cells 1998 Jun;3(6):347-55 Defect in cytokinesis of fission yeast induced by mutation in the WD40 repeat motif of a TFIID subunit. Yamamoto T, Horikoshi M. Department of Cellular Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan. BACKGROUND: TBP-associated factors contain a variety of structural motifs and their related in vivo significance has remained unclear. We have attempted to identify specific biological phenomena linked to a particular domain of a TAF by analysing domain-exchanged chimeric mutants between Schizosaccharomyces pombe (Sp) and Saccharomyces cerevisiae (Sc) counterparts. RESULTS: Contrary to the case of TBP, Sp TAF containing the WD40 repeat cannot be exchanged for its Sc counterpart, despite their highly conserved primary structures. This 'species-specific' function locates in the N-terminal region. The C-terminal region, largely consisting of the WD40 repeat, is exchangeable for the corresponding region of its Sc counterpart. Growth of the strain harbouring this C-terminal chimeric mutant is temperature-sensitive. The chimeric gene product did not disappear at a restrictive temperature, a finding which strongly suggests that the growth defect is caused by an aberration in the interactions through the WD40 repeat structural motif. With temperature elevation, the chimeric mutants underwent drastic morphological changes due to a defect in cytokinesis. CONCLUSIONS: The WD40 repeat of TAF is primarily involved in reactions which might regulate cytokinesis in Sp. PMID: 9734781 [PubMed - indexed for MEDLINE] 930: J Virol 1998 Oct;72(10):8332-7 CREB-2, a cellular CRE-dependent transcription repressor, functions in association with Tax as an activator of the human T-cell leukemia virus type 1 promoter. Gachon F, Peleraux A, Thebault S, Dick J, Lemasson I, Devaux C, Mesnard JM. Laboratoire Infections Retrovirales et Signalisation Cellulaire, CRBM-CNRS UPR 1086, Institut de Biologie, 34060 Montpellier, France. The Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) has been implicated in human T-cell immortalization. The primary function of Tax is to transcriptionally activate the HTLV-1 promoter, but Tax is also known to stimulate expression of cellular genes. It has been reported to associate with several transcription factors, as well as proteins not involved in transcription. To better characterize potential cellular targets of Tax present in infected cells, a Saccharomyces cerevisiae two-hybrid screening was performed with a cDNA library constructed from the HTLV-1-infected MT2 cell line. From this study, we found 158 positive clones representing seven different cDNAs. We focused our attention on the cDNA encoding the transcription factor CREB-2. CREB-2 is an unconventional member of the ATF/CREB family in that it lacks a protein kinase A (PKA) phosphorylation site and has been reported to negatively regulate transcription from the cyclic AMP response element of the human enkephalin promoter. In this study, we demonstrate that CREB-2 cooperates with Tax to enhance viral transcription and that its basic-leucine zipper C-terminal domain is required for both in vitro and in vivo interactions with Tax. Our results confirm that the activation of the HTLV-1 promoter through Tax and factors of the ATF/CREB family is PKA independent. PMID: 9733879 [PubMed - indexed for MEDLINE] 931: J Biol Chem 1998 Sep 18;273(38):24963-71 Transmembrane protein insertion orientation in yeast depends on the charge difference across transmembrane segments, their total hydrophobicity, and its distribution. Harley CA, Holt JA, Turner R, Tipper DJ. Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. The determinants of transmembrane protein insertion orientation at the endoplasmic reticulum have been investigated in Saccharomyces cerevisiae using variants of a Type III (naturally exofacial N terminus (Nexo)) transmembrane fusion protein derived from the N terminus of Ste2p, the alpha-factor receptor. Small positive and negative charges adjacent to the transmembrane segment had equal and opposite effects on orientation, and this effect was independent of N- or C-terminal location, consistent with a purely electrostatic interaction with response mechanisms. A 3:1 bias toward Nexo insertion, observed in the absence of a charge difference, was shown to reflect the Nexo bias conferred by longer transmembrane segments. Orientation correlated best with total hydrophobicity rather than length, but it was also strongly affected by the distribution of hydrophobicity within the transmembrane segment. The most hydrophobic terminus was preferentially translocated. Insertion orientation thus depends on integration of responses to at least three parameters: charge difference across a transmembrane segment, its total hydrophobicity, and its hydrophobicity gradient. Relative signal strengths were estimated, and consequences for topology prediction are discussed. Responses to transmembrane sequence may depend on protein-translocon interactions, but responses to charge difference may be mediated by the electrostatic field provided by anionic phospholipids. PMID: 9733804 [PubMed - indexed for MEDLINE] 932: J Mol Biol 1998 Sep 11;282(1):13-24 Missense translation errors in Saccharomyces cerevisiae. Stansfield I, Jones KM, Herbert P, Lewendon A, Shaw WV, Tuite MF. Research School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK. We describe the development of a novel plasmid-based assay for measuring the in vivo frequency of misincorporation of amino acids into polypeptide chains in the yeast Saccharomyces cerevisiae. The assay is based upon the measurement of the catalytic activity of an active site mutant of type III chloramphenicol acetyl transferase (CATIII) expressed in S. cerevisiae. A His195(CAC)-->Tyr195(UAC) mutant of CATIII is completely inactive, but catalytic activity can be restored by misincorporation of histidine at the mutant UAC codon. The average error frequency of misincorporation of histidine at this tyrosine UAC codon in wild-type yeast strains was measured as 0. 5x10(-5) and this frequency was increased some 50-fold by growth in the presence of paromomycin, a known translational-error-inducing antibiotic. A detectable frequency of misincorporation of histidine at a mutant Ala195 GCU codon was also measured as 2x10(-5), but in contrast to the Tyr195-->His195 misincorporation event, the frequency of histidine misincorporation at Ala195 GCU was not increased by paromomycin, inferring that this error did not result from miscognate codon-anticodon interaction. The His195 to Tyr195 missense error assay was used to demonstrate increased frequencies of missense error at codon 195 in SUP44 and SUP46 mutants. These two mutants have previously been shown to exhibit a translation termination error phenotype and the sup44+ and sup46+ genes encode the yeast ribosomal proteins S4 and S9, respectively. These data represent the first accurate in vivo measurement of a specific mistranslation event in a eukaryotic cell and directly confirm that the eukaryotic ribosome plays an important role in controlling missense errors arising from non-cognate codon-anticodon interactions. Copyright 1998 Academic Press. PMID: 9733638 [PubMed - indexed for MEDLINE] 933: Mol Pharmacol 1998 Sep;54(3):591-8 Pharmacological analysis of sterol delta8-delta7 isomerase proteins with [3H]ifenprodil. Moebius FF, Reiter RJ, Bermoser K, Glossmann H, Cho SY, Paik YK. Institut fur Biochemische Pharmakologie, Universitat Innsbruck, Peter Mayr Str. 1, A-6020 Innsbruck, Austria. Sterol Delta8-Delta7 isomerases (SIs) catalyze the shift of the double bond from C8-9 to C7-8 in the B-ring of sterols. Surprisingly, the isoenzymes in fungi (ERG2p) and vertebrates [emopamil binding protein (EBP)] are structurally completely unrelated, whereas the sigma1 receptor, a mammalian protein of unknown function, bears significant similarity with the yeast ERG2p. Here, we compare the drug binding properties of SIs and related proteins with [3H]ifenprodil as a common high affinity radioligand (Kd = 1.4-19 nM), demonstrating an intimate pharmacological relationship among ERG2p, sigma1 receptor, and EBP. This renders SIs a remarkable example for structurally diverse enzymes with similar pharmacological profiles and the propensity to bind drugs from different chemical groups with high affinity. We identified a variety of experimental drugs with nanomolar affinity for the human EBP (Ki = 0.5-14 nM) such as MDL28815, AY9944, triparanol, and U18666A. These compounds, as well as the fungicide tridemorph and the clinically used drugs tamoxifen, clomiphene, amiodarone, and opipramol, inhibit the in vitro activity of the recombinant human EBP (IC50 = 0.015-54 microM). The high affinity of the human EBP for 3H-tamoxifen (Kd = 3 +/- 2 nM) implies that the EBP carries the previously described microsomal antiestrogen binding site. Interactions of the EBP with structurally diverse lipophilic amines suggest that novel compounds of related structure should be counterscreened for inhibition of the enzyme to avoid interference with sterol Delta8-Delta7 isomerization. PMID: 9730919 [PubMed - indexed for MEDLINE] 934: Biochemistry 1998 Sep 8;37(36):12496-506 The 32- and 14-kilodalton subunits of replication protein A are responsible for species-specific interactions with single-stranded DNA. Sibenaller ZA, Sorensen BR, Wold MS. Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242, USA. Replication protein A (RPA) is a multisubunit single-stranded DNA-binding (ssDNA) protein that is required for cellular DNA metabolism. RPA homologues have been identified in all eukaryotes examined. All homologues are heterotrimeric complexes with subunits of approximately 70, approximately 32, and approximately 14 kDa. While RPA homologues are evolutionarily conserved, they are not functionally equivalent. To gain a better understanding of the functional differences between RPA homologues, we analyzed the DNA-binding parameters of RPA from human cells and the budding yeast Saccharomyces cerevisiae (hRPA and scRPA, respectively). Both yeast and human RPA bind ssDNA with high affinity and low cooperativity. However, scRPA has a larger occluded binding site (45 nucleotides versus 34 nucleotides) and a higher affinity for oligothymidine than hRPA. Mutant forms of hRPA and scRPA containing the high-affinity DNA-binding domain from the 70-kDa subunit had nearly identical DNA binding properties. In contrast, subcomplexes of the 32- and 14-kDa subunits from both yeast and human RPA had weak ssDNA binding activity. However, the binding constants for the yeast and human subcomplexes were 3 and greater than 6 orders of magnitude lower than those for the RPA heterotrimer, respectively. We conclude that differences in the activity of the 32- and 14-kDa subunits of RPA are responsible for variations in the ssDNA-binding properties of scRPA and hRPA. These data also indicate that hRPA and scRPA have different modes of binding to ssDNA, which may contribute to the functional disparities between the two proteins. PMID: 9730822 [PubMed - indexed for MEDLINE] 935: J Biol Chem 1998 Sep 11;273(37):23781-5 Erratum in: J Biol Chem 1998 Oct 16;273(42):27755 A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor IID. Martinez E, Kundu TK, Fu J, Roeder RG. Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021, USA. In yeast, SPT3 is a component of the multiprotein SPT-ADA-GCN5 acetyltransferase (SAGA) complex that integrates proteins with transcription coactivator/adaptor functions (ADAs and GCN5), histone acetyltransferase activity (GCN5), and core promoter-selective functions (SPTs) involving interactions with the TATA-binding protein (TBP). In particular, yeast SPT3 has been shown to interact directly with TBP. Here we report the molecular cloning of a cDNA encoding a human homologue of yeast SPT3. Amino acid sequence comparisons between human SPT3 (hSPT3) and its counterparts in different yeast species reveal three highly conserved domains, with the most conserved 92-amino acid N-terminal domain being 25% identical with human TAFII18. Despite the significant sequence similarity with TAFII18, native hSPT3 is not a bona fide TAFII because it is not associated in vivo either with human TBP/TFIID or with a TFIID-related TBP-free TAFII complex. However, we present evidence that hSPT3 is associated in vivo with TAFII31 and the recently described longer form of human GCN5 (hGCN5-L) in a novel human complex that has histone acetyltransferase activity. We propose that the human SPT3-TAFII31-GCN5-L acetyltransferase (STAGA) complex is a likely homologue of the yeast SAGA complex. PMID: 9726987 [PubMed - indexed for MEDLINE] 936: Mol Biol Cell 1998 Sep;9(9):2349-60 An alpha-tubulin mutant destabilizes the heterodimer: phenotypic consequences and interactions with tubulin-binding proteins. Vega LR, Fleming J, Solomon F. Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. Many effectors of microtubule assembly in vitro enhance the polymerization of subunits. However, several Saccharomyces cerevisiae genes that affect cellular microtubule-dependent processes appear to act at other steps in assembly and to affect polymerization only indirectly. Here we use a mutant alpha-tubulin to probe cellular regulation of microtubule assembly. tub1-724 mutant cells arrest at low temperature with no assembled microtubules. The results of several assays reported here demonstrate that the heterodimer formed between Tub1-724p and beta-tubulin is less stable than wild-type heterodimer. The unstable heterodimer explains several conditional phenotypes conferred by the mutation. These include the lethality of tub1-724 haploid cells when the beta-tubulin-binding protein Rbl2p is either overexpressed or absent. It also explains why the TUB1/tub1-724 heterozygotes are cold sensitive for growth and why overexpression of Rbl2p rescues that conditional lethality. Both haploid and heterozygous tub1-724 cells are inviable when another microtubule effector, PAC2, is overexpressed. These effects are explained by the ability of Pac2p to bind alpha-tubulin, a complex we demonstrate directly. The results suggest that tubulin-binding proteins can participate in equilibria between the heterodimer and its components. PMID: 9725898 [PubMed - indexed for MEDLINE] 937: Nucleic Acids Res 1998 Sep 15;26(18):4137-45 Cooperative interaction of branch signals in the actin intron of Saccharomyces cerevisiae. Castanotto D, Rossi JJ. Molecular Biology Department, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, USA. In pre-mRNA splicing, specific spliceosomal components recognize key intron sequences, but the mechanisms by which splice sites are selected arenot completely understood. In the Saccharomyces cerevisiae actin intron a silent branch point-like sequence (UACUAAG) is located 7 nt upstream of the canonical sequence. Mutation of the canonicalUACUAAC sequence to UAAUAAC reduces utilization of this signal and activates the cryptic UACUAAG. Splicing-dependent beta-galactosidase assays have shown that these two splice signals cooperate to enhance splicing. Analyses of several variants of this double branch point intron demonstrate that the upstream UACUAAG sequence significantly increases usage of the UAAUAAC as a site of lariat formation. This activation is sequence-specific and unidirectional. However the ability of the UACUAAG signal to activate the downstream branch point is dependent on the presence of a short non-conserved sequence located a few nucleotides upstream of the UACUAAG. Mutation of this sequence leads to the disappearance of the cooperative interactions between the two branch signals. Our results show that this non-conserved sequence and the UACUAAG signal must both be present to achieve activation of the downstream branch point and suggest that a specific structure may be necessary to allow efficient recognition of the UAAUAAC. PMID: 9722632 [PubMed - indexed for MEDLINE] 938: J Cell Biochem 1998 Sep 1;70(3):366-75 Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene regulation. Grayson J, Bassel-Duby R, Williams RS. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8573, USA. Previous investigations have demonstrated synergistic interactions in vivo between CCAC and A/T-rich nucleotide sequence motifs as functional components of muscle-specific transcriptional enhancers. Using CCAC and A/T-rich elements from the myoglobin and muscle creatine kinase (MCK) gene enhancers, Sp1 and myocyte-specific enhancer factor-2 (MEF-2) were identified as cognate binding proteins that recognize these sites. Physical interactions between Sp1 and MEF-2 were demonstrated by immunological detection of both proteins in DNA binding complexes formed in vitro by nuclear extracts in the presence of only the A/T sequence motif, by coprecipitation of recombinant MEF-2 in the presence of a glutathione-S-transferase-Sp1 fusion protein bound to glutathione beads, and by a two-hybrid assay in Saccharomyces cerevisiae. The interaction with Sp1 in vitro and in vivo is specific for MEF-2 and was not observed with serum response factor, a related MADS domain protein. Forced expression of Sp1 and MEF-2 in insect cells otherwise lacking these factors promotes synergistic transcriptional activation of a promoter containing binding sites for both proteins. These data expand the repertoire of functional and physical interactions between lineage-restricted (MEF-2) and ubiquitous (Sp1) transcription factors that may be important for myogenic differentiation. PMID: 9706874 [PubMed - indexed for MEDLINE] 939: Mol Endocrinol 1998 Aug;12(8):1172-83 Functional interactions of the AF-2 activation domain core region of the human androgen receptor with the amino-terminal domain and with the transcriptional coactivator TIF2 (transcriptional intermediary factor2). Berrevoets CA, Doesburg P, Steketee K, Trapman J, Brinkmann AO. Department of Endocrinology and Reproduction, Erasmus University, Rotterdam, The Netherlands. Previous studies in yeast and mammalian cells showed a functional interaction between the amino-terminal domain and the carboxy-terminal, ligand-binding domain (LBD) of the human androgen receptor (AR). In the present study, the AR subdomains involved in this in vivo interaction were determined in more detail. Cotransfection experiments in Chinese hamster ovary (CHO) cells and two-hybrid experiments in yeast revealed that two regions in the NH2-terminal domain are involved in the functional interaction with the LBD: an interacting domain at the very NH2 terminus, located between amino acid residues 3 and 36, and a second domain, essential for transactivation, located between residues 370 and 494. Substitution of glutamic acid by glutamine at position 888 (E888Q) in the AF-2 activation domain (AD) core region in the LBD, markedly decreased the interaction with the NH2-terminal domain. This mutation neither influenced hormone binding nor LBD homodimerization, suggesting a role of the AF-2 AD core region in the functional interaction between the NH2-terminal domain and the LBD. The AF-2 AD core region was also involved in the interaction with the coactivator TIF2 (transcriptional intermediary factor 2), as the E888Q mutation decreased the stimulatory effect of TIF2 on AR AF-2 activity. Cotransfection of TIF2 and the AR NH2-terminal domain expression vectors did not result in synergy between both factors in the induction of AR AF-2 activity. TIF2 highly induced AR AF-2 activity on a complex promoter [mouse mammary tumor virus (MMTV)], but it was hardly active on a minimal promoter (GRE-TATA). In contrast, the AR NH2-terminal domain induced AR AF-2 activity on both promoter constructs. These data indicate that both the AR NH2-terminal domain and the coactivator TIF2 functionally interact, either directly or indirectly, with the AF-2 AD core region in the AR-LBD, but the level of transcriptional response induced by TIF2 depends on the promoter context. PMID: 9717843 [PubMed - indexed for MEDLINE] 940: Mycoses 1998;41 Suppl 1:32-8 Cytochromes P450 in fungi. Vanden Bossche H, Koymans L. Department of Anti-infectives Research, Janssen Research Foundation, Beerse, Belgium. The article gives an overview on the history of the discovery of P450 cytochromes and on their occurrence in nature, especially on their interactions with metabolic pathways in fungi. The significance of the P450 cytochromes in the ergosterol synthesis as well as in the inhibitory mechanisms caused by imidazole and triazole antimycotics is described in detail. Publication Types: Review Review, Tutorial PMID: 9717384 [PubMed - indexed for MEDLINE] 941: Genes Dev 1998 Aug 15;12(16):2587-97 Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1. Kaiser P, Sia RA, Bardes EG, Lew DJ, Reed SI. The Scripps Research Institute (TSRI), La Jolla, California 92037 USA. Ubiquitin-mediated proteolysis controls the abundance of many cell cycle regulatory proteins. Recent work in Saccharomyces cerevisiae suggests that a complex consisting of Cdc53, Skp1, and a third component known as an F-box protein (termed SCF) in combination with Cdc34 specifically targets regulatory proteins for degradation, and that substrate specificity is likely to be mediated by the F-box subunit. A screen for genetic interactions with a cdc34 mutation yielded MET30, which encodes an F-box protein. MET30 is an essential gene required for cell cycle progression and met30 mutations interact genetically with mutations in SCF components. Furthermore, physical interactions between Met30, Cdc53, Cdc34, and Skp1 in vivo provide evidence for an SCFMet30 complex. We demonstrate the involvement of Met30 in the degradation of the Cdk-inhibitory kinase Swe1. Swe1 is stabilized in met30 mutants and GST-Met30 pull-down experiments reveal that Met30 specifically binds Swe1 in vivo. Furthermore, extracts prepared from cdc34 or met30 mutants are defective in polyubiquitination of Swe1. Taken together, these data suggest that SCF-mediated proteolysis may contribute to the regulation of entry into mitosis. Our data, in combination with previously published results, also provide evidence for distinct SCF complexes in vivo and support the idea that their F-box subunits mediate SCF substrate specificity. PMID: 9716410 [PubMed - indexed for MEDLINE] 942: Genes Dev 1998 Aug 15;12(16):2574-86 Telomere-mediated chromosome pairing during meiosis in budding yeast. Rockmill B, Roeder GS. Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520-8103 USA. Certain haploid strains of Saccharomyces cerevisiae can undergo meiosis, but meiotic prophase progression and subsequent nuclear division are delayed if these haploids carry an extra chromosome (i. e., are disomic). Observations indicate that interactions between homologous chromosomes cause a delay in meiotic prophase, perhaps to allow time for interhomolog interactions to be completed. Analysis of meiotic mutants demonstrates that the relevant aspect of homolog recognition is independent of meiotic recombination and synaptonemal complex formation. A disome in which the extra chromosome is circular sporulates without a delay, indicating that telomeres are important for homolog recognition. Consistent with this hypothesis, fluorescent in situ hybridization demonstrates that a circular chromosome has a reduced capacity to pair with its homolog, and a telomere-associated meiotic protein (Ndj1) is required to delay sporulation in disomes. A circular dimer containing two copies of the same chromosome delays meiosis to the same extent as two linear homologs, implying that physical proximity bypasses the requirement for telomeres in homolog pairing. Analysis of a disome carrying two linear permuted chromosomes suggests that even nonhomologous chromosome ends can promote homolog pairing to a limited extent. We speculate that telomere-mediated chromosome movement and/or telomere clustering promote homolog pairing. PMID: 9716409 [PubMed - indexed for MEDLINE] 943: Biochim Biophys Acta 1998 Aug 10;1366(1-2):127-37 Bcl-2 family proteins and mitochondria. Reed JC, Jurgensmeier JM, Matsuyama S. The Burnham Institute, Program on Apoptosis and Cell Death Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. jreed@burnham-institute.org The Bcl-2 family of proteins plays a pivotal role in regulating cell life and death. Many of these proteins reside in the outer mitochondrial membrane, oriented towards the cytosol. Cytoprotective Bcl-2 family proteins such as Bcl-2 and Bcl-XL prevent mitochondrial permeability transition pore opening and release of apoptogenic proteins from mitochondria under many circumstances that would otherwise result in either apoptosis or necrosis. In contrast, some pro-apoptotic members of this family such as Bax can induce these destructive changes in mitochondria in both mammalian cells and when expressed exogenously in yeast. The mechanisms by which Bcl-2 family proteins control cell life and death remain elusive, but may include both the ability to form ion channels or pores in membranes and physical interactions with a variety of proteins implicated in apoptosis regulation. PMID: 9714773 [PubMed - indexed for MEDLINE] 944: J Biol Chem 1998 Aug 28;273(35):22589-94 Yeast transcript elongation factor (TFIIS), structure and function. I: NMR structural analysis of the minimal transcriptionally active region. Olmsted VK, Awrey DE, Koth C, Shan X, Morin PE, Kazanis S, Edwards AM, Arrowsmith CH. Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada. TFIIS is a general transcription elongation factor that helps arrested RNA polymerase II elongation complexes resume transcription. We have previously shown that yeast TFIIS (yTFIIS) comprises three structural domains (I-III). The three-dimensional structures of domain II and part of domain III have been previously reported, but neither domain can autonomously stimulate transcription elongation. Here we report the NMR structural analysis of residues 131-309 of yTFIIS which retains full activity and contains all of domains II and III. We confirm that the structure of domain II in the context of fully active yTFIIS is the same as that determined previously for a shorter construct. We have determined the structure of the C-terminal zinc ribbon domain of active yTFIIS and shown that it is similar to that reported for a shorter construct of human TFIIS. The region linking domain II with the zinc ribbon of domain III appears to be conformationally flexible and does not adopt a single defined tertiary structure. NMR analysis of inactive mutants of yTFIIS support a role for the linker region in interactions with the transcription elongation complex. PMID: 9712887 [PubMed - indexed for MEDLINE] 945: J Immunol 1998 Aug 15;161(4):1728-37 Interaction of p59fyn kinase with the dynein light chain, Tctex-1, and colocalization during cytokinesis. Campbell KS, Cooper S, Dessing M, Yates S, Buder A. Basel Institute for Immunology, Switzerland. ks_campbell@fccc.edu The protein tyrosine kinase p59fyn (Fyn) plays important roles in both lymphocyte Ag receptor signaling and cytokinesis of proB cells. We utilized yeast two-hybrid cloning to identify the product of the tctex-1 gene as a protein that specifically interacts with Fyn, but not with other Src family kinases. Tctex-1 was recently identified as a component of the dynein cytoskeletal motor complex. The capacity of a Tctex-1-glutathione S-transferase fusion protein to effectively bind Fyn from cell lysates confirmed the authenticity of this interaction. Tctex-1 binding required the first 19 amino acids of Fyn and integrity of two lysine residues within this sequence that were previously shown to be important for Fyn interactions with the immunoreceptor tyrosine-based activation motifs (ITAMs) of lymphocyte Ag receptors. Expression of tctex-1 mRNA and protein was observed in all lymphoma lines analyzed, and immunofluorescence confocal microscopy localized the protein to the perinuclear region. Analysis of a T cell hybridoma revealed prominent colocalization of Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in cells undergoing cytokinesis. Our results provide a unique insight into a mechanism by which Tctex-1 might mediate specific