Yeast, 2003 Jan 30, 20(2), 133 - 48 Identification of novel protein-protein interactions at the cytosolic surface of the Sec63 complex in the yeast ER membrane; Willer M et al.; Precursors of secretory proteins are targeted to the membrane of the endoplasmic reticulum by specific protein complexes that recognize their signal sequence . All eukaryotic cells investigated so far have been found to possess the signal recognition particle (SRP) that targets the majority of precursors to the translocation machinery . In Saccharomyces cerevisiae a number of proteins are translocated independently of SRP . These precursors rely on a different signal sequence-binding complex, which includes Sec62p, Sec63p, Sec71p and Sec72p . Identifying interactions between individual components of this tetrameric protein complex is important in the understanding of its function . We demonstrate a specific interaction between the only two essential proteins in this complex, Sec62p and Sec63p . Second, we show evidence of homodimerization of Sec72p molecules and further identify the YLR301w gene product as a novel in vivo interacting partner of Sec72p . Finally, we determine the authentic N-terminus of Sec62p and describe interacting subdomains of both Sec62p and Sec63p . Plant Mol Biol, 2002 Dec, 50(6), 855 - 70 Analysing protein-protein interactions with the yeast two-hybrid system; Causier B et al.; Plant research is moving into the post-genomic era . Proteomic-based strategies are now being developed to study functional aspects of the genes predicted from the various genome-sequencing initiatives . All biological processes depend on interactions formed between proteins and the mapping of such interactions on a global scale is providing interesting functional insights . One of the techniques that has proved itself invaluable in the mapping of protein-protein interactions is the yeast two-hybrid system . This system is a sensitive molecular genetic approach for studying protein-protein interactions in vivo . In this review we will introduce the yeast two-hybrid system, discuss modifications of the system that may be of interest to the plant science community and suggest potential applications of the technology. Biosci Rep, 2002 Jun-Aug, 22(3-4), 373 - 94 Nobel Lecture . Yeast and cancer; Hartwell LH; The discovery of genes that control cell division in yeast, and their relation to cancer, is reviewed. Exp Mol Med, 2002 Sep 30, 34(4), 285 - 93 Intracellular trafficking and metabolic turnover of yeast prepro-alpha-factor-SRIF precursors in GH3 cells; Lee MA et al.; Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells . We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi . These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells . A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997) . This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation . Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC . When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that approximately 68% of initially synthesized propeptides were secreted constitutively . About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of approximately 2 h . In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles . All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells. Genes Dev, 2003 Jan 1, 17(1), 77 - 87 Yeast DNA polymerase zeta (zeta) is essential for error-free replication past thymine glycol; Johnson RE et al.; DNA polymerase zeta (Polzeta) promotes the mutagenic bypass of DNA lesions in eukaryotes . Genetic studies in Saccharomyces cerevisiae have indicated that relative to the contribution of other pathways, Polzeta makes only a modest contribution to lesion bypass . Intriguingly, however, disruption of the REV3 gene, which encodes the catalytic subunit of Polzeta, causes early embryonic lethality in mice . Here, we present genetic and biochemical evidence for the requirement of yeast Polzeta for predominantly error-free replication past thymine glycol (Tg), a DNA lesion formed frequently by free radical attack . These results raise the possibility that, as in yeast, in higher eukaryotes also, Polzeta makes a major contribution to the replicative bypass of Tgs as well as other lesions that block synthesis by replicative DNA polymerases . Such a preeminent role of Polzeta in lesion bypass would ensure that rapid cell divisions continue unabated during early embryonic development, thereby minimizing the generation of DNA strand breaks, chromosome aberrations, and the ensuing apoptotic response. Chembiochem, 2003 Jan 3, 4(1), 101 - 7 Analysis of protein tyrosine kinase inhibitors in recombinant yeast lacking the ERG6 gene; Clark DD et al.; Studies of small-molecule-protein interactions in yeast can be hindered by the limited permeability of yeast to small molecules . This diminished permeability is thought to be related to the unique sterol composition of fungal membranes, which are enriched in the steroid ergosterol . We report the construction of the novel Saccharomyces cerevisiae yeast strain DCY250, which is compatible with yeast two-hybrid-based systems and bears a targeted disruption of the ERG6 gene to ablate ergosterol biosynthesis and enhance permeability to small molecules . The small-molecule inhibitors of protein tyrosine kinases (PTKs) PP1, PP2, herbimycin A, and staurosporine were investigated with yeast tribrid systems that detect the activity of the PTKs v-Abl and v-Src . These tribrid systems function by expression of the PTK, a B42 activation domain fused to the phosphotyrosine-binding Grb2 SH2 domain, a DNA-bound LexA-GFP-(AAYANAA)(4) universal PTK substrate, and a lacZ reporter gene . Yeast genetic systems that lack functional ERG6 were found to be as much as 20-fold more sensitive to small-molecule inhibitors of PTKs than systems with ERG6, and these deficient systems may provide a useful platform for the discovery and analysis of small-molecule-protein interactions. J Biol Chem, 2003 Mar 14, 278(11), 9671 - 7 Epub 2003 Jan 02. Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast; Hermand D et al.; Here we report functional characterization of the essential fission yeast Skp1 homologue . We have created a conditional allele of skp1 (skp1-3f) mimicking the mutation in the budding yeast skp1-3 allele . Although budding yeast skp1-3 arrests at the G(1)/S transition, skp1-3f cells progress through S phase and instead display two distinct phenotypes . A fraction of the skp1-3f cells arrest in mitosis with high Cdc2 activity . Other skp1-3f cells as well as the skp1-deleted cells accumulate abnormal thick septa leading to defects in cell separation . Subsequent identification of 16 fission yeast F-box proteins led to identification of the product of pof6 (for pombe F-box) as a Skp1-associated protein . Interestingly, cells deleted for the essential pof6 gene display a similar cell separation defect noted in skp1 mutants, and Pof6 localizes to septa and cell tips . Purification of Pof6 demonstrates association of Skp1, whereas the Pcu1 cullin was absent from the complex . These findings reveal an essential non-Skp1-Cdc53/Cullin-F-box protein function for the fission yeast Skp1 homologue and the F-box protein Pof6 in cell separation. Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 193 - 8 Epub 2002 Dec 30. RNA interference machinery regulates chromosome dynamics during mitosis and meiosis in fission yeast; Hall IM et al.; The regulation of higher-order chromosome structure is central to cell division and sexual reproduction . Heterochromatin assembly at the centromeres facilitates both kinetochore formation and sister chromatid cohesion, and the formation of specialized chromatin structures at telomeres serves to maintain the length of telomeric repeats, to suppress recombination, and to aid in formation of a bouquet-like structure that facilitates homologous chromosome pairing during meiosis . In fission yeast, genes encoding the Argonaute, Dicer, and RNA-dependent RNA polymerase factors involved in RNA interference (RNAi) are required for heterochromatin formation at the centromeres and mating type region . In this study, we examine the effects of deletions of the fission yeast RNAi machinery on chromosome dynamics during mitosis and meiosis . We find that the RNAi machinery is required for the accurate segregation of chromosomes . Defects in mitotic chromosome segregation are correlated with loss of cohesin at centromeres . Although the telomeres of RNAi mutants maintain silencing, length, and localization of the heterochromatin protein Swi6, we discovered defects in the proper clustering of telomeres in interphase mitotic cells . Furthermore, a small proportion of RNAi mutant cells display aberrant telomere clustering during meiotic prophase . This study demonstrates that the fission yeast RNAi machinery is required for the proper regulation of chromosome architecture during mitosis and meiosis. DNA Repair (Amst), 2002 Sep 4, 1(9), 763 - 70 Examining the potential role of DNA polymerases eta and zeta in triplet repeat instability in yeast; Dixon MJ et al.; Triplet repeats undergo frequent mutations in human families afflicted with certain neurodegenerative diseases and also in model organisms . Although the molecular mechanisms of triplet repeat instability are still being identified, it is likely that aberrant DNA synthesis plays an important role . Many DNA polymerases stall at triplet repeat sequences, probably due to the adoption of unusual DNA secondary structures . One possible mechanism to explain triplet repeat contractions is that a triplet repeat hairpin on the template strand inhibits replicative polymerases and that one or more bypass polymerases are recruited for synthesis past the hairpin . If the translesion synthesis is mutagenic, contractions can be generated . To address this possibility, Saccharomyces cerevisiae strains lacking either pol zeta (rev7), pol eta (rad30), or both were tested for trinucleotide repeat (TNR) contractions using three separate, sensitive genetic assays . If these bypass polymerases are important for mutagenesis, then the mutants should show a reduction in the contraction rate . Two genetic tests for triplet repeat contractions showed no significant change for the mutants compared to wild type . A third assay showed a five-fold reduction in contraction rates due to pol eta ablation . Despite this modest decrease, the overall contraction rate was still high, indicating that many deletions still occur in the absence of both polymerases . Expansion rates were also unaffected in the mutant strains . These results indicate that, in yeast, pol eta and pol zeta most likely have little role in triplet repeat mutagenesis. DNA Repair (Amst), 2002 Sep 4, 1(9), 743 - 53 Evidence for sequential action of two ATPase active sites in yeast Msh2-Msh6; Drotschmann K et al.; Bacterial MutS homodimers contain two ATPase active sites that have non-equivalent functions in DNA mismatch repair . The homologous Msh2-Msh6 complex in eukaryotes also has intrinsic ATPase activity that is essential for mismatch repair . Here, we investigate differences in the two putative ATPase active sites by examining the properties of heterodimers containing alanine substituted for an invariant glutamic acid in the active site of either Msh2, Msh6 or both . Mutation rates in wild type versus Glu-->Ala mutant haploid yeast strains indicate that both ATPase active sites are essential for mismatch repair activity in vivo . The properties of purified heterodimers suggest that the ATPase active site in Msh6 binds ATP with higher affinity and hydrolyzes ATP faster and with higher efficiency than does the ATPase active site in Msh2 . This suggests sequential action of the two ATPase active sites, in which ATP binds to Msh6 first to trigger downstream events in mismatch repair. J Environ Monit, 2002 Dec, 4(6), 1040 - 6 Determination of estrogenic activity in landfill leachate by simplified yeast two-hybrid assay; Kawagoshi Y et al.; We previously developed a simplified yeast two-hybrid assay of estrogenic activity . In the present study, the optimal conditions for sensitivity and stability of this assay were decided . The assay could determine the estrogenic activity of 4 x 10(-11) mol dm(-1) 17beta-estradiol and also anti-estrogenic activity by using 4-hydroxytamoxifen as a control . The assay was used to test various chemicals suspected of estrogenicity . Many kinds of chemical, including alkylphenols, naphthols, xylenols, methoxychlor, phthalates, and bisphenol-A, showed estrogenic activity, and several, such as 2,5-xylenol and di-iso-octyl phthalate, showed anti-estrogenic but no estrogenic activity . Copresence of two estrogenic chemicals produced additive activity . The assay was also used to test leachate and water samples from the sea-based landfill of Osaka North Port in Japan . Estrogenic activity was detected in leachate from a municipal waste-dumping site, but not in that from a dredged soil-dumping site, which did however cause inhibition of cell growth. Biochem Biophys Res Commun, 2003 Jan 17, 300(3), 679 - 85 Mammalian and yeast 14-3-3 isoforms form distinct patterns of dimers in vivo; Chaudhri M et al.; The 14-3-3 protein family associates with many proteins involved in intracellular signalling . In many