recruitment of Fyn to the dynein complex in lymphocytes, which may be a critical event in mediating the previously defined role of Fyn in cytokinesis. PMID: 9712037 [PubMed - indexed for MEDLINE] 946: Mol Cell Biol 1998 Sep;18(9):5392-403 High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating type locus HMLalpha. Weiss K, Simpson RT. Department of Biochemistry and Molecular Biology, The Center for Gene Regulation, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. Genetic studies have suggested that chromatin structure is involved in repression of the silent mating type loci in Saccharomyces cerevisiae. Chromatin mapping at nucleotide resolution of the transcriptionally silent HMLalpha and the active MATalpha shows that unique organized chromatin structure characterizes the silent state of HMLalpha. Precisely positioned nucleosomes abutting the silencers extend over the alpha1 and alpha2 coding regions. The HO endonuclease recognition site, nuclease hypersensitive at MATalpha, is protected at HMLalpha. Although two precisely positioned nucleosomes incorporate transcription start sites at HMLalpha, the promoter region of the alpha1 and alpha2 genes is nucleosome free and more nuclease sensitive in the repressed than in the transcribed locus. Mutations in genes essential for HML silencing disrupt the nucleosome array near HML-I but not in the vicinity of HML-E, which is closer to the telomere of chromosome III. At the promoter and the HO site, the structure of HMLalpha in Sir protein and histone H4 N-terminal deletion mutants is identical to that of the transcriptionally active MATalpha. The discontinuous chromatin structure of HMLalpha contrasts with the continuous array of nucleosomes found at repressed a-cell-specific genes and the recombination enhancer. Punctuation at HMLalpha may be necessary for higher-order structure or karyoskeleton interactions. The unique chromatin architecture of HMLalpha may relate to the combined requirements of transcriptional repression and recombinational competence. PMID: 9710623 [PubMed - indexed for MEDLINE] 947: Mol Cell Biol 1998 Sep;18(9):5308-19 Vam7p, a SNAP-25-like molecule, and Vam3p, a syntaxin homolog, function together in yeast vacuolar protein trafficking. Sato TK, Darsow T, Emr SD. Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California at San Diego School of Medicine, La Jolla, California 92093-0668, USA. A genetic screen to isolate gene products required for vacuolar morphogenesis in the yeast Saccharomyces cerevisiae identified VAM7, a gene which encodes a protein containing a predicted coiled-coil domain homologous to the coiled-coil domain of the neuronal t-SNARE, SNAP-25 (Y. Wada and Y. Anraku, J. Biol. Chem. 267:18671-18675, 1992; T. Weimbs, S. H. Low, S. J. Chapin, K. E. Mostov, P. Bucher, and K. Hofmann, Proc. Natl. Acad. Sci. USA 94:3046-3051, 1997). Analysis of a temperature-sensitive-for-function (tsf) allele of VAM7 (vam7(tsf)) demonstrated that the VAM7 gene product directly functions in vacuolar protein transport. vam7(tsf) mutant cells incubated at the nonpermissive temperature displayed rapid defects in the delivery of multiple proteins that traffic to the vacuole via distinct biosynthetic pathways. Examination of vam7(tsf) cells at the nonpermissive temperature by electron microscopy revealed the accumulation of aberrant membranous compartments that may represent unfused transport intermediates. A fraction of Vam7p was localized to vacuolar membranes. Furthermore, VAM7 displayed genetic interactions with the vacuolar syntaxin homolog, VAM3. Consistent with the genetic results, Vam7p physically associated in a complex containing Vam3p, and this interaction was enhanced by inactivation of the yeast NSF (N-ethyl maleimide-sensitive factor) homolog, Sec18p. In addition to the coiled-coil domain, Vam7p also contains a putative NADPH oxidase p40(phox) (PX) domain. Changes in two conserved amino acids within this domain resulted in synthetic phenotypes when combined with the vam3(tsf) mutation, suggesting that the PX domain is required for Vam7p function. This study provides evidence for the functional and physical interaction between Vam7p and Vam3p at the vacuolar membrane, where they function as part of a t-SNARE complex required for the docking and/or fusion of multiple transport intermediates destined for the vacuole. PMID: 9710615 [PubMed - indexed for MEDLINE] 948: Mol Cell Biol 1998 Sep;18(9):5189-98 Genetic, physical, and functional interactions between the triphosphatase and guanylyltransferase components of the yeast mRNA capping apparatus. Ho CK, Schwer B, Shuman S. Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA. We have characterized an essential Saccharomyces cerevisiae gene, CES5, that when present in high copy, suppresses the temperature-sensitive growth defect caused by the ceg1-25 mutation of the yeast mRNA guanylyltransferase (capping enzyme). CES5 is identical to CET1, which encodes the RNA triphosphatase component of the yeast capping apparatus. Purified recombinant Cet1 catalyzes hydrolysis of the gamma phosphate of triphosphate-terminated RNA at a rate of 1 s-1. Cet1 is a monomer in solution; it binds with recombinant Ceg1 in vitro to form a Cet1-Ceg1 heterodimer. The interaction of Cet1 with Ceg1 elicits >10-fold stimulation of the guanylyltransferase activity of Ceg1. This stimulation is the result of increased affinity for the GTP substrate. A truncated protein, Cet1(201-549), has RNA triphosphatase activity, heterodimerizes with and stimulates Ceg1 in vitro, and suffices when expressed in single copy for cell growth in vivo. The more extensively truncated derivative Cet1(246-549) also has RNA triphosphatase activity but fails to stimulate Ceg1 in vitro and is lethal when expressed in single copy in vivo. These data suggest that the Cet1-Ceg1 interaction is essential but do not resolve whether the triphosphatase activity is also necessary. The mammalian capping enzyme Mce1 (a bifunctional triphosphatase-guanylyltransferase) substitutes for Cet1 in vivo. A mutation of the triphosphatase active-site cysteine of Mce1 is lethal. Hence, an RNA triphosphatase activity is essential for eukaryotic cell growth. This work highlights the potential for regulating mRNA cap formation through protein-protein interactions. PMID: 9710603 [PubMed - indexed for MEDLINE] 949: Mol Cell 1998 Jul;2(1):135-40 Circularization of mRNA by eukaryotic translation initiation factors. Wells SE, Hillner PE, Vale RD, Sachs AB. Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA. Communication between the 5' cap structure and 3' poly(A) tail of eukaryotic mRNA results in the synergistic enhancement of translation. The cap and poly(A) tail binding proteins, eIF4E and Pab1p, mediate this effect in the yeast S. cerevisiae through their interactions with different parts of the translation factor eIF4G. Here, we demonstrate the reconstitution of an eIF4E/eIF4G/Pab1p complex with recombinant proteins, and show by atomic force microscopy that the complex can circularize capped, polyadenylated RNA. Our results suggest that formation of circular mRNA by translation factors could contribute to the control of mRNA expression in the eukaryotic cell. PMID: 9702200 [PubMed - indexed for MEDLINE] 950: Mol Vis 1998 Aug 11;4:13 Interaction of phosducin and phosducin isoforms with a 26S proteasomal subunit, SUG1. Zhu X, Craft CM. Doheny Eye Institute and Department of Cell & Neurobiology, University of Southern California School of Medicine, Los Angeles, CA 90033, USA. PURPOSE: Retinal phosducin (Phd) and phosducin-like protein 1 (PhLP1) selectively bind G-protein beta/gamma subunits (Gbetagamma). Our laboratory has recently identified two phosducin-like orphan proteins (PhLOP1 and PhLOP2) that lack the ability to interact with Gbetagamma. In search of potential functional protein partner(s) for these phosducin orphans, we examined their protein-protein interactions using a yeast two-hybrid screen. METHODS: A bovine retina yeast expression cDNA library was screened with the GAL4 DNA binding domain (BD) fusion of PhLOP1. Quantitative analysis of the selected positives with PhLOP1 and other Phd isoforms was assessed by growth and beta-galactosidase activity. Further molecular, biochemical, and immunological detection methods utilizing glutathione S-transferase (GST)-Phd isoform fusion proteins and the potential partner were also performed. RESULTS: A member of the superfamily of putative ATPases was selected in the yeast two hybrid screen. Further characterization identified a direct interaction of this putative ATPase with PhLOP1, as well as Phd and PhLP1, but not with PhLOP2. A database search verified this ATPase as a bovine orthologue of the yeast SUG1 (ySUG1), a putative transcriptional mediator and a subunit of the 26S proteasome complex. Our experiments reveal that the carboxy-terminus of PhLOP1 is essential for the protein-protein interaction with SUG1, but it alone is not sufficient to mediate SUG1 interaction. CONCLUSIONS: Based on these experimental results, Phd, PhLP1 and PhLOP1 have protein-protein interaction with SUG1. PhLOP1, a truncated amino-terminal splice variant of Phd, is the best candidate for the interaction with SUG1 among the four Phd isoforms studied, which suggests a potential function for PhLOP1. PMID: 9701609 [PubMed - indexed for MEDLINE] 951: Biochemistry 1998 Aug 11;37(32):11171-81 Structure determination and characterization of Saccharomyces cerevisiae profilin. Eads JC, Mahoney NM, Vorobiev S, Bresnick AR, Wen KK, Rubenstein PA, Haarer BK, Almo SC. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA. The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 microM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins. The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to the Kd of 2.0 microM for the interaction between yeast profilin and pyrene-labeled yeast actin. The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface. The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar to the reported affinity of Acanthamoeba profilin for poly-L-proline. Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin. The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure. PMID: 9698363 [PubMed - indexed for MEDLINE] 952: Pac Symp Biocomput 1998;:264-78 A computational "genome walk" technique to identify regulatory interactions in gene networks. Wagner A. Santa Fe Institute, NM 87501, USA. aw@santafe.edu To delineate the astronomical number of possible interactions of all genes in a genome is a task for which conventional experimental techniques are ill-suited. Sorely needed are rapid and inexpensive methods that identify candidates for interacting genes, candidates that can be further investigated by experiment. The subject of this paper is the application of a novel method to the genome of the yeast Saccharomyces cerevisiae. The method applies to an important class of gene interactions, that is, transcriptional regulation via transcription factors (TFs) that bind to specific enhancer or silencer sites on DNA. The method addresses the question: which of the genes in a genome are likely to be regulated by one or more TFs with known DNA binding specificity? It takes advantage of the fact that many TFs show cooperativity in transcriptional activation which manifests itself in closely spaced TF binding sites. Such "clusters" of binding sites are very unlikely to occur by chance alone, as opposed to individual sites, which are often abundant both in the genome and in promoter regions. Statistical information about binding site clusters in the genome, can be complemented by information about (i) known biochemical functions of the TF, (ii) the structure of its binding site, and (iii) function of the genes near the cluster, to identify genes likely to be regulated by a given transcription factor. Previously, binding sites of well characterized transcription factors in Saccharomyces cerevisiae were analyzed. Here, the method is applied to a somewhat different situation: the yeast DNA binding activity yE2F, similar to the mammalian transcription factor E2F. yE2F has a DNA binding specificity identical to E2F, and its binding site shows UAS activity in a GAL1-based promoter construct. However, despite its high conservation, the in vivo function of yE2F is unknown. The analysis carried out the here suggests candidate genes for regulation by yE2F. PMID: 9697188 [PubMed - indexed for MEDLINE] 953: Cell 1998 Jul 24;94(2):217-27 Degradation signal masking by heterodimerization of MATalpha2 and MATa1 blocks their mutual destruction by the ubiquitin-proteasome pathway. Johnson PR, Swanson R, Rakhilina L, Hochstrasser M. Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637, USA. Proteolysis by the ubiquitin-proteasome pathway is often regulated, but the mechanisms underlying such regulation remain ill-defined. In Saccharomyces cerevisiae, cell type is controlled by the MAT transcription factors. The alpha2 repressor is a known ubiquitin pathway substrate in alpha haploid cells. We show that a1 is rapidly degraded in a haploids. In a/alpha diploids, alpha2 and a1 are stabilized by heterodimerization. Association depends on N-terminal coiled-coil interactions between a1 and alpha2. Residues in alpha2 important for these interactions overlap a critical determinant of an alpha2 degradation signal, which we delimit by extensive mutagenesis. Our data provide a detailed description of a natural ubiquitin-dependent degradation signal and point to a molecular mechanism for regulated turnover in which proteolytic signals are differentially masked in alternative multiprotein complexes. PMID: 9695950 [PubMed - indexed for MEDLINE] 954: Mol Biol Cell 1998 Aug;9(8):2201-16 A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and Spc98p, components of the yeast gamma-tubulin complex. Nguyen T, Vinh DB, Crawford DK, Davis TN. Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195, USA. The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110-221 allele, which encodes mutations in the amino terminus. The screen identified mutations in SPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98-63 allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, the spc97 alleles are synthetic lethal with spc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97 interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98-63 spc110-221 cells is caused by the failure of Spc98-63p to interact with Spc110-221p. In contrast, the lethal phenotype in spc97-62 spc110-221 cells can be attributed to a decreased interaction between Spc97-62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p. PMID: 9693376 [PubMed - indexed for MEDLINE] 955: J Endocrinol 1998 Jun;157(3):361-71 Post-transcriptional gene regulatory mechanisms in eukaryotes: an overview. Day DA, Tuite MF. Department of Biosciences, University of Kent, Canterbury, UK. Expression of a gene can be controlled at many levels, including transcription, mRNA splicing, mRNA stability, translation and post-translational events such as protein stability and modification. The majority of studies to date have focused on transcriptional control mechanisms, but the importance of post-transcriptional mechanisms in regulating gene expression in eukaryotes is becoming increasingly clear. In this short review, selected examples of post-transcriptional gene regulatory mechanisms operating in both lower and higher eukaryotes will be used to highlight the plethora of such mechanisms already identified. The underlying theme is that post-transcriptional gene regulation relies on specific RNA-protein interactions that either result in the targeted degradation of the mRNA or prevent access of the ribosome to the translation start codon. Such interactions can occur in the 5' or 3' untranslated regions of an mRNA or within the decoded portion of the molecule. The importance of these regulatory mechanisms in a range of biological systems is also illustrated. Publication Types: Review Review, Academic PMID: 9691970 [PubMed - indexed for MEDLINE] 956: Genetics 1998 Aug;149(4):1717-27 Sro7p, a Saccharomyces cerevisiae counterpart of the tumor suppressor l(2)gl protein, is related to myosins in function. Kagami M, Toh-e A, Matsui Y. Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113, Japan. Yeast SRO7 was identified as a multicopy suppressor of a defect in Rho3p, a small GTPase that maintains cell polarity. Sro7p and Sro77p, a homologue of Sro7p, possess domains homologous to the protein that are encoded by the Drosophila tumor suppressor gene lethal (2) giant larvae [l(2)gl]. sro7Delta sro77Delta mutants showed a partial defect of organization of the polarized actin cytoskeleton and a cold-sensitive growth phenotype. A human counterpart of l(2)gl could suppress the sro7Delta sro77Delta defect. Similar to the l(2)gl protein, Sro7p formed a complex with Myo1p, a type II myosin. These results indicate that Sro7p and Sro77p are the yeast counterparts of the l(2)gl protein. Our genetic analysis revealed that deletion of SRO7 and SRO77 showed reciprocal suppression with deletion of MYO1 (i.e., the sro7Delta sro77Delta defect was suppressed by myo1Delta and vice versa). In addition, SRO7 showed genetic interactions with MYO2, encoding an essential type V myosin: Overexpression of SRO7 suppressed a defect in MYO2 and, conversely, overexpression of MYO2 suppressed the cold-sensitive phenotype of sro7Delta sro77Delta mutants. These results indicate that Sro7 function is closely related to both Myo1p and Myo2p. We propose a model in which Sro7 function is involved in the targeting of the myosin proteins to their intrinsic pathways. PMID: 9691031 [PubMed - indexed for MEDLINE] 957: Science 1998 Jul 31;281(5377):698-700 Nucleation of COPII vesicular coat complex by endoplasmic reticulum to Golgi vesicle SNAREs. Springer S, Schekman R. Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3202, USA. Protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus involves specific uptake into coat protein complex II (COPII)-coated vesicles of secretory and of vesicle targeting (v-SNARE) proteins. Here, two ER to Golgi v-SNAREs, Bet1p and Bos1p, were shown to interact specifically with Sar1p, Sec23p, and Sec24p, components of the COPII coat, in a guanine nucleotide-dependent fashion. Other v-SNAREs, Sec22p and Ykt6p, might interact more weakly with the COPII coat or interact indirectly by binding to Bet1p or Bos1p. The data suggest that transmembrane proteins can be taken up into COPII vesicles by direct interactions with the coat proteins and may play a structural role in the assembly of the COPII coat complex. PMID: 9685263 [PubMed - indexed for MEDLINE] 958: Plant J 1998 Jun;14(6):685-92 Isolation of putative plant transcriptional coactivators using a modified two-hybrid system incorporating a GFP reporter gene. Cormack RS, Hahlbrock K, Somssich IE. Max-Planck-Institut fur Zuchtungsforschung, Abteilung Biochemie, Cologne, Germany. Dual hybrid interacting screening in yeast led to the identification of two proteins from Arabidopsis both exhibiting sequence similarity to a family of transcriptional coactivators from a diverse range of organisms. Their discovery constitutes the first description of such plant proteins. A modified yeast two-hybrid approach utilising the green fluorescent protein (GFP) of Aequora victoria was developed and used to clone one of the putative plant transcriptional coactivators from an Arabidopsis cDNA library. The two proteins, designated KIWI and KELP, can associate both hetero- and homomerically and their genes were cloned and mapped on the Arabidopsis genome. Both proteins are believed to play a role in gene activation during pathogen defence and plant development. The involvement of these proteins in general plant transcription as well as the advantages of using GFP as a reporter gene for detecting protein-protein interactions are discussed. PMID: 9681033 [PubMed - indexed for MEDLINE] 959: J Cell Biol 1998 Jul 27;142(2):443-55 Iqg1p, a yeast homologue of the mammalian IQGAPs, mediates cdc42p effects on the actin cytoskeleton. Osman MA, Cerione RA. Department of Pharmacology, Cornell University, Ithaca, New York 14853, USA. The Rho-type GTPase Cdc42p has been implicated in diverse cellular functions including cell shape, cell motility, and cytokinesis, all of which involve the reorganization of the actin cytoskeleton. Targets of Cdc42p that interface the actin cytoskeleton are likely candidates for mediating cellular activities. In this report, we identify and characterize a yeast homologue for the mammalian IQGAP, a cytoskeletal target for Cdc42p. The yeast IQGAP homologue, designated Iqg1p, displays a two-hybrid interaction with activated Cdc42p and coimmunoprecipitates with actin filaments. Deletion of IQG1 results in a temperature-sensitive lethality and causes aberrant morphologies including elongated and round multinucleated cells. This together with its localization at the mother-bud neck, suggest that Iqg1p promotes budding and cytokinesis. At restrictive temperatures, the vacuoles of the mutant cells enlarge and vesicles accumulate in the bud. Interestingly, Iqg1p shows two-hybrid interactions with the ankyrin repeat-containing protein, Akr1p (Kao, L.-R., J. Peterson, J. Ruiru, L. Bender, and A. Bender. 1996. Mol. Cell. Biol. 16:168-178), which inhibits pheromone signaling and appears to promote cytokinesis and/or trafficking. We also show two-hybrid interactions between Iqg1p and Afr1p, a septin-binding protein involved in projection formation (Konopka, J.B., C. DeMattei, and C. Davis. 1995. Mol. Cell. Biol. 15:723-730). We propose that Iqg1p acts as a scaffold to recruit and localize a protein complex involved in actin-based cellular functions and thus mediates the regulatory effects of Cdc42p on the actin cytoskeleton. PMID: 9679143 [PubMed - indexed for MEDLINE] 960: J Cell Biol 1998 Jul 27;142(2):341-54 A functional GTPase domain, but not its transmembrane domain, is required for function of the SRP receptor beta-subunit. Ogg SC, Barz WP, Walter P. Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California School of Medicine, San Francisco, California 94143-0448, USA. The signal recognition particle and its receptor (SR) target nascent secretory proteins to the ER. SR is a heterodimeric ER membrane protein whose subunits, SRalpha and SRbeta, are both members of the GTPase superfamily. Here we characterize a 27-kD protein in Saccharomyces cerevisiae (encoded by SRP102) as a homologue of mammalian SRbeta. This notion is supported (a) by Srp102p's sequence similarity to SRbeta; (b) by its disposition as an ER membrane protein; (c) by its interaction with Srp101p, the yeast SRalpha homologue; and (d) by its role in SRP-dependent protein targeting in vivo. The GTP-binding site in Srp102p is surprisingly insensitive to single amino acid substitutions that inactivate other GTPases. Multiple mutations in the GTP-binding site, however, inactivate Srp102p. Loss of activity parallels a loss of affinity between Srp102p and Srp101p, indicating that the interaction between SR subunits is important for function. Deleting the transmembrane domain of Srp102p, the only known membrane anchor in SR, renders SR soluble in the cytosol, which unexpectedly does not significantly impair SR function. This result suggests that SR functions as a regulatory switch that needs to associate with the ER membrane only transiently through interactions with other components. PMID: 9679135 [PubMed - indexed for MEDLINE] 961: Biotechnology (N Y) 1995 Dec;13(13):1431-4 Comment on: Biotechnology (N Y). 1995 Dec;13(13):1474-8. Fishing for protein interactions with tribrids. Paul J, Trueheart J. Cadus Pharmaceutical Corporation, Tarrytown, NY 10591-6704, USA. 75-763-2141@compuserve. com Publication Types: Comment PMID: 9678924 [PubMed - indexed for MEDLINE] 962: Adv Genet 1998;38:185-218 DNA breakage and repair. Jeggo PA. MRC Cell Mutation Unit, University of Sussex, Brighton, United Kingdom. For many years it has been evident that mammalian cells differ dramatically from yeast and rejoin the majority of their DNA DSBs by a nonhomologous mechanism, recently termed NHEJ. In the last few years a number of genes and proteins have been identified that operate in the pathway providing insights into the mechanism. These proteins include the three components of DNA-PK, DNA ligase IV, and XRCC4. In yeast Sir2, -3, and -4 proteins are also involved in the process and therefore are likely to play a role in higher organisms. Studies with yeast suggest that NHEJ is an error-free mechanism. Although the process is far from understood, it is likely that the DNA-PK complex or Ku alone acts in a complex with the Sir proteins possibly protecting the ends and preventing random rejoining. Further work is required to establish the details of this mechanism and to determine whether this represents an accurate rejoining process for a complex break induced by ionizing radiation. It will be intriguing to discover how the cell achieves efficient and accurate rejoining without the use of homology. Interactions between the components of DNA-PK and other proteins playing a central role in damage response mechanisms are beginning to emerge. Interestingly, there is evidence that DNA repair and damage response mechanisms overlap in lower organisms. The overlapping defects of the yeast Ku mutants, tell mutants, and AT cell lines in telomere maintenance further suggest overlapping functions or interacting mechanisms. A challenge for the future will be to establish how these different damage response mechanisms overlap and interact. Publication Types: Review Review, Academic PMID: 9677708 [PubMed - indexed for MEDLINE] 963: J Biol Chem 1998 Jul 31;273(31):19792-6 A family of Arf effectors defined as suppressors of the loss of Arf function in the yeast Saccharomyces cerevisiae. Zhang CJ, Cavenagh MM, Kahn RA. Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322-3050, USA. Arf proteins are ubiquitous, eukaryotic regulators of virtually every step of vesicular membrane traffic. ADP-ribosylation factors are essential in yeast and the lethality resulting from either overexpression or underexpression (deletion) of Arf genes has previously been ascribed to dysregulation of the secretory process. We have identified a family of four genes (Suppressors of Arf ts, SAT) as high copy suppressors of a loss of function allele of ARF1 (arf1-3). Those proteins with SAT activity were found to contain a minimal consensus motif, including a C2C2H2 cluster with a novel and specific spacing. Genetic interactions between members of this family and with ARF1 are consistent with each sharing a common cellular pathway. Included in this family is Gcs1, a protein previously described (Poon, P. P., Wang, X., Rotman, M., Huber, I., Cukierman, E., Cassel, D., Singer, R. A., and Johnston, G. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10074-10077) to possess Arf GTPase-activating protein (GAP) activity, demonstrating a direct interaction between Arf and at least one of these suppressors. The suppression of the loss of Arf function by overexpression of Gcs1 and demonstration of direct, preferential binding of Gcs1 to the activated form of Arf (Arf.GTP) lead us to conclude that the biological role of Gcs1 is as an effector of the essential function of Arf in mitotic growth, rather than a down-regulator as implied by the biochemical (Arf GAP) activity. Suppression of the growth defect of arf1(-3) cells was observed under conditions that did not alter the secretory defect associated with arf1(-) mutation, indicating that the essential role of Arf in eukaryotes can be distinguished from role(s) in the secretory pathway and appear to employ distinct pathways and effectors. PMID: 9677411 [PubMed - indexed for MEDLINE] 964: Yeast 1998 Jun 15;14(8):733-46 The C-terminal hydrophobic repeat of Schizosaccharomyces pombe heat shock factor is not required for heat-induced DNA-binding. Saltsman KA, Prentice HL, Kingston RE. Department of Molecular Biology, Massachusetts General Hospital, Boston 02114, USA. The C-terminal hydrophobic repeat (CTR) of heat shock transcription factor (HSF) has been proposed to regulate DNA binding by intramolecular interactions with the leucine zipper motifs present in the HSF trimerization domain. Schizosaccharomyces pombe provides a useful model organism for the study of the regulation of HSF DNA binding because, unlike Saccharomyces cerevisiae, S. pombe hsf is highly heat shock inducible for DNA binding and contains a clear homology to the CTR. We examined the role that the CTR plays in the regulation of S. pombe hsf by constructing isogenic strains bearing deletion and point mutations in the chromosomal copy of hsf. Surprisingly, we found that point mutation of key hydrophobic amino acids within the CTR, as well as full deletion of it, yielded factors that show normal binding at normal growth temperatures and full levels of heat-induced binding. Deletion of the CTR did, however, slightly lower the temperature required for maximal activation. In contrast, a large deletion of the C-terminus, which removes close to a third of the coding sequence, was deregulated and bound DNA at control temperature. Several of the deletion mutants were significantly reduced in their level of expression, yet they showed wild-type levels of DNA binding activity following heat shock. These experiments demonstrate that appropriate regulation of the DNA binding activity of S. pombe hsf is not solely dependent upon the CTR, and imply that a feedback mechanism exists that establishes proper levels of DNA binding following heat shock despite mutations that significantly alter levels of total hsf. PMID: 9675818 [PubMed - indexed for MEDLINE] 965: Cell 1998 Jul 10;94(1):73-82 Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Glover JR, Lindquist S. Howard Hughes Medical Institute and Department of Molecular Genetics and Cell Biology, The University of Chicago, Illinois 60637, USA. Hsp104 is a stress tolerance factor that promotes the reactivation of heat-damaged proteins in yeast by an unknown mechanism. Herein, we demonstrate that Hsp104 functions in this process directly. Unlike other chaperones, Hsp104 does not prevent the aggregation of denatured proteins. However, in concert with Hsp40 and Hsp70, Hsp104 can reactivate proteins that have been denatured and allowed to aggregate, substrates refractory to the action of other chaperones. Hsp104 cooperates with the chaperones present in reticulocyte lysates but not with DnaK of E. coli. We conclude that Hsp104 has a protein remodeling activity that acts on trapped, aggregated proteins and requires specific interactions with conventional chaperones to promote refolding of the intermediates it produces. PMID: 9674429 [PubMed - indexed for MEDLINE] 966: Nucleic Acids Res 1998 Aug 1;26(15):3577-83 Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids: expressionvectors with bi-directional promoters for use in Saccharomyces cerevisiae. Miller CA 3rd, Martinat MA, Hyman LE. Environmental Health Sciences Department and Tulane-Xavier Center for Bioenvironmental Research,Tulane University School of Public Health and Tropical Medicine, 1430 Tulane Avenue, New Orleans,LA 70112, USA. The pBEVY (bi-directional expression vectors for yeast) plasmids were designed with constitutive and galactose-induced bi-directional promoters to direct the expression of multiple proteins in Saccharomyces cerevisiae . Using human estrogen receptor as a test gene, relatively balanced expression levels from each side of a bi-directional promoter were observed. Expression of a functional heterodimeric transcription factor composed of human aryl hydrocarbon receptor (Ahr) and aryl hydrocarbon receptor nuclear translocator (Arnt) proteins was accomplished using a single pBEVY plasmid. Previous studies suggest that inhibitory cross-talk between the estrogen receptor and the Ahr/Arnt complex may occur and that Hsp90-Ahr complex formation is important for Ahr-mediated signal transduction. Evidence for functional interaction among these proteins was investigated using pBEVY plasmids in a yeast system. No inhibitory cross-talk was observed in signaling assays performed with yeast that co-expressed Ahr, Arnt and estrogen receptor. In contrast, Ahr/Arnt-mediated signal transduction was reduced by 80% in a temperature-sensitive Hsp90 strain grown under non-permissive conditions. We conclude that pBEVY plasmids facilitate the examination of multiple protein interactions in yeast model systems. PMID: 9671822 [PubMed - indexed for MEDLINE] 967: Mol Cell Biol 1998 Aug;18(8):4899-913 Disruption of PML subnuclear domains by the acidic IE1 protein of human cytomegalovirus is mediated through interaction with PML and may modulate a RING finger-dependent cryptic transactivator function of PML. Ahn JH, Brignole EJ 3rd, Hayward GS. Molecular Virology Laboratories, Departments of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. Both of the major immediate-early (IE) proteins IE1 and IE2 of human cytomegalovirus (HCMV) as well as input viral DNA and sites of viral IE transcription colocalize with or adjacent to punctate PML domains (PML oncogenic domains [PODs] or nuclear domain 10) in the nucleus within the first few hours after infection of permissive human fibroblasts. However, colocalization of IE1 and PML in PODs is only transient, with both proteins subsequently redistributing into a nuclear diffuse form. These processes are believed to promote efficient viral IE transcription and initiation of DNA synthesis especially at low multiplicities of infection. To examine the mechanism of PML displacement by IE1, we carried out indirect immunofluorescence assay experiments with plasmids expressing intact or deleted forms of PML and IE1 in DNA-transfected cells. The results demonstrated that deletion of the C-terminal acidic region of IE1 uncouples the requirements for displacement of both endogenous and coexpressed PML from those needed to target to the PODs. Mutant PML proteins containing either a Cys point mutation within the N-terminal RING finger domain or a small deletion (of positions 281 to 304) within the coiled-coil region did not localize to the PODs but instead gave a nuclear diffuse distribution, similar to that produced by intact PML in the presence of IE1. Endogenous PML also colocalized with IE1 in metaphase chromosomes in HCMV or recombinant adenovirus type 5-IE1-infected HF cells undergoing mitosis, implying that there may be a direct physical interaction between IE1 and PML. Indeed, a specific interaction between IE1 and PML was observed in a yeast two-hybrid assay, and the strength of this interaction was comparable to that of IE2 with the retinoblastoma protein. The RING finger mutant form of PML showed a threefold-lower interaction with IE1 in the yeast system, and deletion of the N-terminal RING finger domain of PML abolished the interaction. Consistent with the IFA results, a mutant IE1 protein that lacks the C-terminal acidic region was sufficient for interaction with PML in the yeast system. The two-hybrid interaction assay also showed that both the N-terminal RING finger domain and the intact coiled-coil region of PML are required cooperatively for efficient self-interactions involving dimerization or oligomerization. Furthermore, truncated or deleted GAL4/PML fusion proteins that retained the RING finger domain but lacked the intact coiled-coil region displayed an unmasked cryptic transactivator function in both yeast and mammalian cells, and the RING finger mutation abolished this transactivation property of PML. Therefore, we suggest that a direct interaction between IE1 and the N-terminal RING finger domain of PML may inhibit oligomerization and protein-protein complex formation by PML, leading to displacement of PML and IE1 from the PODs, and that this interaction may also modulate a putative conditional transactivator function of PML. PMID: 9671498 [PubMed - indexed for MEDLINE] 968: Mol Cell Biol 1998 Aug;18(8):4935-46 Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5. Phan L, Zhang X, Asano K, Anderson J, Vornlocher HP, Greenberg JR, Qin J, Hinnebusch AG. Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA. Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of approximately 600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3. PMID: 9671501 [PubMed - indexed for MEDLINE] 969: EMBO J 1998 Jul 15;17(14):3981-9 The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome-nascent chain complex. Pfund C, Lopez-Hoyo N, Ziegelhoffer T, Schilke BA, Lopez-Buesa P, Walter WA, Wiedmann M, Craig EA. Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706, USA. The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular chaperones. The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70 found associated with translating ribosomes. To understand better the function of Ssb in association with ribosomes, the Ssb-ribosome interaction was characterized. Incorporation of the aminoacyl-tRNA analog puromycin by translating ribosomes caused the release of Ssb concomitant with the release of nascent chains. In addition, Ssb could be cross-linked to nascent chains containing a modified lysine residue with a photoactivatable cross-linker. Together, these results suggest an interaction of Ssb with the nascent chain. The interaction of Ssb with the ribosome-nascent chain complex was stable, as demonstrated by resistance to treatment with high salt; however, Ssb interaction with the ribosome in the absence of nascent chain was salt sensitive. We propose that Ssb is a core component of the translating ribosome which interacts with both the nascent polypeptide chain and the ribosome. These interactions allow Ssb to function as a chaperone on the ribosome, preventing the misfolding of newly synthesized proteins. PMID: 9670014 [PubMed - indexed for MEDLINE] 970: Biotechniques 1998 Jul;25(1):85-8, 90-2 Development of a yeast trihybrid screen using stable yeast strains and regulated protein expression. Fuller KJ, Morse MA, White JH, Dowell SJ, Sims MJ. Immunology Unit, Glaxo Wellcome Medicines Research Centre, Stevenage, Hertfordshire, UK. We describe a yeast trihybrid system that facilitates rapid screening of cDNA libraries. Novel yeast vectors were developed that direct integration of cDNA encoding the bait and third protein component into the yeast chromosome. A recombinant yeast strain is thus generated (screening strain) and is available for library transformation. Transformation with the library DNA is a single, efficient transformation event, allowing the cDNA library to be represented in one step. Recovery of the library plasmid from the yeast is also simplified, since it is the only episomal plasmid. Assay of trihybrid interaction and identification of positive clones is facilitated by regulating expression of the third protein component using the yeast MET3 promoter, which is repressed in the presence of exogenous methionine. Trihybrid interactions are detected only on media lacking methionine. This trihybrid system uses the standard E. coli LacZ and yeast HIS3 reporter genes and is compatible with most available Gal4 activation domain cDNA libraries. We describe the successful application of this yeast trihybrid system to the study of phosphoprotein interactions involved in T-cell signaling. Publication Types: Technical Report PMID: 9668981 [PubMed - indexed for MEDLINE] 971: Methods Mol Biol 1998;84:201-22 Two-hybrid analysis of Ras-Raf interactions. Van Aelst L. Cold Spring Harbor Laboratory, NY, USA. PMID: 9666451 [PubMed - indexed for MEDLINE] 972: Gene 1998 Jul 17;215(1):143-52 Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map. Hua SB, Luo Y, Qiu M, Chan E, Zhou H, Zhu L. Gene, Net Group, CLONTECH Laboratories Inc., 1020 East Meadow Circle, Palo Alto, CA 94303, USA. sbhua@clontech.com Identification of all human protein-protein interactions will lead to a global human protein linkage map that will provide important information for functional genomics studies. The yeast two-hybrid system is a powerful molecular genetic approach for studying protein-protein interactions. To apply this technology to generate a human protein linkage map, the first step is to construct two-hybrid cDNA libraries that cover the entire human genome. With a homologous recombination-mediated approach, we have constructed a modular human EST-derived yeast two-hybrid library in the Gal4 activation domain-based vector, pACT2. Quality analysis of this library indicated that the approach of constructing two-hybrid cDNA libraries from individually arrayed human EST clones is feasible, and such a two-hybrid library is suitable for detecting protein-protein interactions. This is also the first time that a comprehensive two-hybrid system cDNA library has been constructed from a collection of individually arrayed EST clones. PMID: 9666106 [PubMed - indexed for MEDLINE] 973: Methods Mol Biol 1998;93:251-61 Analysis of protein interactions between protein phosphatase 1 and noncatalytic subunits using the yeast two-hybrid assay. Ramaswamy NT, Dalley BK, Cannon JF. Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, USA. PMID: 9664543 [PubMed - indexed for MEDLINE] 974: J Cell Biol 1998 Jul 13;142(1):39-49 Assembly of the yeast vacuolar H+-ATPase occurs in the endoplasmic reticulum and requires a Vma12p/Vma22p assembly complex. Graham LA, Hill KJ, Stevens TH. Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA. Three previously identified genes from Saccharomyces cerevisiae, VMA12, VMA21, and VMA22, encode proteins localized to the endoplasmic reticulum (ER). These three proteins are required for the biogenesis of a functional vacuolar ATPase (V-ATPase), but are not part of the final enzyme complex. Subcellular fractionation and chemical cross-linking studies have revealed that Vma12p and Vma22p form a stable membrane associated complex. Cross-linking analysis also revealed a direct physical interaction between the Vma12p/Vma22p assembly complex and Vph1p, the 100-kD integral membrane subunit of the V-ATPase. The interaction of the Vma12p/Vma22p complex with Vph1p was transient (half-life of approximately 5 min), reflecting trafficking of this V-ATPase subunit through the ER en route to the vacuolar membrane. Analysis of these protein-protein interactions in ER-blocked sec12 mutant cells indicated that the Vph1p-Vma12p/Vma22p interactions are quite stable when transport of the V-ATPase out of the ER is blocked. Fractionation of solubilized membrane proteins on a density gradient revealed comigration of Vma22p and Vma12p, indicating that they form a complex even in the absence of cross-linker. Vma12p and Vma22p migrated to fractions separate from Vma21p. Loss of Vph1p caused the Vma12p/Vma22p complex to sediment to less dense fractions, consistent with association of Vma12p/ Vma22p with nascent Vph1p in ER membranes. This is the first evidence for a dedicated assembly complex in the ER required for the assembly of an integral membrane protein complex (V-ATPase) as it is transported through the secretory pathway. PMID: 9660861 [PubMed - indexed for MEDLINE] 975: J Biol Chem 1998 Jul 17;273(29):18573-85 Complex formation by all five homologues of mammalian translation initiation factor 3 subunits from yeast Saccharomyces cerevisiae. Asano K, Phan L, Anderson J, Hinnebusch AG. Laboratory of Eukaryotic Gene Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA. The PRT1, TIF34, GCD10, and SUI1 proteins of Saccharomyces cerevisiae were found previously to copurify with eukaryotic translation initiation factor 3 (eIF3) activity. Although TIF32, NIP1, and TIF35 are homologous to subunits of human eIF3, they were not known to be components of the yeast factor. We detected interactions between PRT1, TIF34, and TIF35 by the yeast two-hybrid assay and in vitro binding assays. Discrete segments (70-150 amino acids) of PRT1 and TIF35 were found to be responsible for their binding to TIF34. Temperature-sensitive mutations mapping in WD-repeat domains of TIF34 were isolated that decreased binding between TIF34 and TIF35 in vitro. The lethal effect of these mutations was suppressed by increasing TIF35 gene dosage, suggesting that the TIF34-TIF35 interaction is important for TIF34 function in translation. Pairwise in vitro interactions were also detected between PRT1 and TIF32, TIF32 and NIP1, and NIP1 and SUI1. Furthermore, PRT1, NIP1, TIF34, TIF35, and a polypeptide with the size of TIF32 were specifically coimmunoprecipitated from the ribosomal salt wash fraction. We propose that all five yeast proteins homologous to human eIF3 subunits are components of a stable heteromeric complex in vivo and may comprise the conserved core of yeast eIF3. PMID: 9660829 [PubMed - indexed for MEDLINE] 976: J Biol Chem 1998 Jul 17;273(29):18490-8 Characterization of Pak2p, a pleckstrin homology domain-containing, p21-activated protein kinase from fission yeast. Sells MA, Barratt JT, Caviston J, Ottilie S, Leberer E, Chernoff J. Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. p21-activated kinases (PAKs) bind to and are activated by Rho family GTPases such as Cdc42 and Rac. Since these GTPases play key roles in regulating cell polarity, stress responses, and cell cycle progression, the ability of PAK to affect these processes has been examined. We previously showed that fission yeast pak1+ encodes an essential protein that affects mating and cell polarity. Here, we characterize a second pak gene (pak2+) from Schizosaccharomyces pombe. Like the Saccharomyces cerevisiae proteins Cla4p and Skm1p, fission yeast Pak2p contains an N-terminal pleckstrin homology domain in addition to a p21-binding domain and a protein kinase domain that are common to other members of the PAK family. Unlike pak1+, pak2(+) is not essential for vegetative growth or for mating in S. pombe. Overexpression of the wild-type pak2+ allele suppresses the lethal growth defect associated with deletion of pak1+, and this suppression requires both the pleckstrin homology- and the p21-binding domains of Pak2p, as well as kinase activity. A substantial fraction of Pak2p is associated with membranous components, an association mediated both by the pleckstrin homology- and by the p21-binding domains. These results show that S. pombe encodes at least two pak genes with distinct functions and suggest that the membrane localization of Pak2p, directed by its interactions with membrane lipids and Cdc42p, is critical to its biological activity. PMID: 9660818 [PubMed - indexed for MEDLINE] 977: J Virol 1998 Aug;72(8):6944-9 Binding of the human immunodeficiency virus type 1 Gag protein to the viral RNA encapsidation signal in the yeast three-hybrid system. Bacharach E, Goff SP. Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA. We have used the yeast three-hybrid system (D. J. SenGupta, B. Zhang, B. Kraemer, P. Pochart, S. Fields, and M. Wickens, Proc. Natl. Acad. Sci. USA 93:8496-8501, 1996) to study binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein to the HIV-1 RNA encapsidation signal (HIVPsi). Interaction of these elements results in the activation of a reporter gene in the yeast Saccharomyces cerevisiae. Using this system, we have shown that the HIV-1 Gag protein binds specifically to a 139-nucleotide fragment of the HIVPsi signal containing four stem-loop structures. Mutations in either the Gag protein or the encapsidation signal that have been shown previously to impair this interaction reduced the activation of the reporter gene. Interestingly, the nucleocapsid portion of Gag retained the RNA binding activity but lost its specificity compared to the full-length Gag. These results demonstrate the utility of this system and suggest that a variety of genetic analyses could be performed to study Gag-encapsidation signal interactions. PMID: 9658151 [PubMed - indexed for MEDLINE] 978: J Virol 1998 Aug;72(8):6732-41 Complete protein linkage map of poliovirus P3 proteins: interaction of polymerase 3Dpol with VPg and with genetic variants of 3AB. Xiang W, Cuconati A, Hope D, Kirkegaard K, Wimmer E. Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-5222, USA. Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3Dpol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3Dpol interaction. The viral proteinase 3Cpro was not found to interact with other 3Cpro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CDpro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed. PMID: 9658121 [PubMed - indexed for MEDLINE] 979: Science 1998 Jul 10;281(5374):262-6 Structure of the Escherichia coli RNA polymerase alpha subunit amino-terminal domain. Zhang G, Darst SA. Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. The 2.5 angstrom resolution x-ray crystal structure of the Escherichia coli RNA polymerase (RNAP) alpha subunit amino-terminal domain (alphaNTD), which is necessary and sufficient to dimerize and assemble the other RNAP subunits into a transcriptionally active enzyme and contains all of the sequence elements conserved among eukaryotic alpha homologs, has been determined. The alphaNTD monomer comprises two distinct, flexibly linked domains, only one of which participates in the dimer interface. In the alphaNTD dimer, a pair of helices from one monomer interact with the cognate helices of the other to form an extensive hydrophobic core. All of the determinants for interactions with the other RNAP subunits lie on one face of the alphaNTD dimer. Sequence alignments, combined with secondary-structure predictions, support proposals that a heterodimer of the eukaryotic RNAP subunits related to Saccharomyces cerevisiae Rpb3 and Rpb11 plays the role of the alphaNTD dimer in prokaryotic RNAP. PMID: 9657722 [PubMed - indexed for MEDLINE] 980: Biochim Biophys Acta 1998 Jun 29;1385(2):271-86 Subunit structure, function and organisation of pyruvate decarboxylases from various organisms. Konig S. Institut fur Biochemie, Fachbereich Biochemie/Biotechnologie, Martin-Luther-Universitat Halle-Wittenberg, D-06099 Halle/Saale, Germany. The nature of the environment of macromolecules influences and determines the state of their overall structure and the extent of binding of specific (cofactors, substrates) or unspecific ligands. How these interactions between enzyme molecules and ligands influence their quaternary structures and, in this way, the realisation of high catalytic activity will be discussed here for the enzyme pyruvate decarboxylase from various organisms: brewer's yeast, brewer's yeast strain, recombinant wild type and site-specific mutants of Saccharomyces cerevisiae, the recombinant wild type of the bacterium Zymomonas mobilis and germinating seeds of the plant Pisum sativum from a structural point of view including both high resolution models from crystal structure analysis and low resolution models from small angle X-ray solution scattering with synchrotron radiation. Publication Types: Review Review, Tutorial PMID: 9655918 [PubMed - indexed for MEDLINE] 981: Eur J Biochem 1998 May 1;253(3):560-75 Identification of Man alpha1-3Man alpha1-2Man and Man-linked phosphate on O-mannosylated recombinant leech-derived tryptase inhibitor produced by Saccharomyces cerevisiae and determination of the solution conformation of the mannosylated polypeptide. Bergwerff AA, Stark W, Fendrich G, Knecht R, Blommers MJ, Maerki W, Kragten EA, van Oostrum J. Core Technology Area, Novartis AG, Basle, Switzerland. The production of recombinant leech-derived tryptase inhibitor (rLDTI) by two different strains of Saccharomyces cerevisiae resulted in the secretion of non-glycosylated and glycosylated rLTDI. Monosaccharide analysis and a-mannosidase treatment demonstrated that glycosylated rLDTI was exclusively alpha-mannosylated. A trypsin digest of reduced and S-carboxymethylated glycosylated rLDTI was separated on a reverse-phase HPLC column. Glycopeptides identified by a combination of matrix-assisted laser desorption mass spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed the presence of different glycoforms. It was found that Ser24, Ser33 and Ser36 were partially glycosylated with a single mannose residue, whereas Thr42 in glycosylated rLDTI from both strains was fully occupied with manno-oligosaccharides with a degree of polymerization ranging over 1-3 and 1-13 depending on the yeast strain. In phosphorylated rLDTI a single phosphate group was predominantly located at the innermost Man residue of units of mannobiose, mannotriose, mannotetraose and mannopentaose at Thr42. Oligosaccharides released by alkaline treatment were reduced by sodium borohydride and separated by high-pH anion-exchange chromatography on a CarboPac MA1 column, and analyzed by one- and two-dimensional 1H-NMR spectroscopy. Besides the major oligosaccharide Man alpha1-2Man-ol, the (for yeast protein O-glycosylation) unusual Man alpha1-3Man alpha1-2Man-ol was determined. The solution conformation of glycosylated rLDTI was investigated by two-dimensional NMR spectroscopy. Structure calculations by means of distance geometry showed that glycosylated rLDTI is compactly folded and contained small secondary structure elements. Analysis of the chemical shifts showed that amino acids Val32-Ser33, Ser36-Ser39 and Thr42 were affected by the O-mannosylation. In addition, changes in chemical shift were observed within the beta-hairpin peptide regions Val13-Ser16 and Gly18-Tyr21 attributed to direct interactions of the mannose residue at Ser36. Furthermore, the protein-linked oligosaccharides were spatially grouped in a position opposite of the canonical binding loop. PMID: 9654051 [PubMed - indexed for MEDLINE] 982: Mol Cell 1998 Jun;1(7):1051-5 A single amino acid change in the yeast retrotransposon Ty5 abolishes targeting to silent chromatin. Gai X, Voytas DF. Department of Zoology and Genetics, Iowa State University, Ames 50011, USA. Many retrotransposons and retroviruses are thought to select integration sites through interactions with specific chromosomal proteins. In yeast, the Ty5 retrotransposon integrates preferentially with regions bound by silent chromatin, namely the telomeres and the HMR and HML mating loci. A Ty5 mutant (M3) was identified with an approximately 20-fold decrease in targeted integration as measured by a plasmid-based targeting assay. Often chromosomal insertions generated by M3, none were located at the telomeres or silent mating loci. A single amino acid change at the boundary of integrase and reverse transcriptase is responsible for the mutant phenotype. We predict that this mutation lies within a targeting domain that mediates Ty5 target choice by interacting with a component of silent chromatin. PMID: 9651588 [PubMed - indexed for MEDLINE] 983: Mol Gen Genet 1998 May;258(3):215-21 Expression of the yeast BFR2 gene is regulated at the transcriptional level and through degradation of its product. Chabane S, Kepes F. Service de Biochimie et de Genetique Moleculaire, DBCM/DSV, CEA/Saclay, Gif, France. The essential Saccharomyces cerevisiae gene BFR2 has been isolated as a high-copy suppressor of the growth defects induced by Brefeldin A, a drug that disrupts the Golgi apparatus and its protein influx. Furthermore, BFR2 has been found to display genetic interactions with four mutations affecting protein transport to the Golgi apparatus. Here we show that the level of BFR2 mRNA rapidly increased over fivefold in response to cold shock, and over threefold following nutrient replenishment by dilution of cells from exhausted to fresh minimal medium. During subsequent growth, the transcript level returned to its basal values, except for a transient drop toward the end of the exponential phase. The early burst of transcription was not caused by toxic compounds in the fresh medium, or by synchrony among cells that had simultaneously entered their first cell cycle. The BFR2 gene product (Bfr2p) was synthesized following the early burst of mRNA, and was no longer produced when the mRNA was back to basal level. Bfr2p was finally degraded after growth became limited, and reached undetectable levels in exhausted medium. Under steady-state conditions of lengthened exponential phase, the intracellular level of Bfr2p remained constant. This peculiar pattern of gene expression suggests that Bfr2p is essential for mass growth or cell proliferation, whereas