cases, there is a distinct preference for a particular isoform(s) of 14-3-3 . A specific repertoire of 14-3-3 dimer formation may therefore influence which of the interacting proteins could be brought together . We have analysed the pattern of dimer formation for two of the most abundant isoforms of 14-3-3, epsilon ( epsilon ) and gamma (gamma), following their stable expression . This revealed a distinct preference for particular dimer combinations that is largely independent of cellular conditions . gamma 14-3-3 occurred as homodimers and also formed heterodimers, mainly with epsilon 14-3-3 (In PC12 and Cos cells) . The epsilon isoform formed heterodimers with 14-3-3 beta, gamma, zeta, and eta, but no homodimers were detected . The two 14-3-3 homologues, BMH1 and BMH2 from Saccharomyces cerevisiae, were mainly heterodimers. Biosci Biotechnol Biochem, 2002 Nov, 66(11), 2515 - 9 Purification and characterization of beta-1,6-glucanase of Streptomyces rochei application in the study of yeast cell wall proteins; Wu H et al.; A beta-1,6-glucanase was purified to apparent homogeneity from a commercial yeast digestive enzyme prepared from Streptomyces rochei by a series of column chromatographies . The molecular mass of the purified enzyme was 60 kDa by SDS-PAGE . The purified enzyme had an optimum pH range from 4.0 to 6.0 and was stable in the same pH range . The enzyme was stable under 50 degrees C but lost almost all activity at 60 degrees C . The enzyme was specific to beta-1,6-glucan and had little activity towards beta-1,3-glucan and beta-1,4-glucan . When the beta-1,6-glucan was hydrolyzed with the purified enzyme for 5 h, the reaction products contained 20% glucose, 36% gentiobiose, and 44% other oligosaccharides, suggesting that the enzyme is an endo-type glucanase . When the purified enzyme was used for the digestion of the cell wall of Saccharomyces cerevisiae, cell-wall proteins covalently bound to the cell-wall glucan were recovered as soluble forms, suggesting that this enzyme is useful for analysis of yeast-cell wall proteins. Tsitologiia, 2002, 44(8), 788 - 91 {Effect of melanins from black yeast fungi on cultured human cells . II . Differentiation of keratinocytes in vitro}; Blinova MI et al.; Data on the influence of the black yeast melanin (3 samples) on the in vitro differentiation of human keratinocytes are presented . The effect of melanins was estimated by the morphological state of keratinocytes using electron microscopy . The obtained differences in the state of the formed multilayer keratinocyte sheets depended on the melanin sample. Tsitologiia, 2002, 44(8), 780 - 7 {Effect of melanins from black yeast fungi on cultured human cells . I . Proliferation of keratinocytes and fibroblasts}; Blinova MI et al.; Results of screening of the influence exerted by yeast black melanin on the proliferation of human skin keratinocytes and embryonic fibroblasts are presented . The optimal concentration of the investigated melanins was found to be within 0.005 and 0.0001 mg/ml . 17 samples of DHN-melanin from black yeast and 2 commercial samples of {symbol: see text}OPA-melanin (natural and synthetic) were investigated . It was established that keratinocyte proliferation was inhibited by 3 black yeast melanin samples; the influence of other 14 samples was the same as in the control . Keratinocyte proliferation was stimulated only by a commercial sample of natural {symbol: see text}OPA-melanin at concentration 0.005 mg/ml . The synthetic melanin at concentrations 0.005 and 0.001 mg/ml inhibited keratinocyte proliferation . Of the 17 investigated black yeast melanin samples, only one sample stimulated fibroblast proliferation at concentration 0.005 mg/ml . Three other samples inhibited the proliferation; of these one sample did it at all used concentrations, and two samples at concentration 0.0001 mg/ml . The rest 13 samples of black yeast DHN-melanins and the synthetic {symbol: see text}OPA-melanin did not differ in either action from the control. J Immunol Methods, 2003 Jan 15, 272(1-2), 235 - 46 A yeast display system for engineering functional peptide-MHC complexes; Brophy SE et al.; In a cellular immune response, antigenic peptides derived by intracellular processing of foreign pathogens are bound to the class I major histocompatability complex (MHC I) and presented to CD8(+) cytotoxic T cells . Although the crystal structures of several different MHC products have been solved, many MHC molecules, including some associated with diseases, have not been amenable to biochemical and structural studies . The variability in this success is based largely on the fact that peptide-MHC complexes vary extensively in their stability . These properties also are intimately tied to the biological activity of the complexes . The ability to apply the techniques of directed evolution to this system in order to engineer stable complexes has been complicated by the trimeric structure of peptide-MHC complexes, requiring association of three polypeptides: the heavy chain, beta2-microglubulin (beta2m), and a short peptide . We show here that single-chain forms of peptide-MHC complexes can be expressed as Aga-2 fusions on the surface of yeast . Three different complexes, SIYRYYGL-K(b)-beta2m (SIYR-K(b)), EQYKFYSV-K(b)-beta2m (dEV8-K(b)), and SIINFEKL-K(b)-beta2m (OVA-K(b)), were expressed on yeast and detected by flow cytometry with a conformation-specific anti-K(b) antibody (B.8.24.3) . In addition, yeast displaying K(b) loaded with exogenous SIYR and OVA peptides were recognized by a high-affinity T cell receptor that is specific for SIYR-K(b) and by an antibody (25.D1-16) that is specific for OVA-K(b), respectively . Finally, yeast that display the SIYRYYGL-K(b) also directly stimulated CD69 up-regulation on naive 2C T cells . Hence, yeast display represents a technology that can be used for directed evolution of any of the components of the trimeric pep-MHC complex. Arch Biochem Biophys, 2003 Jan 15, 409(2), 341 - 8 N-Terminal modifications of the 19S regulatory particle subunits of the yeast proteasome; Kimura Y et al.; The yeast (Saccharomyces cerevisiae) contains three N-acetyltransferases, NatA, NatB, and NatC, each of which acetylates proteins with different N-terminal regions . The 19S regulatory particle of the yeast 26S proteasome consists of 17 subunits, 12 of which are N-terminally modified . By using nat1, nat3, and mak3 deletion mutants, we found that 8 subunits, Rpt4, Rpt5, Rpt6, Rpn2, Rpn3, Rpn5, Rpn6, and Rpn8, were NatA substrates, and that 2 subunits, Rpt3 and Rpn11, were NatB substrates . Mass spectrometric analysis revealed that the initiator Met of Rpt2 precursor polypeptide was processed and a part of the mature Rpt2 was N-myristoylated . The crude extracts from the normal strain and the nat1 deletion mutant were similar in chymotrypsin-like activity in the presence of ATP in vitro and in the accumulation level of the 26S proteasome . These characteristics were different from those of the 20S proteasome: the chymotrypsin-like activity and accumulation level of 20S proteasome were appreciably higher from the nat1 deletion mutant than from the normal strain. Mol Cell, 2002 Dec, 10(6), 1319 - 30 Asymmetric sorting of ash1p in yeast results from inhibition of translation by localization elements in the mRNA; Chartrand P et al.; ASH1 mRNA localizes at the bud tip of late-anaphase yeast, resulting in accumulation of Ash1p in the daughter nucleus . We show that disruption of the secondary structure, but not the protein coding, of all four ASH1 localization elements resulted in RNA and protein delocalization . Localization of both was incrementally restored by replacement of each of the four elements . However, transposition of the elements to the 3'UTR reinstated the RNA, but not the protein, localization . Interestingly, the mutant ASH1 mRNA was translated more efficiently, suggesting that asymmetry of Ash1p resulted from translational inhibition by the localization elements . In support of this, Ash1p asymmetry could be rescued by slowing its translation. J Recept Signal Transduct Res, 2002 Feb-Nov, 22(1-4), 471 - 81 Genetic approaches to the identification of interactions between membrane proteins in yeast; Auerbach D et al.; The recent sequencing of entire eukaryotic genomes has renewed the interest in identifying and characterizing all gene products that are expressed in a given organism . The characterization of unknown gene products is facilitated by the knowledge of its binding partners . Thus, a novel protein may be classified by identifying previously characterized proteins that interact with it . If such an approach is carried out on a large scale, it may allow the rapid characterization of the thousands of predicted open reading frames identified by recent sequencing projects . Currently, the yeast two-hybrid system is the most widely used genetic assay for the detection of protein-protein interactions . The yeast two-hybrid system has become popular because it requires little individual optimization and because, as compared to conventional biochemical methods, the identification and characterization of protein-protein interactions can be completed in a relatively short time span . In this review, we briefly discuss the yeast two-hybrid system and its application to large scale screening studies that aim at deciphering all protein-protein interactions taking place in a given cell type or organism . We then focus on a class of proteins that is unsuitable for conventional yeast two-hybrid systems, namely integral membrane proteins and membrane-associated proteins, and describe several novel genetic systems that combine the advantages of the yeast two-hybrid system with the potential to identify interaction partners of membrane-associated proteins in their natural setting. Genes Dev, 2002 Dec 15, 16(24), 3158 - 72 Receptor-mediated endoproteolytic activation of two transcription factors in yeast; Andreasson C et al.; Yeast possess a plasma membrane sensor of external amino acids that functions as a ligand-activated receptor . This multimeric sensor, dubbed the SPS sensor, initiates signals that regulate the expression of genes required for proper amino acid uptake . Stp1p and Stp2p are transcription factors that bind to specific sequences within the promoters of SPS-sensor-regulated genes . These factors exhibit redundant and overlapping abilities to activate transcription . We have found that Stp1p and Stp2p are synthesized as latent cytoplasmic precursors . In response to extracellular amino acids, the SPS sensor induces the rapid endoproteolytic processing of Stp1p and Stp2p . The processing of Stp1p/Stp2p occurs independently of proteasome function and without the apparent involvement of additional components . The shorter forms of these transcription factors, lacking N-terminal inhibitory domains, are targeted to the nucleus, where they transactivate SPS-sensor target genes . These results define a completely unique and streamline metabolic control pathway that directly routes environmental signals initiated at the plasma membrane to transcriptional activation in the nucleus of yeast. Ceska Slov Farm, 2002 Nov, 51(6), 297 - 300 {Anti-yeast activity of ethanol extracts of Lilium candidum L.}; Mucaji P et al.; The paper deals with anti-yeast activity of ethanolic extracts from the flowers and bulbs of Lilium candidum L., Liliaceae, as well as some compounds isolated from these extracts . Several different methods were used for the determination of anti-yeast activity: Lowry method of protein determination, dilution and cultivation method . The extract from the bulbs was shown to be more active than the extract from the flowers, while isolated compounds were inactive against the tested yeasts. Mol Biol (Mosk), 2002 Nov-Dec, 36(6), 1026 - 34 {The context organization of functional regions in yeast genes with high-level expression}; Kochetov AV et al.; With the example of yeast genes, context organization was compared for functional gene regions (promoter, 5'-UTR, 3'-UTR) and tested for association with the level of gene expression . Several parameters (nucleotide composition, dinucletoide content bias) proved to correlate with expression level, each functional region having its specific features . Context optimization of a functional region was assumed to be essential for highly efficient interaction with the expression system of the cell . Specific context features were considered as dispersed signals important for high-level gene expression. Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 127 - 35 Epub 2002 Dec 19. Structure of a yeast hypothetical protein selected by a structural genomics approach; Eswaramoorthy S et al.; Yeast hypothetical protein YBL036C (SWISS-PROT P38197), initially thought to be a member of an 11-protein family, was selected for crystal structure determination since no structural or functional information was available . The structure has been determined independently by MIR and MAD methods to 2.0 A resolution . The MAD structure was determined largely through automated model building . The protein folds as a TIM barrel beginning with a long N-terminal helix, in contrast to the classic triose phosphate isomerase (TIM) structure, which begins with a beta-strand . A cofactor, pyridoxal 5'-phosphate, is covalently bound near the C-terminal end of the barrel, the usual active site in TIM-barrel folds . A single-domain monomeric molecule, this yeast protein resembles the N-terminal domain of alanine racemase or ornithine decarboxylase, both of which are two-domain dimeric proteins . The yeast protein has been shown to have amino-acid racemase activity . Although selected as a member of a protein family having no obvious relationship to proteins of known structure, the protein fold turned out to be a well known and widely distributed fold . This points to the need for a more comprehensive base of structural information and better structure-modeling tools before the goal of structure prediction from amino-acid sequences can be realised . In this case, similarity to a known structure allowed inferences to be made about the structure and function of a widely distributed protein family. J Cell Biol, 2002 Dec 23, 159(6), 971 - 82 Epub 2002 Dec 23. Ribonucleoprotein-dependent localization of the yeast class V myosin Myo4p; Kruse C et al.; Class V myosins are motor proteins with functions in vesicle transport, organelle segregation, and RNA localization . Although they have been extensively studied, only little is known about the regulation of their spatial distribution . Here we demonstrate that a GFP fusion protein of the budding yeast class V myosin Myo4p accumulates at the bud cortex and is a component of highly dynamic cortical particles . Bud-specific enrichment depends on Myo4p's association with its cargo, a ribonucleoprotein complex containing the RNA-binding protein She2p . Cortical accumulation of Myo4p at the bud tip can be explained by a transient retention mechanism that requires SHE2 and, apparently, localized mRNAs bound to She2p . A mutant She2 protein that is unable to recognize its cognate target mRNA, ASH1, fails to localize Myo4p . Mutant She2p accumulates inside the nucleus, indicating that She2p shuttles between the nucleus and cytoplasm and is exported in an RNA-dependent manner . Consistently, inhibition of nuclear mRNA export results in nuclear accumulation of She2p and cytoplasmic Myo4p mislocalization . Loss of She2p can be complemented by direct targeting of a heterologous lacZ mRNA to a complex of Myo4p and its associated adaptor She3p, suggesting that She2p's function in Myo4p targeting is to link an mRNA to the motor complex. Curr Biol, 2002 Dec 23, 12(24), 2076 - 89 Live imaging of telomeres: yKu and Sir proteins define redundant telomere-anchoring pathways in yeast; Hediger F et al.; BACKGROUND: The positioning of chromosomal domains within interphase nuclei is thought to facilitate transcriptional repression in yeast . Although this is particularly well characterized for telomeres, the molecular basis of their specific subnuclear organization is poorly understood . The use of live fluorescence imaging overcomes limitations of in situ staining on fixed cells and permits the analysis of chromatin dynamics in relation to stages of the cell cycle . RESULTS: We have characterized the dynamics of yeast telomeres and their associated domains of silent chromatin by using rapid time-lapse microscopy . In interphase, native telomeres are highly dynamic but remain within a restricted volume adjacent to the nuclear envelope . This constraint is lost during mitosis . A quantitative analysis of selected mutants shows that the yKu complex is necessary for anchoring some telomeres at the nuclear envelope (NE), whereas the myosin-like proteins Mlp1 and Mlp2 are not . We are able to correlate increased telomeric repression with increased anchoring and show that silent chromatin is tethered to the NE in a Sir-dependent manner in the absence of the yKu complex . Sir-mediated anchoring is S phase specific, while the yKu-mediated pathway functions throughout interphase . Subtelomeric elements of yeast telomere structure influence the relative importance of the yKu- and Sir-dependent mechanisms . CONCLUSIONS: Interphase positioning of telomeres can be achieved through two partially redundant mechanisms . One requires the heterodimeric yKu complex, but not Mlp1 and Mlp2 . The second requires Silent information regulators, correlates with transcriptional repression, and is specific to S phase. J Biol Chem, 2003 Mar 14, 278(11), 9938 - 43 Epub 2002 Dec 18. Nep98p is a component of the yeast spindle pole body and essential for nuclear division and fusion; Nishikawa S et al.; During the mating of yeast Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus . This process requires the functions of BiP/Kar2p, a member of the Hsp70 family in the endoplasmic reticulum, and its partner protein, Jem1p . To investigate further the role of BiP and Jem1p in nuclear fusion, we screened for partner proteins for Jem1p by the yeast two-hybrid system and identified Nep98p . Nep98p is an essential integral membrane protein of the nuclear envelope and is enriched in the spindle pole body (SPB), the sole microtubule-organizing center in yeast . Temperature-sensitive nep98 mutant cells contain abnormal SPBs lacking the half-bridge, suggesting the essential role of Nep98p in the organization of the normal SPB . Additionally, nep98 mutant cells show defects in mitotic nuclear division and nuclear fusion during mating . Because Jem1p is not required for nuclear division, Nep98p probably has dual functions in Jem1p-dependent karyogamy and in Jem1p-independent nuclear division. J Biol Chem, 2003 Feb 28, 278(9), 6985 - 91 Epub 2002 Dec 18. Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3; Anand M et al.; The translation elongation machinery in fungi differs from other eukaryotes in its dependence upon eukaryotic elongation factor 3 (eEF3) . eEF3 is essential in vivo and required for each cycle of the translation elongation process in vitro . Models predict eEF3 affects the delivery of cognate aminoacyl-tRNA, a function performed by eEF1A, by removing deacylated tRNA from the ribosomal Exit site . To dissect eEF3 function and its link to the A-site activities of eEF1A, we have identified a temperature-sensitive allele of the YEF3 gene . The F650S substitution, located between the two ATP binding cassettes, reduces both ribosome-dependent and intrinsic ATPase activities . In vivo this mutation increases sensitivity to aminoglycosidic drugs, causes a 50% reduction of total protein synthesis at permissive temperatures, slows run-off of polyribosomes, and reduces binding to eEF1A . Reciprocally, excess eEF3 confers synthetic slow growth, increased drug sensitivity, and reduced translation in an allele specific fashion with an E122K mutation in the GTP binding domain of eEF1A . In addition, this mutant form of eEF1A shows reduced binding of eEF3 . Thus, optimal in vivo interactions between eEF3 and eEF1A are critical for protein synthesis. J Nutr Sci Vitaminol (Tokyo), 2002 Aug, 48(4), 298 - 304 Comparison of ingestive effects of brewer's yeast, casein, and soy protein on bioavailability of dietary iron; Tuyet Mai TT et al.; The effects of brewer's yeast, casein, and soy protein intakes on the absorption and retention as well as the incorporation into hemoglobin and systemic iron stores of dietary iron were examined in an animal experiment with growing rats . Relative biological values (RBV) of iron in the rats fed casein (C), soy protein (SP), and yeast (Y) diets were 1.00, 0.31, and 1.77, respectively . The apparent absorption of iron in Y-diet-fed rats was significantly higher than that in C- or SP-diet-fed rats . The hemoglobin regeneration efficiency (HRE) of iron in Y group was significantly higher than those in C and SP groups . As a result of search for iron-absorptive enhancers (IAE) in yeast, RBV and HRE of the yeast-cell-wall-including diet turned out to be significantly higher than those of its lacking diet . These results suggest that IAE occurring in the yeast cell wall may be effective for iron absorption. Yeast, 2003 Jan 15, 20(1), 25 - 30 Real-time imaging of the surface topography of living yeast cells by atomic force microscopy; Ahimou F et al.; Atomic force microscopy (AFM) was used to image the surface topography of living Saccharomyces cerevisiae cells at high resolution and to monitor enzyme digestion of the cell wall in real time . Apart from the presence of bud scars, the surface of native cells imaged in aqueous solution was homogeneous and smooth . Topographic images of the surface were recorded to a lateral resolution of 2 nm without significant modification of the surface morphology . Successive images of single cells were collected at fixed time intervals following addition of protease and amyloglucosidase solutions . Protease caused a progressive increase of surface roughness . Large depressions surrounded by protruding edges, approximately 50 nm in height, were formed and attributed to the erosion of the mannoprotein outer layer . By contrast, no modification of the cell surface was noted upon addition of amyloglucosidase, which was consistent with the cell wall biochemical composition . These results indicate that AFM is a complementary tool to electron microscopy in that it allows the surface of living cells to be explored directly in real time . J Med Food, 2002 Summer, 5(2), 85 - 90 Growth characteristics and selenium status changes of yeast cells with inorganic and organic selenium supplementation: selenium, a chemopreventive agent; Bansal MP et al.; We attempted to determine the level and form of selenium (Se) that yielded the maximum Se status of yeast cells, for their evaluation as a source of Se for chemopreventive action . The influence of various Se concentrations from organic (selenomethionine) and inorganic (sodium selenite) Se compounds on growth pattern and cell viability and the alterations in the antioxidant enzyme system of yeast were evaluated . A continuous decrease in cell and colony-forming units counts was observed with increasing concentrations of Se from either source . Increasing Se status of yeast cells was found with increasing concentrations of Se with both forms, with much greater uptake for organic Se at maximum Se concentrations . A continuous increase in glutathione peroxidase (GSH-Px) activity with increasing Se concentrations in both forms revealed an active Se response in terms of antioxidant activity, with a more pronounced percentage increase with selenomethionine . A highly significant increase in total glutathione was observed with selenomethionine supplementation, compared with sodium selenite . A decreasing trend in reduced glutathione was observed with increasing organic or inorganic Se concentrations . An increasing trend in glutathione-S-transferase activity was observed with increasing Se concentrations for both forms . Significantly higher values of glutathione-S-transferase were associated with the organic form at higher Se concentrations . There was normal activity of Se in mammalian cells . The results showed that an organic Se source more greatly enhances the Se status of yeast cells and hence could help in chemoprevention if consumed by the population. J Biol Chem, 2003 Feb 21, 278(8), 6337 - 45 Epub 2002 Dec 16. A short regulatory domain restricts glycerol transport through yeast Fps1p; Tamas MJ et al.; The controlled export of solutes is crucial for cellular adaptation to hypotonic conditions . In the yeast Saccharomyces cerevisiae glycerol export is mediated by Fps1p, a member of the major intrinsic protein (MIP) family of channel proteins . Here we describe a short regulatory domain that restricts glycerol transport through Fps1p . This domain is required for retention of cellular glycerol under hypertonic stress and hence acquisition of osmotolerance . It is located in the N-terminal cytoplasmic extension close to the first transmembrane domain . Several residues within that domain and its precise position are critical for channel control while the proximal residues 13-215 of the N-terminal extension are not required . The sequence of the regulatory domain and its position are perfectly conserved in orthologs from other yeast species . The regulatory domain has an amphiphilic character, and structural predictions indicate that it could fold back into the membrane bilayer . Remarkably, this domain has structural similarity to the channel forming loops B and E of Fps1p and other glycerol facilitators . Intragenic second-site suppressor mutations of the sensitivity to high osmolarity conferred by truncation of the regulatory domain caused diminished glycerol transport, confirming that elevated channel activity is the cause of the osmosensitive phenotype. J Cell Biol, 2002 Dec 23, 159(6), 945 - 56 Epub 2002 Dec 16. Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p; Jaspersen SL et al.; Accurate duplication of the Saccharomyces cerevisiae spindle pole body (SPB) is required for formation of a bipolar mitotic spindle . We identified mutants in SPB assembly by screening a temperature-sensitive collection of yeast for defects in SPB incorporation of a fluorescently marked integral SPB component, Spc42p . One SPB assembly mutant contained a mutation in a previously uncharacterized open reading frame that we call MPS3 (for monopolar spindle) . mps3-1 mutants arrest in mitosis with monopolar spindles at the nonpermissive temperature, suggesting a defect in SPB duplication . Execution point experiments revealed that MPS3 function is required for the first step of SPB duplication in G1 . Like cells containing mutations in two other genes required for this step of SPB duplication (CDC31 and KAR1), mps3-1 mutants arrest with a single unduplicated SPB that lacks an associated half-bridge . MPS3 encodes an essential integral membrane protein that localizes to the SPB half-bridge . Genetic interactions between MPS3 and CDC31 and binding of Cdc31p to Mps3p in vitro, as well as the fact that Cdc31p localization to the SPB is partially dependent on Mps3p function, suggest that one function for Mps3p during SPB duplication is to recruit Cdc31p, the yeast centrin homologue, to the half-bridge. J Cell Biol, 2002 Dec 23, 159(6), 1039 - 49 Epub 2002 Dec 16. Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes; Sciorra VA et al.; Phospholipase D (PLD) generates lipid signals that coordinate membrane trafficking with cellular signaling . PLD activity in vitro and in vivo is dependent on phosphoinositides with a vicinal 4,5-phosphate pair . Yeast and mammalian PLDs contain an NH2-terminal pleckstrin homology (PH) domain that has been speculated to specify both subcellular localization and regulation of PLD activity through interaction with phosphatidylinositol 4,5-bisphosphate (PI{4,5}P2) . We report that mutation of the PH domains of yeast and mammalian PLD enzymes generates catalytically active PI(4,5)P2-regulated enzymes with impaired biological functions . Disruption of the PH domain of mammalian PLD2 results in relocalization of the protein from the PI(4,5)P2-containing plasma membrane to endosomes . As a result of this mislocalization, mutations within the PH domain render the protein unresponsive to activation in vivo . Furthermore, the integrity of the PH domain is vital for yeast PLD function in both meiosis and secretion . Binding of PLD2 to model membranes is enhanced by acidic phospholipids . Studies with PLD2-derived peptides suggest that this binding involves a previously identified polybasic motif that mediates activation of the enzyme by PI(4,5)P2 . By comparison, the PLD2 PH domain binds PI(4,5)P2 with lower affinity but sufficient selectivity to function in concert with the polybasic motif to target the protein to PI(4,5)P2-rich membranes . Phosphoinositides therefore have a dual role in PLD regulation: membrane targeting mediated by the PH domain and stimulation of catalysis mediated by the polybasic motif. J Gen Appl Microbiol, 2000 Jun, 46(3), 113 - 117 Molecular breeding of yeast with higher metal-adsorption capacity by expression of histidine-repeat insertion in the protein anchored to the cell wall; Kambe-Honjoh H et al.; A fusion protein of hexa-histidine repeat (His) and glycosylphosphatidylinositol (GPI)-anchor region of Saccharomyces cerevisiae Cwp1 with Aspergillus oryzae Taka-amylase A (TAA) was expressed on the yeast cell surface . The expressed fusion protein (TAA-His-Cwp1) was localized on the cell wall and demonstrated amylolytic activity . In comparison with the TAA-Cwp1 expressing strain, these cells exhibited 1.6- to 2.8-fold higher adsorbing capacity for Cu(2+), Ni(2+), and Zn(2+). J Gen Appl Microbiol, 2000 Dec, 46(6), 311 - 316 Simple detection of a yeast mitochondrial DNA-binding protein, Abf2p, on SDS-DNA gels; Miyakawa I et al.; Abf2p, a mitochondrial DNA-binding protein of yeast Saccharomyces cerevisiae, was selectively detected among mitochondrial nucleoid proteins by SDS-DNA polyacrylamide gel electrophoresis (SDS-DNA PAGE) followed by ethidium bromide staining . This method is simple and specific for the detection of Abf2p, and it may be used to identify an Abf2p-like protein that is present in mitochondrial nucleoids from other yeasts. Nat Cell Biol, 2003 Jan, 5(1), 77 - 81 Yeast epsin-related proteins required for Golgi-endosome traffic define a gamma-adaptin ear-binding motif; Duncan MC et al.; Clathrin-coated vesicles (CCVs) are a central component of endocytosis and traffic between the trans-Golgi network (TGN) and endosomes . Although endocytic CCV formation is well characterized, much less is known about CCV formation at internal membranes . Here we describe two epsin amino-terminal homology (ENTH) domain-containing proteins, Ent3p and Ent5p, that are intimately involved in clathrin function at the Golgi . Both proteins associate with the clathrin adaptor Gga2p in vivo; Ent5p also interacts with the clathrin adaptor complex AP-1 and clathrin . A novel, conserved motif that mediates the interaction of Ent3p and Ent5p with gamma-ear domains of Gga2p and AP-1 is defined . Ent3p and Ent5p colocalize with clathrin, and cells lacking both Ent proteins exhibit defects in clathrin localization and traffic between the Golgi and endosomes . The findings suggest that Ent3p and Ent5p constitute a functionally related pair that co-operate with Gga proteins and AP-1 to recruit clathrin and promote formation of clathrin coats at the Golgi/endosomes . On the basis of our results and the established roles of epsin and epsin-related proteins in endocytosis, we propose that ENTH-domain-containing proteins are a universal component of CCV formation. Mol Cell Biol, 2003 Jan, 23(1), 349 - 58 Activator-independent functions of the yeast mediator sin4 complex in preinitiation complex formation and transcription reinitiation; Reeves WM et al.; RNA polymerase II (Pol II) Mediator plays an essential role in both basal and activated transcription . Previously, subunits of the Sin4 Mediator complex (Sin4, Pgd1, Gal11, and Med2) have been implicated in both positive and negative transcriptional regulation . Furthermore, it was proposed that this subcomplex constitutes an activator-binding domain . A yeast nuclear-extract system was used to investigate the biochemical role of the Sin4 complex . In contrast to previous findings, we found at least two general activator-independent roles for the Sin4 complex . First, mutations in sin4 and pgd1 destabilized the Pol II-Med complex, leading to a reduced rate and extent of preinitiation complex (PIC) formation both in the presence and absence of activators . Although reduced in amount compared with the wild type, PICs that are formed lacking the Sin4 complex are stable and can initiate transcription normally . Second, mutation of pgd1 causes partial disruption of the Sin4 complex and leads to a defect in transcription reinitiation . This defect is caused by dissociation of mutant Mediator from promoters after initiation, leading to nonfunctional Scaffold complexes . These results show that function of the Sin4 complex is not essential for transcription activation in a crude in vitro system but that it plays key roles in the general transcription mechanism. Mol Cell Biol, 2003 Jan, 23(1), 80 - 91 Yeast Isw1p forms two separable complexes in vivo; Vary JC Jr et al.; There are several classes of ATP-dependent chromatin remodeling complexes, which modulate the structure of chromatin to regulate a variety of cellular processes . The budding yeast, Saccharomyces cerevisiae, encodes two ATPases of the ISWI class, Isw1p and Isw2p . Previously Isw1p was shown to copurify with three other proteins . Here we identify these associated proteins and show that Isw1p forms two separable complexes in vivo (designated Isw1a and Isw1b) . Biochemical assays revealed that while both have equivalent nucleosome-stimulated ATPase activities, Isw1a and Isw1b differ in their abilities to bind to DNA and nucleosomal substrates, which possibly accounts for differences in specific activities in nucleosomal spacing and sliding . In vivo, the two Isw1 complexes have overlapping functions in transcriptional regulation of some genes yet distinct functions at others . In addition, these complexes show different contributions to cell growth at elevated temperatures. Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16648 - 53 Epub 2002 Dec 13. Dicer is required for chromosome segregation and gene silencing in fission yeast cells; Provost P et al.; RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs . Here we report a role for Dicer in chromosome segregation of fission yeast . Deletion of the Dicer (dcr1+) gene caused slow growth, sensitivity to thiabendazole, lagging chromosomes during anaphase, and abrogated silencing of centromeric repeats . As Dicer in other species, Dcr1p degraded double-stranded RNA into approximately 23 nucleotide fragments in vitro, and dcr1Delta cells were partially rescued by expression of human Dicer, indicating evolutionarily conserved functions . Expression profiling demonstrated that dcr1+ was required for silencing of two genes containing a conserved motif. J Cell Sci, 2003 Jan 15, 116(Pt 2), 259 - 71 Regulation of meiotic progression by the meiosis-specific checkpoint kinase Mek1 in fission yeast; Perez-Hidalgo L et al.; During the eukaryotic cell cycle, accurate transmission of genetic information to progeny is ensured by the operation of cell cycle checkpoints . Checkpoints are regulatory mechanisms that block cell cycle progression when key cellular processes are defective or chromosomes are damaged . During meiosis, genetic recombination between homologous chromosomes is essential for proper chromosome segregation at the first meiotic division . In response to incomplete recombination, the pachytene checkpoint (also known as the meiotic recombination checkpoint) arrests or delays meiotic cell cycle progression, thus preventing the formation of defective gametes . Here, we describe a role for a meiosis-specific kinase, Mek1, in the meiotic recombination checkpoint in fission yeast . Mek1 belongs to the Cds1/Rad53/Chk2 family of kinases containing forkhead-associated domains, which participate in a number of checkpoint responses from yeast to mammals . We show that defects in meiotic recombination generated by the lack of the fission yeast Meu13 protein lead to a delay in entry into meiosis I owing to inhibitory phosphorylation of the cyclin-dependent kinase Cdc2 on tyrosine 15 . Mutation of mek1(+) alleviates this checkpoint-induced delay, resulting in the formation of largely inviable meiotic products . Experiments involving ectopic overexpression of the mek1(+) gene indicate that Mek1 inhibits the Cdc25 phosphatase, which is responsible for dephosphorylation of Cdc2 on tyrosine 15 . Furthermore, the meiotic recombination checkpoint is impaired in a cdc25 phosphorylation site mutant . Thus, we provide the first evidence of a connection between an effector kinase of the meiotic recombination checkpoint and a crucial cell cycle regulator and present a model for the operation of this meiotic checkpoint in fission yeast. J Biol Chem, 2003 Feb 21, 278(8), 5821 - 7 Epub 2002 Dec 12. The first putative transmembrane segment of subunit c" (Vma16p) of the yeast V-ATPase is not necessary for function; Nishi T et al.; The yeast vacuolar ATPase (V-ATPase) contains three proteolipid subunits: c (Vma3p), c' (Vma11p), and c" (Vma16p) . Each subunit contains a buried glutamate residue that is essential for function, and these subunits are not able to substitute for each other in supporting activity . Subunits c and c' each contain four putative transmembrane segments (TM1-4), whereas subunit c" is predicted to contain five . To determine whether TM1 of subunit c" serves an essential function, a deletion mutant of Vma16p was constructed lacking TM1 (Vma16p-Delta TM1) . Although this construct does not complement the loss of Vma3p or Vma11p, it does complement the loss of full-length Vma16p . Vacuoles isolated from the strain expressing Vma16p-Delta TM1 showed V-ATPase activity and proton transport greater than 80% relative to wild type and displayed wild type levels of subunits A and a, suggesting normal assembly of the V-ATPase complex . These results suggest that TM1 of Vma16p is dispensable for both activity and assembly of the V-ATPase . To obtain information about the topology of Vma16p, labeling of single cysteine-containing mutants using the membrane-permeable reagent 3-(N-maleimidylpropionyl)biocytin (MPB) and the -impermeable reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) was tested . Both the Cys-less form of Vma16p and eight single cysteine-containing mutants retained greater than 80% of wild type levels of activity . Of the eight mutants tested, two (S5C and S178C) were labeled by MPB . MPB-labeling of S5C was blocked by AMS in intact vacuoles, whereas S178C was blocked by AMS only in the presence of permeabilizing concentrations of detergent . In addition, a hemagglutinin epitope tag introduced into the C terminus of Vma16p was recognized by an anti-hemagglutinin antibody in intact vacuolar membranes, suggesting a cytoplasmic orientation for the C terminus . These results suggest that subunit c" contains four rather than five transmembrane segments with both the N and C terminus on the cytoplasmic side of the membrane. FEBS Lett, 2002 Dec 18, 532(3), 450 - 4 Mouse Apg10 as an Apg12-conjugating enzyme: analysis by the conjugation-mediated yeast two-hybrid method; Mizushima N et al.; Autophagosome formation is a central event in macroautophagy . The Apg12-Apg5 conjugate, which is essential in this process, is generated by a ubiquitin-like protein conjugation system . In yeast, Apg12, following activation by the E1-like Apg7, forms a thioester with Apg10 (E2-like) . Apg12 is finally conjugated to Apg5 via an isopeptide bond . The possible requirement of an E3-like protein for the conjugation, however, has not yet been confirmed . The Apg12 system is conserved among eukaryotes, although a mammalian counterpart of Apg10 has not yet been identified . Here, we report the identification and characterization of the mouse Apg10 ortholog . A yeast two-hybrid screen using the mouse Apg5 (mApg5) as bait identified a novel protein with 19% identity to yeast Apg10 . We designated this protein mouse Apg10 (mApg10) . We demonstrated by a modified yeast two-hybrid assay that mApg10 mediates the conjugation of mApg12 and mApg5 . The in vivo interaction of mApg12 with mApg10 in HeLa cells suggests that mApg10 is an Apg12-conjugating enzyme, likely serving as an Apg5-recognition molecule in the Apg12 system . This novel two-hybrid method, which we have named 'conjugation-mediated yeast two-hybrid', proves to be a simple and useful technique with which to analyze protein-protein conjugation. J Biol Chem, 2003 Feb 21, 278(8), 6330 - 6 Epub 2002 Dec 11. Molecular architecture of the phosphorylation region of the yeast plasma membrane H+-ATPase; Valiakhmetov A et al.; The molecular architecture of the yeast plasma membrane H(+)-ATPase phosphorylation region was explored by Fe(2+)-catalyzed cleavage . An ATP-Mg(2+).Fe(2+) complex was found to act as an affinity cleavage reagent in the presence of dithiothreitol/H(2)O(2) . Selective enzyme cleavage required bound adenine nucleotide, either ATP or ADP, in the presence of Mg(2+) . The fragment profile included a predominant N-terminal 61-kDa fragment, a minor 37-kDa fragment, and three prominent C-terminal fragments of 39, 36, and 30 kDa . The 61-kDa N-terminal and 39-kDa C-terminal fragments were predicted to originate from cleavage within the conserved MLT(558)GDAVG sequence . The 37-kDa fragment was consistent with cleavage within the S4/M4 sequence PVGLPA(340)V, while the 30-kDa and 36-kDa C-terminal fragments appeared to originate from cleavage in or around sequences D(646)TGIAVE and DMPGS(595)ELADF, respectively . The latter are spatially close to the highly conserved motif GD(634)GVND(638)APSL and conserved residues Thr(558) and Lys(615), which have been implicated in coordinating Mg(2+) and ATP . Overall, these results demonstrate that Fe(2+) associated with ATP and Mg(2+) acts as an affinity cleavage agent of the H(+)-ATPase with backbone cleavage occurring in conserved regions known to coordinate metal-nucleotide complexes . This study provides support for a three-dimensional organization of the phosphorylation region of the yeast plasma membrane H(+)-ATPase that is consistent with, but not identical to, typical P-type enzymes. J Biol Chem, 2003 Feb 21, 278(8), 6002 - 11 Epub 2002 Dec 11. Redox properties of human endothelial nitric-oxide synthase oxygenase and reductase domains purified from yeast expression system; Du M et al.; Characterization of the redox properties of endothelial nitric-oxide synthase (eNOS) is fundamental to understanding the complicated reaction mechanism of this important enzyme participating in cardiovascular function . Yeast overexpression of both the oxygenase and reductase domains of human eNOS, i.e . eNOS(ox) and eNOS(red), has been established to accomplish this goal . UV-visible and electron paramagnetic resonance (EPR) spectral characterization for the resting eNOS(ox) and its complexes with various ligands indicated a standard NOS heme structure as a thiolate hemeprotein . Two low spin imidazole heme complexes but not the isolated eNOS(ox) were resolved by EPR indicating slight difference in heme geometry of the dimeric eNOS(ox) domain . Stoichiometric titration of eNOS(ox) demonstrated that the heme has a capacity for a reducing equivalent of 1-1.5 . Additional 1.5-2.5 reducing equivalents were consumed before heme reduction occurred indicating the presence of other unknown high potential redox centers . There is no indication for additional metal centers that could explain this extra electron capacity of eNOS(ox) . Ferrous eNOS(ox), in the presence of l-arginine, is fully functional in forming the tetrahydrobiopterin radical upon mixing with oxygen as demonstrated by rapid-freeze EPR measurements . Calmodulin binds eNOS(red) at 1:1 stoichiometry and high affinity . Stoichiometric titration and computer simulation enabled the determination for three redox potential separations between the four half-reactions of FMN and FAD . The extinction coefficient could also be resolved for each flavin for its semiquinone, oxidized, and reduced forms at multiple wavelengths . This first redox characterization on both eNOS domains by stoichiometric titration and the generation of a high quality EPR spectrum for the BH(4) radical intermediate illustrated the usefulness of these tools in future detailed investigations into the reaction mechanism of eNOS. Eukaryot Cell, 2002 Dec, 1(6), 1032 - 40 Role of Ptc2 type 2C Ser/Thr phosphatase in yeast high-osmolarity glycerol pathway inactivation; Young C et al.; Three type 2C Ser/Thr phosphatases (PTCs) are negative regulators of the yeast Saccharomyces cerevisiae high-osmolarity glycerol mitogen-activated protein kinase (MAPK) pathway . Ptc2 and Ptc3 are 75% identical to each other and differ from Ptc1 in having a noncatalytic domain . Previously, we showed that Ptc1 inactivates the pathway by dephosphorylating the Hog1 MAPK; Ptc1 maintains low basal Hog1 activity and dephosphorylates Hog1 during adaptation . Here, we examined the function of Ptc2 and Ptc3 . First, deletion of PTC2 and/or PTC3 together with PTP2, encoding the protein tyrosine phosphatase that inactivates Hog1, produced a strong growth defect at 37 degrees C that was dependent on HOG1, providing further evidence that PTC2 and PTC3 are negative regulators . Second, overexpression of PTC2 inhibited Hog1 activation but did not affect Hog1-Tyr phosphorylation, suggesting that Ptc2 inactivates the pathway by dephosphorylating the Hog1 activation loop phosphothreonine (pThr) residue . Indeed, in vitro studies confirmed that Ptc2 was specific for Hog1-pThr . Third, deletion of both PTC2 and PTC3 led to greater Hog1 activation upon osmotic stress than was observed in wild-type strains, although no obvious change in Hog1 inactivation during adaptation was seen . These results indicate that Ptc2 and Ptc3 differ from Ptc1 in that they limit maximal Hog1 activity . The function of the Ptc2 noncatalytic domain was also examined . Deletion of this domain decreased V(max) by 1.6-fold and increased K(m) by 2-fold . Thus Ptc2 requires an additional amino acid sequence beyond the catalytic domain defined for PTCs for full activity. Eukaryot Cell, 2002 Dec, 1(6), 978 - 86 Loss of compartmentalization causes misregulation of lysine biosynthesis in peroxisome-deficient yeast cells; Breitling R et al.; To characterize the metabolic role of peroxisomes in yeast cells under physiological conditions, we performed a comprehensive meta-analysis of published microarray data . Previous studies of yeast peroxisomes have mainly been focused on the function of peroxisomes under extreme conditions, such as growth on oleate or methanol as the sole carbon source, and may therefore not be representative of the normal physiological role of yeast peroxisomes . Surprisingly, our analysis of the microarray data reveals that the only pathway responding to peroxisome deficiency in mid-log phase is lysine biosynthesis, whereas classical peroxisomal pathways such as beta-oxidation are unaffected . We show that the upregulation of lysine biosynthesis genes in peroxisome-deficient yeasts shares many characteristics with the physiological response to lysine starvation . We provide data that suggest that this is the result of a "pathological" stimulation of the Lys14p transcriptional activator by the pathway intermediate aminoadipate semialdehyde . Mistargeting of the peroxisomal lysine pathway to the cytosol increases the active concentration of aminoadipate semialdehyde, which is no longer contained in the peroxisome and can now activate Lys14p at much lower levels than in wild-type yeasts . This is the first well-documented example of pathway misregulation in response to peroxisome deficiency and will be useful in understanding the phenotypic details of human peroxisome-deficient patients (Zellweger syndrome). Eukaryot Cell, 2002 Dec, 1(6), 915 - 25 A multigene family that interacts with the amino terminus of plasmodium MSP-1 identified using the yeast two-hybrid system; Mello K et al.; Merozoite surface protein 1 (MSP-1) is a high-molecular-weight protein expressed on the surface of the malaria merozoite in a noncovalent complex with other protein molecules . MSP-1 undergoes a series of proteolytic processing events, but no precise biological role for the various proteolytic fragments of MSP-1 or for the additional proteins present in the complex is known . Through the use of the yeast two-hybrid system, we have isolated genes encoding proteins that interact with a region of the amino-terminal proteolytic fragment of MSP-1 from the mouse parasite Plasmodium yoelii . This analysis has led to the isolation of two sequence-related molecules, one of which is the P . yoelii homologue of MSP-7 originally described in Plasmodium falciparum . BLAST analysis of the P . falciparum database has revealed that there are six related protein molecules present in this species encoded near each other on chromosome 13 . In P . falciparum, we designated these molecules MSRP-1 to -5 . Analysis of the P . yoelii database indicates a similar chromosomal organization for the two genes in the mouse parasite species . The three P . falciparum sequences with the highest degree of homology to the P . yoelii sequences isolated in the two-hybrid screen have been characterized at the molecular level (MSRP-1 to -3) . Expression analysis indicated that the mRNAs are expressed at various levels in the different asexual stages . Immunofluorescence studies colocalized the expression of the MSRP molecules and the amino-terminal portion of MSP-1 to the surfaces of trophozoites . In vitro binding experiments confirmed the interaction between MSRP-1, MSRP-2, and the amino-terminal region of P . falciparum MSP-1. J Biol Chem, 2003 Feb 14, 278(7), 4862 - 74 Epub 2002 Dec 10. Molecular interactions of yeast frequenin (Frq1) with the phosphatidylinositol 4-kinase isoform, Pik1; Huttner IG et al.; Frq1, a 190-residue N-myristoylated calcium-binding protein, associates tightly with the N terminus of Pik1, a 1066-residue phosphatidylinositol 4-kinase . Deletion analysis of an Frq1-binding fragment, Pik1-(10-192), showed that residues within 80-192 are necessary and sufficient for Frq1 association in vitro . A synthetic peptide (residues 151-199) competed for binding of {(35)S}Pik1-(10-192) to bead-immobilized Frq1, whereas shorter peptides (164-199 and 174-199) did not . Correspondingly, a deletion mutant, Pik1(delta152-191), did not co-immunoprecipitate efficiently with Frq1 and did not support growth at elevated temperature . Site-directed mutagenesis of Pik1-(10-192) suggested that recognition determinants lie over an extended region . Titration calorimetry demonstrated that binding of an 83-residue fragment, Pik1-(110-192), or the 151-199 peptide to Frq1 shows high affinity (K(d) approximately 100 nm) and is largely entropic, consistent with hydrophobic interaction . Stoichiometry of Pik1-(110-192) binding to Frq1 was 1:1, as judged by titration calorimetry, by changes in NMR spectrum and intrinsic tryptophan fluorescence, and by light scattering . In cell extracts, Pik1 and Frq1 exist mainly in a heterodimeric complex, as shown by size exclusion chromatography . Cys-15 in Frq1 is not S-palmitoylated, as assessed by mass spectrometry; a Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A) double mutant rescued the inviability of frq1Delta cells . This study defines the segment of Pik1 required for high affinity binding of Frq1. Mol Biol Cell, 2002 Dec, 13(12), 4429 - 42 Lipid-dependent subcellular relocalization of the acyl chain desaturase in yeast; Tatzer V et al.; The degree of acyl chain desaturation of membrane lipids is a critical determinant of membrane fluidity . Temperature-sensitive mutants of the single essential acyl chain desaturase, Ole1p, of yeast have previously been isolated in screens for mitochondrial inheritance mutants (Stewart, L.C . and Yaffe, M.P . (1991) . J . Cell Biol . 