it is either toxic or not required during nutrient-limited growth. PMID: 9645427 [PubMed - indexed for MEDLINE] 984: Arch Virol 1998;143(5):981-96 Erratum in: Arch Virol 1998;143(10):2064 In vivo interactions among rotavirus nonstructural proteins. Gonzalez RA, Torres-Vega MA, Lopez S, Arias CF. Departamento de Genetica y Fisiologia Molecular, Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos, Mexico. The rotavirus genome encodes six nonstructural (NS) proteins, five of which (NSP1, NSP2, NSP3, NSP5, and NSP6) have been suggested to be involved in a variety of events, such as genome replication, regulation of gene expression, and gene assortment. These NS proteins have been found to be associated with replication complexes that are precursors of the viral core, however, little information is available about the intermolecular interactions that may exist among them. Using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible combinations among the rotavirus NS proteins were tested, and several interactions were observed. NSP1 interacted with the other four proteins tested; NSP3 associated with itself; and NSP5 was found to form homodimers and to interact with NSP6. Co-immunoprecipitation of proteins from rotavirus-infected cells, using hyperimmune sera monospecific for the NS proteins, showed the same interactions for NSP1 as those observed in yeast. Immunofluorescence co-localization analysis of virus-infected epithelial cells revealed that the intracellular distribution of proteins that were seen to interact in yeast had patterns of distribution that would allow such intermolecular interactions to occur. These findings should contribute to the understanding of the role these proteins play in different aspects of the virus replication cycle. PMID: 9645203 [PubMed - indexed for MEDLINE] 985: Mol Med 1998 May;4(5):299-323 Convergence and divergence of the signaling pathways for insulin and phosphoinositolglycans. Muller G, Wied S, Piossek C, Bauer A, Bauer J, Frick W. Hoechst Marion Roussel Deutschland GmbH, Frankfurt am Main, Germany. guenter.mueller@hmrag.com Phosphoinositolglycan molecules isolated from insulin-sensitive mammalian tissues have been demonstrated in numerous in vitro studies to exert partial insulin-mimetic activity on glucose and lipid metabolism in insulin-sensitive cells. However, their ill-defined structures, heterogeneous nature, and limited availability have prohibited the analysis of the underlying molecular mechanism. Phosphoinositolglycan-peptide (PIG-P) of defined and homogeneous structure prepared in large scale from the core glycan of a glycosyl-phosphatidylinositol-anchored membrane protein from Saccharomyces cerevisiae has recently been shown to stimulate glucose transport as well as a number of glucose-metabolizing enzymes and pathways to up to 90% (at 2 to 10 microns) of the maximal insulin effect in isolated rat adipocytes, cardiomyocytes, and diaphragms (G. Muller et al., 1997, Endocrinology 138: 3459-3476). Consequently, we used this PIG-P for the present study in which we compare its intracellular signaling with that of insulin. The activation of glucose transport by both PIG-P and insulin in isolated rat adipocytes and diaphragms was found to require stimulation of phosphatidylinositol (PI) 3-kinase but to be independent of functional p70S6kinase and mitogen-activated protein kinase. The increase in glycerol-3-phosphate acyltransferase activity in rat adipocytes in response to PIG-P and insulin was dependent on both PI 3-kinase and p70S6kinase. This suggest that the signaling pathways for PIG-P and insulin to glucose transport and metabolism converage at the level of PI 3-kinase. A component of the PIG-P signaling pathway located up-stream of PI 3-kinase was identified by desensitization of isolated rat adipocytes for PIG-P action by combined treatment with trypsin and NaCl under conditions that preserved cell viability and the insulin-mimetic activity of sodium vanadate but completely blunted the insulin response. Incubation of the cells with either trypsin or NaCl alone was ineffective. The desensitized adipocytes were reconstituted for stimulation of lipogenesis by PIG-P by addition of the concentrated trypsin/salt extract. The reconstituted adipocytes exhibited 65-75% of the maximal PIG-P response and similar EC50 values for PIG-P (2 to 5 microns) compared with control cells. A proteinaceous N-ethylmaleimide (NEM)-sensitive component contained in the trypsin/salt extract was demonstrated to bind in a functional manner to the adipocyte plasma membrane of desensitized adipocytes via bipolar interactions. An excess of trypsin/salt extract inhibited PIG-P action in untreated adipocytes in a competitive fashion compatible with a receptor function for PIG-P of this protein. The presence of the putative PIG-P receptor protein in detergent-insoluble complexes prepared from isolated rat adipocytes suggests that caveolae/detergent-insoluble complexes of the plasma membrane may play a role in insulin-mimetic signaling by PIG-P. Furthermore, treatment of isolated rat diaphragms and adipocytes with PIG-P as well as with other agents exerting partially insulin-mimetic activity, such as PI-specific phospholipase C (PLC) and the sulfonylurea glimepiride, triggered tyrosine phosphorylation of the caveolar marker protein caveolin, which was apparently correlated with stimulation of lipogenesis. Strikingly, in adipocytes subjected to combined trypsin/salt treatment, PIG-P, PI-specific PLC, and glimepiride failed completely to provoke insulin-mimetic effects. A working model is presented for a signaling pathway in insulin-sensitive cells used by PIG(-P) molecules which involves GPI structures, the trypsin/salt- and NEM-sensitive receptor protein for PIG-P, and additional proteins located in caveolae/detergent-insoluble complexes. PMID: 9642681 [PubMed - indexed for MEDLINE] 986: Mol Cell Biol 1998 Jul;18(7):4400-6 Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA. Hays SL, Firmenich AA, Massey P, Banerjee R, Berg P. Department of Biochemistry, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford University, Stanford, California 94305, USA. The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication. A Saccharomyces cerevisiae strain carrying the rfa1-44 allele displays a number of impaired recombination and repair phenotypes, all of which are suppressible by overexpression of RAD52. We demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placing RFA1 and RAD52 in the same genetic pathway. Furthermore, two-hybrid analysis indicates the existence of interactions between Rad52 and all three subunits of RPA. The nature of this Rad52-RPA interaction was further explored by using two different mutant alleles of rad52. Both mutations lie in the amino terminus of Rad52, a region previously defined as being responsible for its DNA binding ability (U. H. Mortenson, C. Beudixen, I. Sunjeuaric, and R. Rothstein, Proc. Natl. Acad. Sci. USA 93:10729-10734, 1996). The yeast two-hybrid system was used to monitor the protein-protein interactions of the mutant Rad52 proteins. Both of the mutant proteins are capable of self-interaction but are unable to interact with Rad51. The mutant proteins also lack the ability to interact with the large subunit of RPA, Rfa1. Interestingly, they retain their ability to interact with the medium-sized subunit, Rfa2. Given the location of the mutations in the DNA binding domain of Rad52, a model incorporating the role of DNA in the protein-protein interactions involved in the repair of DNA double-strand breaks is presented. PMID: 9632824 [PubMed - indexed for MEDLINE] 987: Mol Cell Biol 1998 Jul;18(7):3752-61 Interaction of TATA-binding protein with upstream activation factor is required for activated transcription of ribosomal DNA by RNA polymerase I in Saccharomyces cerevisiae in vivo. Steffan JS, Keys DA, Vu L, Nomura M. Department of Biological Chemistry, University of California-Irvine, Irvine, California 92697-1700, USA. Previous in vitro studies have shown that initiation of transcription of ribosomal DNA (rDNA) in the yeast Saccharomyces cerevisiae involves an interaction of upstream activation factor (UAF) with the upstream element of the promoter, forming a stable UAF-template complex; together with TATA-binding protein (TBP), UAF then recruits an essential factor, core factor (CF), to the promoter, forming a stable preinitiation complex. TBP interacts with both UAF and CF in vitro. In addition, a subunit of UAF, Rrn9p, interacts with TBP in vitro and in the two-hybrid system, suggesting the possible importance of this interaction for UAF function. Using the yeast two-hybrid system, we have identified three mutations in RRN9 that abolish the interaction of Rrn9p with TBP without affecting its interaction with Rrn10p, another subunit of UAF. Yeast cells containing any one of these individual mutations, L110S, L269P, or L274Q, did not show any growth defects. However, cells containing a combination of L110S with one of the other two mutations showed a temperature-sensitive phenotype, and this phenotype was suppressed by fusing the mutant genes to SPT15, which encodes TBP. In addition, another mutation (F186S), which disrupts both Rrn9p-TBP and Rrn9p-Rrn10p interactions in the two-hybrid system, abolished UAF function in vivo, and this mutational defect was suppressed by fusion of the mutant gene to SPT15 combined with overexpression of Rrn10p. These experiments demonstrate that the interaction of UAF with TBP, which is presumably achieved by the interaction of Rrn9p with TBP, is indeed important for high-level transcription of rDNA by RNA polymerase I in vivo. PMID: 9632758 [PubMed - indexed for MEDLINE] 988: J Cell Biol 1998 Jun 15;141(6):1371-81 Interaction between mitochondria and the actin cytoskeleton in budding yeast requires two integral mitochondrial outer membrane proteins, Mmm1p and Mdm10p. Boldogh I, Vojtov N, Karmon S, Pon LA. Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA. Transfer of mitochondria to daughter cells during yeast cell division is essential for viable progeny. The actin cytoskeleton is required for this process, potentially as a track to direct mitochondrial movement into the bud. Sedimentation assays reveal two different components required for mitochondria-actin interactions: (1) mitochondrial actin binding protein(s) (mABP), a peripheral mitochondrial outer membrane protein(s) with ATP-sensitive actin binding activity, and (2) a salt-inextractable, presumably integral, membrane protein(s) required for docking of mABP on the organelle. mABP activity is abolished by treatment of mitochondria with high salt. Addition of either the salt-extracted mitochondrial peripheral membrane proteins (SE), or a protein fraction with ATP-sensitive actin-binding activity isolated from SE, to salt-washed mitochondria restores this activity. mABP docking activity is saturable, resistant to high salt, and inhibited by pre-treatment of salt-washed mitochondria with papain. Two integral mitochondrial outer membrane proteins, Mmm1p (Burgess, S.M., M. Delannoy, and R.E. Jensen. 1994. J.Cell Biol. 126:1375-1391) and Mdm10p, (Sogo, L.F., and M.P. Yaffe. 1994. J.Cell Biol. 126:1361- 1373) are required for these actin-mitochondria interactions. Mitochondria isolated from an mmm1-1 temperature-sensitive mutant or from an mdm10 deletion mutant show no mABP activity and no mABP docking activity. Consistent with this, mitochondrial motility in vivo in mmm1-1 and mdm10Delta mutants appears to be actin independent. Depolymerization of F-actin using latrunculin-A results in loss of long-distance, linear movement and a fivefold decrease in the velocity of mitochondrial movement. Mitochondrial motility in mmm1-1 and mdm10Delta mutants is indistinguishable from that in latrunculin-A-treated wild-type cells. We propose that Mmm1p and Mdm10p are required for docking of mABP on the surface of yeast mitochondria and coupling the organelle to the actin cytoskeleton. PMID: 9628893 [PubMed - indexed for MEDLINE] 989: Mutat Res 1998 Mar;407(2):135-45 Genetic interactions between mutants of the 'error-prone' repair group of Saccharomyces cerevisiae and their effect on recombination and mutagenesis. Liefshitz B, Steinlauf R, Friedl A, Eckardt-Schupp F, Kupiec M. Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Israel. We have created an isogenic series of yeast strains that carry genetic systems to monitor different types of recombination and mutation [B. Liefshitz, A. Parket, R. Maya, M. Kupiec, The role of DNA repair genes in recombination between repeated sequences in yeast, Genetics 140 (1995) 1199-1211.]. In the present study we characterize the effect of mutations in genes of the 'error-prone' or postreplicative repair group on recombination and mutation. We show that rad5 and rad18 strains have elevated levels of spontaneous recombination, both of ectopic gene conversion and of recombination between direct repeats. The increase in recombination levels is similar in both mutants and in the rad5 rad18 double mutant, suggesting that the RAD5 and RAD18 gene products act together with respect to spontaneous recombination. In contrast, RAD5 and RAD18 play alternative roles in mutagenic repair: mutations in each of these genes elevate spontaneous forward mutation at the CAN1 locus, but when both genes are deleted, a low level of spontaneous mutagenesis is seen. The RAD5/RAD18 pathway of mutagenic repair is dependent on the REV3-encoded translesion polymerase. We analyze the interactions between the RAD5 and RAD18 gene products and other repair genes. The high recombination levels seen in rad5 and rad18 mutants is dependent on the RAD1, RAD51, RAD52, and RAD57 genes. The Srs2 helicase plays an important role in creating the recombinogenic substrate(s) processed by the RAD5 and RAD18 gene products. PMID: 9637242 [PubMed - indexed for MEDLINE] 990: Biotechnology (N Y) 1995 Dec;13(13):1474-8 Comment in: Biotechnology (N Y). 1995 Dec;13(13):1431-4. The yeast tribrid system--genetic detection of trans-phosphorylated ITAM-SH2-interactions. Osborne MA, Dalton S, Kochan JP. Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche, Inc., USA. Protein-protein interactions are often dependent on the post-translational modification of one component of a complex. To facilitate the study of these interactions in signal transduction, we have developed the yeast tribrid system, a modification of the yeast two-hybrid system. We demonstrate that the interactions are dependent upon the presence of a tyrosine kinase, an SH2 domain and a tyrosine containing substrate. Using the gamma subunit of the high-affinity IgE receptor, Fc epsilon RI, this approach has been used to isolate a novel SH2-containing family member. The mRNA encoding this novel protein is differentially expressed in rat tissues. The yeast tribrid system can be readily adapted for the characterization of novel tyrosine kinases or substrates, as well as the study of protein-protein interactions which involve other post-translational modifications. PMID: 9636306 [PubMed - indexed for MEDLINE] 991: EMBO J 1998 May 1;17(9):2494-503 TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion. Sacher M, Jiang Y, Barrowman J, Scarpa A, Burston J, Zhang L, Schieltz D, Yates JR 3rd, Abeliovich H, Ferro-Novick S. Howard Hughes Medical Institute and the Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA. We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport. To gain insight into the function of Bet3p, we added three c-myc tags to its C-terminus and immunopurified this protein from a clarified detergent extract. Here we report that Bet3p is a member of a large complex ( approximately 800 kDa) that we call TRAPP (transport protein particle). We propose that TRAPP plays a key role in the targeting and/or fusion of ER-to-Golgi transport vesicles with their acceptor compartment. The localization of Bet3p to the cis-Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis. TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins. PMID: 9564032 [PubMed - indexed for MEDLINE] 992: Biochemistry 1998 May 26;37(21):7834-43 The structure of the N-terminus of striated muscle alpha-tropomyosin in a chimeric peptide: nuclear magnetic resonance structure and circular dichroism studies. Greenfield NJ, Montelione GT, Farid RS, Hitchcock-DeGregori SE. Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635, USA. Tropomyosins (TMs) are highly conserved, coiled-coil, actin binding regulatory proteins found in most eukaryotic cells. The amino-terminal domain of 284-residue TMs is among the most conserved and functionally important regions. The first nine residues are proposed to bind to the carboxyl-terminal nine residues to form the "overlap" region between successive TMs, which bind along the actin filament. Here, the structure of the N-terminus of muscle alpha-TM, in a chimeric peptide, TMZip, has been solved using circular dichroism (CD) and two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy. Residues 1-14 of TMZip are the first 14 N-terminal residues of rabbit striated alpha-TM, and residues 15-32 of TMZip are the last 18 C-terminal residues of the yeast GCN4 transcription factor. CD measurements show that TMZip forms a two-stranded coiled-coil alpha-helix with an enthalpy of folding of -34 +/- 2 kcal/mol. In 2D1H NMR studies at 15 degrees C, pH 6.4, the peptide exhibits 123 sequential and medium range intrachain NOE cross peaks per chain, characteristic of alpha-helices extending from residue 1 to residue 29, together with 85 long-range NOE cross peaks arising from interchain interactions. The three-dimensional structure of TMZip has been determined using these data plus an additional 509 intrachain constraints per chain. The coiled-coil domain extends to the N-terminus. Amide hydrogen exchange studies, however, suggest that the TM region is less stable than the GCN4 region. The work reported here is the first atomic-resolution structure of any region of TM and it allows insight into the mechanism of the function of the highly conserved N-terminal domain. PMID: 9601044 [PubMed - indexed for MEDLINE] 993: J Cell Biol 1998 May 18;141(4):887-94 The beta subunit of the Sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation. Kalies KU, Rapoport TA, Hartmann E. Abteilung Biochemie II, Zentrum Biochemie und Molekulare Zellbiologie, Georg-August-Universitat, 37073 Gottingen, Germany. The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane. We have addressed the role of the beta subunit (Sec61beta) during cotranslational protein translocation. With a reconstituted system, we show that a Sec61 complex lacking Sec61beta is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time. The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel. However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal. Thus Sec61beta kinetically facilitates cotranslational translocation, but is not essential for it. Using chemical cross-linking we show that Sec61beta not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61beta and SPC25 and between Sec61beta and Sec61alpha depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity. PMID: 9585408 [PubMed - indexed for MEDLINE] 994: Mol Cell Biol 1998 Jun;18(6):3173-81 Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria. Nargang FE, Rapaport D, Ritzel RG, Neupert W, Lill R. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. frank.nargang@ualberta.ca TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required. PMID: 9584158 [PubMed - indexed for MEDLINE] 995: J Biol Chem 1998 May 15;273(20):12567-75 Jak2-Stat5 interactions analyzed in yeast. Barahmand-Pour F, Meinke A, Groner B, Decker T. Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria. Many cytokine receptors employ Janus protein tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats) for nuclear signaling. Here, we have established yeast strains in which an autoactivated Jak2 kinase induces tyrosine phosphorylation, dimerization, nuclear translocation, and DNA binding of a concomitantly expressed Stat5 protein. Transcriptional activity of Stat5 on a stably integrated, Stat-dependent reporter gene required the C-terminal fusion of the VP16 transactivation domain. In such yeast strains, the interaction between Jak2 and Stat5 was analyzed without interference by other mammalian proteins involved in regulating Jak-Stat signaling, and mutant versions of both proteins were analyzed for their ability to productively interact. Complexes between Jak2 and Stat5 were found to be stable under stringent co-immunoprecipitation conditions. Deletion of the Jak homology regions 2-7 (JH2-JH7) of Jak2, leaving only the kinase domain (JH1) intact, reduced the ability of the kinase to phosphorylate Stat5, whereas deletion of the JH2 domain caused an increased enzymatic activity. A site-directed R618K mutation in the Stat5 SH2 domain abolished the phosphorylation by Jak2, while deletion of the C terminus led to Stat5 hyperphosphorylation. A single phosphotyrosine-SH2 domain interaction was sufficient for the dimerization of Stat5, but such dimers bound to DNA very inefficiently. Together, our data show that yeast cells are appropriate tools for studying Jak-Stat or Stat-Stat interactions. Our mutational analysis suggests that the Stat5 SH2 domain is essential for the interaction with Jak2 and that the kinase domain of Jak2 is sufficient for Jak2-Stat5 interaction. Therefore, the Jak kinase domain may be all that is needed to cause Stat phosphorylation in situations where receptor docking is dispensable. PMID: 9575217 [PubMed - indexed for MEDLINE] 996: Nucleic Acids Res 1998 May 1;26(9):2252-3 A recombination based method to rapidly assess specificity of two-hybrid clones in yeast. Petermann R, Mossier BM, Aryee DN, Kovar H. Children's Cancer Research Institute, St. Anna Kinderspital, Kinderspitalgasse 6, A-1090 Vienna, Austria. The yeast two-hybrid system is frequently used to identify protein-protein interactions. Confirming the specificity of candidate clones requires separation and isolation of yeast plasmids, propagation in bacteria and testing combinations of DNA-binding and activation domain hybrids in yeast. In order to simplify this procedure, we developed a rapid method based on PCR amplification of library insert DNAs and in vivo cloning into the activation domain hybrid vector. Reporter gene activity is assayed in parallel for combinations with different DNA-binding domain hybrids. Further characterization of inserts does not require plasmid isolation and intermediate hosts. PMID: 9547290 [PubMed - indexed for MEDLINE] 997: Curr Opin Struct Biol 1998 Apr;8(2):177-85 Intermediate filament assembly: fibrillogenesis is driven by decisive dimer-dimer interactions. Herrmann H, Aebi U. Division of Cell Biology, German Cancer Research Center, Heidelberg, Germany. H.Herrmann@DKFZ-Heidelberg.de Intermediate filaments are built from one to several members of a multigene family encoding fibrous proteins that share a highly conserved hierarchic assembly plan for the formation of multistranded filaments from distinctly structured extended coiled coils. Despite the rather low primary sequence identity, intermediate filaments form apparently similar filaments with regard to their spatial dimensions and physical properties. Over the past few years, substantial progress has been made in the elucidation of the complex expression patterns and clinically relevant phenotypes of intermediate filaments. The key question of how these filaments assemble and what the molecular architecture of their distinct assembly intermediates comprises, however, has still not been answered to the extent that has been achieved for microfilaments and microtubules. Publication Types: Review Review, Tutorial PMID: 9631290 [PubMed - indexed for MEDLINE] 998: Nat Biotechnol 1996 Apr;14(4):481-4 Comment in: Nat Biotechnol. 1996 Apr;14(4):436. A fusion protein designed for noncovalent immobilization: stability, enzymatic activity, and use in an enzyme reactor. Stempfer G, Holl-Neugebauer B, Kopetzki E, Rudolph R. Boehringer Mannheim Therapeutics, Pennzberg, Germany. We have designed a new method for enzyme immobilization using a fusion protein of yeast alpha-glucosidase containing at its C-terminus a polycationic hexa-arginine fusion peptide. This fusion protein can be directly adsorbed from crude cell extracts on polyanionic matrices in a specific, oriented fashion. Upon noncovalent immobilization by polyionic interactions, the stability of the fusion protein is not affected by pH-, urea-, or thermal-denaturation. Furthermore, the enzymatic properties (specific activity at increasing enzyme concentration, Michaelis constant, or activation energy of the enzymatic reaction) are not influenced by this noncovalent coupling. The operational stability of the coupled enzyme under conditions of continuous substrate conversion is, however, increased significantly compared to the soluble form. Fusion proteins containing polyionic peptide sequences are proposed as versatile tools for the production of immobilized enzyme catalysts. PMID: 9630924 [PubMed - indexed for MEDLINE] 999: Nat Biotechnol 1996 Mar;14(3):329-34 Improved refolding of an immobilized fusion protein. Stempfer G, Holl-Neugebauer B, Rudolph R. Boehringer Mannheim Therapeutics, Penzberg, Germany. Fusion proteins of monomeric alpha-glucosidase from Saccharomyces cerevisiae containing N- or C-terminal hexa-arginie peptides were expressed in the cytosol of Escherichia coli in soluble form. The polycationic peptide moieties allow noncovalent binding of the denatured fusion proteins to a polyanionic solid support. Upon removal of the denaturant, refolding of the matrix-bound protein can proceed without perturbation by aggregation. However, nonspecific interactions of the denatured polypeptide, or of folding intermediates, with the matrix cause a drastic decrease in renaturation under suboptimal folding conditions. At low salt concentrations, ionic interactions of the refolding polypeptide with the matrix result in lower yields of renaturation. At higher salt concentrations, renaturation is prevented by hydrophobic interactions with the matrix. Apart from ionic strength, renaturation of the denatured matrix-bound fusion protein must be optimized with respect to pH, temperature, cosolvents, and matrix material used. Under optimum conditions, immobilized alpha-glucosidase can be renatured with a high yield at protein concentrations up to 5 mg/ml, whereas folding of the wild-type enzyme in solution is feasible only at an extremely low protein concentration (15 micrograms/ml). Thus, folding of the immobilized alpha-glucosidase allows an extremely high yield of the renaturated model protein. The technology should be applicable to other proteins that tend to aggregate during refolding. PMID: 9630895 [PubMed - indexed for MEDLINE] 1000: Biochim Biophys Acta 1998 Jun 22;1403(2):158-68 Protein-protein interactions between keratin polypeptides expressed in the yeast two-hybrid system. Schnabel J, Weber K, Hatzfeld M. Department of Biochemistry, Max-Planck-Institute for Biophysical Chemistry, D-37070 Gottingen, Germany. Keratin filaments are obligatory heteropolymers of type I and type II keratin polypeptides. Specific type I/type II pairs are coexpressed in vivo. In contrast, all type I/type II pairs assemble into filaments in vitro, but the different pairs have different stabilities as demonstrated by treatment with increasing concentrations of urea. We have used the yeast two-hybrid system to analyse type I/type II interactions in a cellular context. We measured interactions between two different keratin pairs and we confirm the findings that K6+K17 form very stable heterodimers whereas K8+K18 interactions were weaker. The deletion of head domains did not reduce the strength of type I/type II interactions. Rather, the affinities were increased and the differences between the two pairs were retained in headless mutants. These findings argue against a major role of the head domains in directing heterodimer interactions and in defining heterodimer stabilities. PMID: 9630597 [PubMed - indexed for MEDLINE]