115, 1249-1257) . We now report that the mutant desaturase relocalizes from its uniform ER distribution to a more punctuate localization at the cell periphery upon inactivation of the enzyme . This relocalization takes place within minutes at nonpermissive conditions, a time scale at which mitochondrial morphology and inheritance is not yet affected . Relocalization of the desaturase is fully reversible and does not affect the steady state localization of other ER resident proteins or the kinetic and fidelity of the secretory pathway, indicating a high degree of selectivity for the desaturase . Relocalization of the desaturase is energy independent but is lipid dependent because it is rescued by supplementation with unsaturated fatty acids . Relocalization of the desaturase is also observed in cells treated with inhibitors of the enzyme, indicating that it is independent of temperature-induced alterations of the enzyme . In the absence of desaturase function, lipid synthesis continues, resulting in the generation of lipids with saturated acyl chains . A model is discussed in which the accumulation of saturated lipids in a microdomain around the desaturase could induce the observed segregation and relocalization of the enzyme. Mol Biol Cell, 2002 Dec, 13(12), 4414 - 28 A specific structural requirement for ergosterol in long-chain fatty acid synthesis mutants important for maintaining raft domains in yeast; Eisenkolb M et al.; Fungal sphingolipids contain ceramide with a very-long-chain fatty acid (C26) . To investigate the physiological significance of the C26-substitution on this lipid, we performed a screen for mutants that are synthetically lethal with ELO3 . Elo3p is a component of the ER-associated fatty acid elongase and is required for the final elongation cycle to produce C26 from C22/C24 fatty acids . elo3delta mutant cells thus contain C22/C24- instead of the natural C26-substituted ceramide . We now report that under these conditions, an otherwise nonessential, but also fungal-specific, structural modification of the major sterol of yeast, ergosterol, becomes essential, because mutations in ELO3 are synthetically lethal with mutations in ERG6 . Erg6p catalyzes the methylation of carbon atom 24 in the aliphatic side chain of sterol . The lethality of an elo3delta erg6delta double mutant is rescued by supplementation with ergosterol but not with cholesterol, indicating a vital structural requirement for the ergosterol-specific methyl group . To characterize this structural requirement in more detail, we generated a strain that is temperature sensitive for the function of Erg6p in an elo3delta mutant background . Examination of raft association of the GPI-anchored Gas1p and plasma membrane ATPase, Pma1p, in the conditional elo3delta erg6(ts) double mutant, revealed a specific defect of the mutant to maintain raft association of preexisting Pma1p . Interestingly, in an elo3delta mutant at 37 degrees C, newly synthesized Pma1p failed to enter raft domains early in the biosynthetic pathway, and upon arrival at the plasma membrane was rerouted to the vacuole for degradation . These observations indicate that the C26 fatty acid substitution on lipids is important for establishing raft association of Pma1p and stabilizing the protein at the cell surface . Analysis of raft lipids in the conditional mutant strain revealed a selective enrichment of ergosterol in detergent-resistant membrane domains, indicating that specific structural determinants on both sterols and sphingolipids are required for their association into raft domains. Mol Biol Cell, 2002 Dec, 13(12), 4130 - 40 Selective protein exit from yeast endoplasmic reticulum in absence of functional COPII coat component Sec13p; Fatal N et al.; Sec13p has been thought to be an essential component of the COPII coat, required for exit of proteins from the yeast endoplasmic reticulum (ER) . We show herein that normal function of Sec13p was not required for ER exit of the Hsp150 glycoprotein . Hsp150 was secreted to the medium under restrictive conditions in a sec13-1 mutant . The COPII components Sec23p and Sec31p and the GTP/GDP exchange factor Sec12p were required in functional form for secretion of Hsp150 . Hsp150 leaves the ER in the absence of retrograde COPI traffic, and the responsible determinant is a peptide repeated 11 times in the middle of the Hsp150 sequence . Herein, we localized the sorting determinant for Sec13p-independent ER exit to the C-terminal domain . Sec13p-dependent invertase left the ER in the absence of normal Sec13p function, when fused to the C-terminal domain of Hsp150, demonstrating that this domain contained an active mediator of Sec13p-independent secretion . Thus, Hsp150 harbors two different signatures that regulate its ER exit . Our data show that transport vesicles lacking functional Sec13p can carry out ER-to-Golgi transport, but select only specific cargo protein(s) for ER exit. J Pathol, 2003 Jan, 199(1), 4 - 7 The yeast two-hybrid system for identifying protein-protein interactions; Coates PJ et al.; The yeast two-hybrid assay is a system for identifying and analysing protein-protein interactions . Since the original description in 1989, the technique has provided insight into many biological pathways . A variety of adaptations to the technique have been developed that allow analysis of protein-DNA, protein-RNA, or small molecule-protein interactions . Recent developments now allow the use of these technologies to perform global analyses of all such interactions that occur in cells . The information gained from these approaches is uncovering many aspects of the complex networks that underlie normal cellular processes and how they are perturbed in disease states . J Cell Biol, 2002 Dec 9, 159(5), 807 - 19 Epub 2002 Dec 09. The yeast nuclear pore complex functionally interacts with components of the spindle assembly checkpoint; Iouk T et al.; Aphysical and functional link between the nuclear pore complex (NPC) and the spindle checkpoint machinery has been established in the yeast Saccharomyces cerevisiae . We show that two proteins required for the execution of the spindle checkpoint, Mad1p and Mad2p, reside predominantly at the NPC throughout the cell cycle . There they are associated with a subcomplex of nucleoporins containing Nup53p, Nup170p, and Nup157p . The association of the Mad1p-Mad2p complex with the NPC requires Mad1p and is mediated in part by Nup53p . On activation of the spindle checkpoint, we detect changes in the interactions between these proteins, including the release of Mad2p (but not Mad1p) from the NPC and the accumulation of Mad2p at kinetochores . Accompanying these events is the Nup53p-dependent hyperphosphorylation of Mad1p . On the basis of these results and genetic analysis of double mutants, we propose a model in which Mad1p bound to a Nup53p-containing complex sequesters Mad2p at the NPC until its release by activation of the spindle checkpoint . Furthermore, we show that the association of Mad1p with the NPC is not passive and that it plays a role in nuclear transport. Curr Opin Cell Biol, 2002 Dec, 14(6), 676 - 83 Transcriptional regulatory networks and the yeast cell cycle; Futcher B; A large amount of microarray gene expression data relevant to the yeast cell cycle has been collected, and several hundred genes have been placed into a model transcriptional control network . Genome-wide studies of the location of cell cycle transcription factors, and a variety of computational approaches, have allowed refinement of the model, and at the same time show how other genome-wide data sets may be organised into model networks. J Biochem (Tokyo), 2002 Dec, 132(6), 975 - 82 Affinity selection of DNA-binding proteins from yeast genomic DNA libraries by improved lambda phage display vector; Hagiwara H et al.; Phage display is a useful means of identifying and selecting proteins of interest that bind specific targets . In order to examine the potential of phage display for the genome-wide screening of DNA-binding proteins, we constructed yeast genomic libraries using lambda foo-based vectors devised in this work . After affinity selection using GAL4 UAS(G) as a probe, phages expressing GAL4 were enriched approximately 5 x 10(5)-fold from the library . Approximately 90% of polypeptides encoded in correct translation reading frames by the selected phages were known or putative polynucleotide-binding proteins . This result clearly indicates that the modified lambda phage display vector in combination with our enrichment technique has great potential for the enrichment of DNA-binding proteins in a sequence-specific manner. Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16922 - 7 Epub 2002 Dec 06. Genetic analysis of iron citrate toxicity in yeast: implications for mammalian iron homeostasis; Chen OS et al.; Deletion of the yeast homologue of frataxin, YFH1, results in mitochondrial iron accumulation and respiratory deficiency (petite formation) . We used a genetic screen to identify mutants that modify iron-associated defects in respiratory activity in Deltayfh1 cells . A deletion in the peroxisomal citrate synthase CIT2 in Deltayfh1 cells decreased the rate of petite formation . Conversely, overexpression of CIT2 in Deltayfh1 cells increased the rate of respiratory loss . Citrate toxicity in Deltayfh1 cells was dependent on iron but was independent of mitochondrial respiration . Citrate toxicity was not restricted to iron-laden mitochondria but also occurred when iron accumulated in cytosol because of impaired vacuolar iron storage . These results suggest that high levels of citrate may promote iron-mediated tissue damage. Biochem Biophys Res Commun, 2002 Dec 20, 299(5), 723 - 9 The function of the nuclear matrix attachment region of silkworm rDNA as an autonomously replicating sequence in plasmid and chromosomal replication origin in yeast; Chen Y et al.; Nuclear matrix attachment regions (MARs) play a crucial role in chromatin architecture, gene expression, and DNA replication . Although it is well known that yeast autonomously replicating sequences (ARSs) bind nuclear matrix and MARs also function as ARS elements in yeast, whether a heterologous MAR or ARS element acts as a replication origin in the chromosome has not been elucidated . We previously identified a MAR (rMAR) located in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA . We report here that this rMAR contains 10 copies of ARS consensus sequence (ACS) and several DNA unwinding regions . The rMAR employs ARS activity in yeast and a rARS element locates in the 3(') region of the rMAR . Furthermore, we have also revealed that either the rMAR or the rARS element functions as a replication origin in the chromosome . Our results provide the first direct evidence to demonstrate that heterologous rMAR and rARS display chromosomal origin activity, suggesting that the chromosome structure and replication origin of rDNA reserve some common features during evolution. Proteomics, 2002 Dec, 2(12), 1715 - 23 Interaction and domain networks of yeast; Wuchty S; Data of currently available protein-protein interaction sets and protein domain sets of yeast are used to set up protein and domain interaction and domain sequence networks . All of them are far from being random or regular networks . In fact, they turn out to be sparse and locally well clustered indicating so-called scale-free and partially small-world topology . These subtle topologies display considerable indirect properties which are measured with a newly introduced transitivity coefficient . Fairly small sets of highly connected proteins and domains shape the topologies of the underlying networks, emphasizing a kind of backbone the nets are based on . The biological nature of these particular nodes is further investigated . Since highly connected proteins and domains accumulated a significant higher number of links by their important involvement in certain cellular aspects, their mutational effect on the cell is considered by a perturbation analysis . In comparison to domains of yeast, what factors force domains to accumulate links to other domains in protein sequences of higher eukaryotes are investigated. J Gen Appl Microbiol, 2002 Feb, 48(1), 17 - 23 Dioszegia zsoltii sp . nov., a new ballistoconidium-forming yeast species with two varieties; Bai FY et al.; As a result of conventional characterization of yeasts isolated from various plant leaves collected in Yunnan, China, six ballistoconidium-forming strains with orange-colored colonies were grouped together . Molecular phylogenetic analysis based on 18S rDNA sequencing showed that two representative strains of this group of yeasts, CH 2.068 and CH 2.497, were closely related to the species in the genus Dioszegia and had signature sequences typical of this genus . However, the six strains from Yunnan differed from the described Dioszegia species remarkably (14.5-17.7% nucleotide divergences) in the ITS (internal transcribed spacer) region sequences, which indicated that they represent a distinct species . Furthermore, among the six strains studied, the ITS region sequence comparison allowed the recognition of two subgroups represented by CH 2.068 and CH 2.497, which differ from each other in three bases in the ITS 2 region . DNA-DNA relatedness revealed that the two subgroups represent two varieties of a new species in the genus Dioszegia, for which Dioszegia zsoltii sp . nov . var . zsoltii and Dioszegia zsoltii var . yunnanensis var . nov . are proposed. J Biol Chem, 2003 Feb 7, 278(6), 3831 - 9 Epub 2002 Dec 04. Functional interactions within yeast mediator and evidence of differential subunit modifications; Balciunas D et al.; It is possible to recruit RNA polymerase II to a target promoter and, thus, activate transcription by fusing Mediator subunits to a DNA binding domain . To investigate functional interactions within Mediator, we have tested such fusions of the lexA DNA binding domain to Med1, Med2, Gal11, Srb7, and Srb10 in wild type, med1, med2, gal11, sin4, srb8, srb10, and srb11 strains . We found that lexA-Med2 and lexA-Gal11 are strong activators that are independent of all Mediator subunits tested . lexA-Srb10 is a weak activator that depends on Srb8 and Srb11 . lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4 . An unexpected finding was that lexA-VP16 differs from Gal4-VP16 in that it is independent of the activator binding Mediator module . Both lexA-Med1 and lexA-Srb7 are stably associated with Med4 and Med8, which suggests that they are incorporated into Mediator . Med4 and Med8 exist in two mobility forms that differ in their association with lexA-Med1 and lexA-Srb7 . Within purified Mediator, Med4 is present as a phosphorylated lower mobility form . Taken together, these results suggest that assembly of Mediator is a multistep process that involves conversion of both Med4 and Med8 to their low mobility forms. Mol Biochem Parasitol, 2002 Nov-Dec, 125(1-2), 103 - 12 Characterization and comparative functional analysis in yeast of a Schistosoma mansoni Rho1 GTPase gene; Santos TM et al.; Low-molecular weight GTP-binding proteins (LMWGPs) of the Ras superfamily are believed to play a role in Schistosoma mansoni female development and egg production . Here we describe the characterization of a novel S . mansoni gene (SMRHO1), highly homologous to Rho-type LMWGPs from several other organisms and encoding a polypeptide with 193 amino acids and an estimated molecular mass of 21.8 kDa . SMRHO1 complemented a Saccharomyces cerevisiae rho1 null mutant strain even in restrictive temperature and calcium concentration, in contrast with the human RHOA GTPase that was not able to provide complementation in such conditions . Comparison of the amino acid sequence of the alpha3-helix loop7 regions of the two proteins allowed the identification of the proline 96 and threonine 100 amino acid residues of human RHOA as the most probable determinants of the complementation differences . We generated SMRHO1 mutants (smrho1(E97P), smrho1(L101T) and smrho1(E97P,) L101T) by site directed mutagenesis and reproduced the conditional lethality phenotype at high temperature, providing strong evidence that the related amino acid positions (Gln(101) and Ile(105)) in the Rho1 GTPase are indeed important for regulation of the cell wall synthesis performed by this protein in yeast . The observation that specific amino acid positions seem to be important for the different functions performed by the Rho GTPases leads to the idea that SMRHO1 might be a useful target in the development of new anti-schistosomiasis drugs, although it does share high sequence homology with the human RhoA GTPase. Appl Microbiol Biotechnol, 2002 Dec, 60(4), 449 - 54 Epub 2002 Oct 12. Stimulation of chymosin secretion by simultaneous expression with chymosin-binding llama single-domain antibody fragments in yeast; Harmsen MM et al.; We studied the effect of coexpression of chymosin and chymosin-binding llama single-domain antibody fragments (VHHs) on the secretion of chymosin by Saccharomyces cerevisiae cells . A VHH expression library containing chymosin-specific VHHs was obtained by immunization of a llama and coexpressed with chymosin in yeast . From this library, we obtained two VHH clones that stimulated chymosin secretion by screening colonies for the level of chymosin secreted . These VHHs bound biotinylated chymosin in an immunoblot procedure but failed to bind chymosin in ELISA, suggesting that their interaction with chymosin was of low affinity . In a second approach, chymosin-specific VHHs were first selected using phage display and then coexpressed with chymosin in yeast cells . Screening yeast cells for higher levels of chymosin secretion resulted in 11 VHHs . Sequence analysis revealed that these 11 VHHs formed four sets of related VHHs that were different from the previously isolated two VHHs . Although binding of VHHs to chymosin could not be demonstrated in ELISA using soluble VHHs, it could be unambiguously demonstrated for clones isolated by phage display, using phage-displayed VHHs . Finally, quantitative Western blot analysis of chymosin amounts demonstrated that coexpression with VHH domains can stimulate the level of secreted chymosin 1.5- to 6-fold. Nucleic Acids Res, 2002 Dec 1, 30(23), 5129 - 35 Difference between deoxyribose- and tetrahydrofuran-type abasic sites in the in vivo mutagenic responses in yeast; Otsuka C et al.; We have analyzed the mutagenic specificity of an abasic site in DNA using the yeast oligonucleotide transformation assay . Oligonucleotides containing an abasic site or its analog were introduced into B7528 or its derivatives, and nucleotide incorporation opposite abasic sites was analyzed . Cytosine was most frequently incorporated opposite a natural abasic site (O) ('C-rule'), followed by thymine . Deletion of REV1 decreased the transformation efficiency and the incorporation of cytosine nearly to a background level . In contrast, deletion of RAD30 did not affect them . We compared the mutagenic specificity with that of a tetrahydrofuran abasic site (F), an abasic analog used widely . Its mutation spectrum was clearly different from that of O . Adenine, not cytosine, was most favorably incorporated . However, deletion of REV1 decreased the transformation efficiency with F-containing oligonucleotide as in the case of O . These results suggest that the bypass mechanism of F is different from that of O, although the bypasses in both cases are dependent on REV1 . We also found that the mutagenic specificity of F can be affected by not only the adjacent bases, but also a base located two positions away from F. J Chromatogr A, 2002 Nov 8, 976(1-2), 409 - 22 Development of new analytical methods for selenium speciation in selenium-enriched yeast material; Chassaigne H et al.; A sequential extraction allowing the discrimination of water-soluble and non-soluble selenium fractions has been developed to evaluate the availability of selenium (Se) in an Se-enriched yeast candidate reference material . The fractionation of selenium-containing compounds in the extracts was achieved on preparative grade 200 Superdex 75 and columns . It showed that water-soluble selenium is present in several fractions with a large mass distribution . Low-molecular- (< or = 10,000) and high-molecular-mass selenocompounds (range 10,000-100,000) were considered separately for further experiments . The analytical approach for low-molecular-mass selenocompounds was based onanion-exchange HPLC with on-line inductively coupled plasma (ICP) MS for quantitative analysis . Selenocystine, selenomethionine, selenite and selenate were quantified in the fractions isolated in preparative chromatography . The study revealed the existence of various unidentified Se species in yeast material . The Se-containing proteins in the yeast material have been further separated and selenium quantified by the combination of gel electrophoresis and electrothermal vaporization-ICP-MS . This new approach allows the separation of the proteins with high resolution by sodium dodecylsulfate-polyacrylamide gel electrophoresis and the sensitive determination of selenium in the protein bands. Proc Natl Acad Sci U S A, 2002 Dec 10, 99 Suppl 4, 16446 - 53 Epub 2002 Dec 02. Changes in the middle region of Sup35 profoundly alter the nature of epigenetic inheritance for the yeast prion {PSI+}; Liu JJ et al.; The yeast prion {PSI(+)} provides an epigenetic mechanism for the inheritance of new phenotypes through self-perpetuating changes in protein conformation . {PSI(+)} is a nonfunctional, ordered aggregate of the translation termination factor Sup35p that influences new Sup35 proteins to adopt the same state . The N-terminal region of Sup35p plays a central role in prion induction and propagation . The C-terminal region provides translation termination activity . The function of the highly charged, conformationally flexible middle region (M) is unknown . An M deletion mutant was capable of existing in either the prion or the nonprion state, but in either case it was mostly insoluble . Substituting a charged synthetic polypeptide for M restored solubility, but the prions formed by this variant were mitotically very unstable . Substituting charged flexible regions from two other proteins for M created variants that acquired prion states (defined as self-perpetuating changes in function transferred to them from wild-type {PSI(+)} elements), but had profoundly different properties . One was soluble in both the prion and the nonprion form, mitotically stable but meiotically unstable, and cured by guanidine HCl but not by alterations in heat shock protein 104 (Hsp104p) . The other could only maintain the prion state in the presence of wild-type protein, producing Mendelian segregation patterns . The unique character of these M variants, all carrying the same N-terminal prion-determining region, demonstrate the importance of M for {PSI(+)} and suggest that a much wider range of epigenetic phenomena might be based on self-perpetuating, prion-like changes in protein conformation than suggested by our current methods for defining prion states. J Mol Biol, 2002 Dec 6, 324(4), 807 - 22 Solution structure and ligand recognition of the WW domain pair of the yeast splicing factor Prp40; Wiesner S et al.; The yeast splicing factor pre-mRNA processing protein 40 (Prp40) comprises two N-terminal WW domains, separated by a ten-residue linker, and six consecutive FF domains . In the spliceosome, the Prp40 WW domains participate in cross-intron bridging by interacting with proline-rich regions present in the branch-point binding protein (BBP) and the U5 small nuclear ribonucleoprotein component Prp8 . Furthermore, binding of Prp40 to the phosphorylated C-terminal domain (CTD) of the largest subunit of RNA polymerase II is thought to link splicing to transcription . To gain insight into this complex interaction network we have determined the solution structure of the tandem Prp40 WW domains by NMR spectroscopy and performed chemical shift mapping experiments with different proline-rich peptides . The WW domains each adopt the characteristic triple-stranded beta-sheet structure and are connected by a stable alpha-helical linker . On the basis of a detailed analysis of residual dipolar couplings (RDC) and 15N relaxation data we show that the tandem Prp40 WW domains behave in solution as a single folded unit with unique alignment and diffusion tensor, respectively . Using {1H-15N}-RDCs, we were able to accurately define the relative orientation of the WW domains revealing that the binding pockets of each domain face opposite sides of the structure . Furthermore, we found that both Prp40 WW domains interact with PPxY motifs (where x is any residue) present in peptides derived from the splicing factors BBP and Prp8 . Moreover, the Prp40 WW domains are shown to bind proline-rich peptides devoid of aromatic residues, which are also recognised by the Abl-SH3 domain and the WW domain of the mammalian Prp40 orthologue formin binding protein 11 . In contrast, no interaction was observed between the Prp40 WW domains and the CTD repeats used in this work. Science, 2002 Nov 29, 298(5599), 1773 - 5 Hybrid speciation in experimental populations of yeast; Greig D et al.; Most models of speciation require gradual change and geographic or ecological isolation for new species to arise . Homoploid hybrid speciation occurred readily between Saccharomyces cerevisiae and Saccharomyces paradoxus . Hybrids had high self-fertility (about 82%), low fertility when backcrossed to either parental species (about 7.5%), and vigorous growth under different thermal environments that favored one or the other of the parental species . Extensive karyotypic changes (tetrasomy) were observed in the hybrids, although genic incompatibilities accounted for 50% of the variation in self-fertility. Trends Pharmacol Sci, 2002 Dec, 23(12), 544 - 7 From genetics and genomics to drug discovery: yeast rises to the challenge; Melese T et al.; Yeast expands its role from eukaryotic genetics and genomics to drug discovery. Curr Opin Microbiol, 2002 Dec, 5(6), 602 - 7 Stationary phase in yeast; Herman PK; Eukaryotic cell proliferation is controlled by specific growth factors and the availability of essential nutrients . If either of these signals is lacking, cells may enter into a specialized nondividing resting state, known as stationary phase or G(0) . The entry into such resting states is typically accompanied by a dramatic decrease in the overall growth rate and an increased resistance to a variety of environmental stresses . Since most cells spend most of their life in these quiescent states, it is important that we develop a full understanding of the biology of the stationary phase/G(0) cell . This knowledge would provide important insights into the control of two of the most fundamental aspects of eukaryotic cell biology: cell proliferation and long-term cell survival . This review will discuss some recent advances in our understanding of the stationary phase of growth in the budding yeast, Saccharomyces cerevisiae. Curr Opin Microbiol, 2002 Dec, 5(6), 564 - 74 Microfilaments and microtubules: the news from yeast; Schott D et al.; New evidence that cortical actin patches and the endocytic machinery share components supports the idea that actin patches are in fact transient membrane coats at the initial stage of endocytosis . Recent studies of actin cables have identified formins as the core of a novel actin-filament-assembling machine . Meanwhile, microtubule-binding proteins have been found in the kinetochore, and factors affecting microtubule dynamic instability have been identified. Eukaryot Cell, 2002 Aug, 1(4), 526 - 37 Isolation and characterization of YlBEM1, a gene required for cell polarization and differentiation in the dimorphic yeast Yarrowia lipolytica; Hurtado CA et al.; The ability to switch between a unicellular yeast form and different filamentous forms (fungal dimorphism) is an important attribute of most pathogenic fungi . Dimorphism involves a series of events that ultimately result in dramatic changes in the polarity of cell growth in response to environmental factors . We have isolated and characterized YlBEM1, a gene encoding a protein of 639 amino acids that is essential for the yeast-to-hypha transition in the yeast Yarrowia lipolytica and whose transcription is significantly increased during this event . Cells with deletions of YlBEM1 are viable but show substantial alterations in morphology, disorganization of the actin cytoskeleton, delocalization of cortical actin and chitin deposition, multinucleation, and loss of mating ability, thus pointing to a major role for YlBEM1 in the regulation of cell polarity and morphogenesis in this fungus . This role is further supported by the localization of YlBemlp, which, like cortical actin, appears to be particularly abundant at sites of growth of yeast, hyphal, and pseudohyphal cells . In addition, the potential involvement of YlBem1p in septum formation and/or cytokinesis is suggested by the concentration of a green fluorescent protein-tagged version of this protein at the mother-bud neck during the last stages of cell division . Interestingly, overexpression of MHY1, YlRAC1, or YlSEC31, three genes involved in filamentous growth of Y . lipolytica, induced hyphal growth of bem1 null mutant cells. Eukaryot Cell, 2002 Jun, 1(3), 440 - 7 G-protein signaling mediates asexual development at 25 degrees C but has no effect on yeast-like growth at 37 degrees C in the dimorphic fungus Penicillium mameffei; Zuber S et al.; The ascomycete Penicillium marneffei is an opportunistic human pathogen exhibiting a temperature-dependent dimorphic switch . At 25 degrees C, P . marneffei grows as filamentous multinucleate hyphae and undergoes asexual development, producing uninucleate spores . At 37 degrees C, it forms uninucleate yeast cells which divide by fission . We have cloned a gene encoding a G alpha subunit of a heterotrimeric G protein from P . marneffei named gasA with high similarity to fadA in Aspergillus nidulans . Through the characterization of a delta gasA strain and mutants carrying a dominant activating or a dominant interfering gasA allele, we show that GasA is a key regulator of asexual development but seems to play no role in the regulation of growth . A dominant activating gasA mutant whose mutation results in a G42-to-R change (gasA(G42R)) does not express brlA, the conidiation-specific regulatory gene, and is locked in vegetative growth, while a dominant interfering gasA(G203R) mutant shows inappropriate brlA expression and conidiation . Interestingly, the gasA mutants have no apparent defect in dimorphic switching or yeast-like growth at 37 degrees C . Growth tests on dibutyryl cyclic AMP (dbcAMP) and theophylline suggest that a cAMP-protein kinase A cascade may be involved in the GasA signaling pathway. Eukaryot Cell, 2002 Feb, 1(1), 56 - 65 Yeast Rpi1 is a putative transcriptional regulator that contributes to preparation for stationary phase; Sobering AK et al.; The RPI1 gene of Saccharomyces cerevisiae was identified initially as a dosage suppressor of the heat shock sensitivity associated with overexpression of RAS2 (J . Kim and S . Powers, Mol . Cell . Biol . 11:3894-3904, 1991) . Based on its failure to suppress mutationally activated RAS2, RPII was proposed to be a negative regulator of the Ras/cyclic AMP (cAMP) pathway that functions at a point upstream of Ras . We isolated RPI1 as a high-copy-number suppressor of the cell lysis defect associated with a null mutation in the MPK1 gene, which encodes the mitogen-activated protein kinase of the cell wall integrity-signaling pathway . Although the sequence of Rpil is not informative about its function, we present evidence that this protein resides in the nucleus, possesses a transcriptional activation domain, and affects the mRNA levels of several cell wall metabolism genes . In contrast to the previous report, we found that RPI1 overexpression suppresses defects associated with mutational hyperactivation of the Ras/cAMP pathway at all points including constitutive mutations in the cAMP-dependent protein kinase . We present additional genetic and biochemical evidence that Rpil functions independently of and in opposition to the Ras/cAMP pathway to promote preparations for the stationary phase . Among these preparations is a fortification of the cell wall that is antagonized by Ras pathway activity . This observation reveals a novel link between the Ras/cAMP pathway and cell wall integrity . Finally, we propose that inappropriate expression of RPI1 during log phase growth drives fortification of the cell wall and that this behavior is responsible for suppression of the mpkl cell lysis defect. Eukaryot Cell, 2002 Apr, 1(2), 163 - 73 Heat stress activates the yeast high-osmolarity glycerol mitogen-activated protein kinase pathway, and protein tyrosine phosphatases are essential under heat stress; Winkler A et al.; The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress . In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1 . The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses . The possible function of Hog1 activation during heat stress was examined, and it was found that the hog1 delta strain does not recover as rapidly from heat stress as well as the wild type . It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress . First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation . Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways. Eukaryot Cell, 2002 Oct, 1(5), 830 - 42 The yeast pafl-rNA polymerase II complex is required for full expression of a subset of cell cycle-regulated genes; Porter SE et al.; We have previously described an alternative form of RNA polymerase II in yeast lacking the Srb and Med proteins but including Pafl, Cdc73, Hprl, and Ccr4 . The Pafl-RNA polymerase II complex (Paf1 complex) acts in the same pathway as the Pkc1-mitogen-activated protein kinase cascade and is required for full expression of many cell wall biosynthetic genes . The expression of several of these cell integrity genes, as well as many other Paf1-requiring genes identified by differential display and microarray analyses, is regulated during the cell cycle . To determine whether the Paf1 complex is required for basal or cyclic expression of these genes, we assayed transcript abundance throughout the cell cycle . We found that transcript abundance for a subset of cell cycle-regulated genes, including CLN1, HO, RNR1, and FAR1, is reduced from 2- to 13-fold in a paf1delta strain, but that this reduction is not promoter dependent . Despite the decreased expression levels, cyclic expression is still observed . We also examined the possibility that the Paf1 complex acts in the same pathway as either SBF (Swi4/Swi6) or MBF (Mbp1/Swi6), the partially redundant cell cycle transcription factors . Consistent with the possibility that they have overlapping essential functions, we found that loss of Paf1 is lethal in combination with loss of Swi4 or Swi6 . In addition, overexpression of either Swi4 or Mbp1 suppresses some paf1delta phenotypes . These data establish that the Paf1 complex plays an important role in the essential regulatory pathway controlled by SBF and MBF. Eukaryot Cell, 2002 Oct, 1(5), 663 - 72 Amino acid-dependent Gcn4p stability regulation occurs exclusively in the yeast nucleus; Pries R et al.; The c-Jun-like transcriptional activator Gcn4p controls biosynthesis of translational precursors in the yeast Saccharomyces cerevisiae . Protein stability is dependent on amino acid limitation and cis signals within Gcn4p which are recognized by cyclin-dependent protein kinases, including Pho85p . The Gcn4p population within unstarved yeast consists of a small relatively stable cytoplasmic fraction and a larger less stable nuclear fraction . Gcn4p contains two nuclear localization signals (NLS) which function independently of the presence or absence of amino acids . Expression of NLS-truncated Gcn4p results in an increased cytoplasmic fraction and an overall stabilization of the protein . The same effect is achieved for the entire Gcn4p in a yrb1 yeast mutant strain impaired in the nuclear import machinery . In the presence of amino acids, controlled destabilization of Gcn4p is triggered by the phosphorylation activity of Pho85p . A pho85delta mutation stabilizes Gcn4p without affecting nuclear import . Pho85p is localized within the nucleus in the presence or absence of amino acids . Therefore, there is a strict spatial separation of protein synthesis and degradation of Gcn4p in yeast . Control of protein stabilization which antagonizes Gcn4p function is restricted to the nucleus. Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2127 - 30 Epub 2002 Nov 23. Crystallographic studies of a novel DNA-binding domain from the yeast transcriptional activator Ndt80; Montano SP et al.; The Ndt80 protein is a transcriptional activator that plays a key role in the progression of the meiotic divisions in the yeast Saccharomyces cerevisiae . Ndt80 is strongly induced during the middle stages of the sporulation pathway and binds specifically to a promoter element called the MSE to activate transcription of genes required for the meiotic divisions . Here, the preliminary structural and functional studies to characterize the DNA-binding activity of this protein are reported . Through deletion analysis and limited proteolysis studies of Ndt80, a novel 32 kDa DNA-binding domain that is sufficient for DNA-binding in vitro has been defined . Crystals of the DNA-binding domain of Ndt80 in two distinct lattices have been obtained, for which diffraction data extend to 2.3 A resolution. Genetics, 2002 Nov, 162(3), 1131 - 45 Alleles of the yeast Pms1 mismatch-repair gene that differentially affect recombination- and replication-related processes; Welz-Voegele C et al.; Mismatch-repair (MMR) systems promote eukaryotic genome stability by removing errors introduced during DNA replication and by inhibiting recombination between nonidentical sequences (spellchecker and antirecombination activities, respectively) . Following a common mismatch-recognition step effected by MutS-homologous Msh proteins, homologs of the bacterial MutL ATPase (predominantly the Mlh1p-Pms1p heterodimer in yeast) couple mismatch recognition to the appropriate downstream processing steps . To examine whether the processing steps in the spellchecker and antirecombination pathways might differ, we mutagenized the yeast PMS1 gene and screened for mitotic separation-of-function alleles . Two alleles affecting only the antirecombination function of Pms1p were identified, one of which changed an amino acid within the highly conserved ATPase domain . To more specifically address the role of ATP binding/hydrolysis in MMR-related processes, we examined mutations known to compromise the ATPase activity of Pms1p or Mlh1p with respect to the mitotic spellchecker and antirecombination activities and with respect to the repair of mismatches present in meiotic recombination intermediates . The results of these analyses confirm a differential requirement for the Pms1p ATPase activity in replication vs . recombination processes, while demonstrating that the Mlh1p ATPase activity is important for all examined MMR-related functions. Genetics, 2002 Nov, 162(3), 1079 - 89 The yeast ubiquitin protease, Ubp3p, promotes protein stability; Brew CT et al.; Stu1p is a microtubule-associated protein required for spindle assembly . In this article we show that the temperature-sensitive stu1-5 allele is synthetically lethal in combination with ubp3, gim1-gim5, and kem1 mutations . The primary focus of this article is on the stu1-5 ubp3 interaction . Ubp3 is a deubiquitination enzyme and a member of a large family of cysteine proteases that cleave ubiquitin moieties from protein substrates . UBP3 is the only one of 16 UBP genes in yeast whose loss is synthetically lethal with stu1-5 . Stu1p levels in stu1-5 cells are several-fold lower than the levels in wild-type cells and the stu1-5 temperature sensitivity can be rescued by additional copies of stu1-5 . These results indicate that the primary effect of the stu1-5 mutation is to make the protein less stable . The levels of Stu1p are even lower in ubp3Delta stu1-5 cells, suggesting that Ubp3p plays a role in promoting protein stability . We also found that ubp3Delta produces growth defects in combination with mutations in other genes that decrease protein stability . Overall, these data support the idea that