Yeasts

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 Ubp3p has a general role in the reversal of protein ubiquitination.

J Cutan Pathol, 2002 Nov, 29(10), 616 - 8
Cutaneous lesions showing giant yeast forms of Blastomyces dermatitidis; Walker K et al.; BACKGROUND: The yeast forms of Blastomyces dermatitidis usually range from 8 to 15-20 micro m in diameter . Larger yeast forms have previously been reported only twice in immunosuppressed patients . In both patients these large forms were seen within the lung . CASE REPORT: We present a 14-year-old cardiac transplant patient, who presented 36 days following his transplantation with acute respiratory distress followed a few days later by erythematous cutaneous papules . RESULTS: Biopsy of a skin lesion showed yeast forms, some greater than 40 micro m in diameter, within and surrounding dermal vessels . Cultures later grew Blastomyces dermatitidis . CONCLUSIONS: To our knowledge this is the first reported case of giant forms of Blastomyces dermatitidis within the skin . With increased iatrogenic immunosuppression, we may expect to see more diverse morphologic forms with deep fungal infections.

Mol Microbiol, 2002 Dec, 46(5), 1429 - 40
The yeast zinc finger regulators Pdr1p and Pdr3p control pleiotropic drug resistance (PDR) as homo- and heterodimers in vivo; Mamnun YM et al.; The transcription factors Pdr1p and Pdr3p from Saccharomyces cerevisiae mediate pleiotropic drug resistance (PDR) by controlling expression of ATP-binding cassette (ABC) transporters such as Pdr5p, Snq2p and Yor1p . Previous in vitro studies demonstrated that Pdr1p and Pdr3p recognize so-called pleiotropic drug resistance elements (PDREs) in the promoters of target genes . In this study, we show that both Pdr1p and Pdr3p are phosphoproteins; Pdr3p isoforms migrate as two bands in gel electrophoresis, reflecting two distinct phosphorylation states . Most importantly, native co-immunoprecipitation experiments, using functional epitope-tagged Pdr1p/Pdr3p variants, demonstrate that Pdr1p and Pdr3p can form both homo- and heterodimers in vivo . Furthermore, in vivo footprinting of PDRE-containing promoters demonstrate that Pdr1p/Pdr3p constitutively occupy both perfect and degenerate PDREs in vivo . Thus, in addition to interaction with other regulators, differential dimerization provides a plausible explanation for the observation that Pdr3p and Pdr1p can both positively and negatively control PDR promoters with different combinations of perfect and degenerate PDREs.

Pigment Cell Res, 2002 Dec, 15(6), 405 - 19
Hermansky-Pudlak syndrome: vesicle formation from yeast to man; Huizing M et al.; The disorders known as Hermansky-Pudlak syndrome (HPS) are a group of genetic diseases resulting from abnormal formation of intracellular vesicles . In HPS, dysfunction of melanosomes results in oculocutaneous albinism, and absence of platelet dense bodies causes a bleeding diathesis . In addition, some HPS patients suffer granulomatous colitis or fatal pulmonary fibrosis, perhaps due to mistrafficking of a subset of lysosomes . The impaired function of specific organelles indicates that the causative genes encode proteins operative in the formation of certain vesicles . Four such genes, HPS1, ADTB3A, HPS3, and HPS4, are associated with the four known subtypes of HPS, i.e . HPS-1, HPS-2, HPS-3, and HPS-4 . ADTB3A codes for the beta 3 A subunit of adaptor complex-3, known to assist in vesicle formation from the trans-Golgi network or late endosome . However, the functions of the HPS1, HPS3, and HPS4 gene products remain unknown . These three genes arose with the evolution of mammals and have no homologs in yeast, reflecting their specialized function . In contrast, all four known HPS-causing genes have homologs in mice, a species with 14 different models of HPS, i.e . hypopigmentation and a platelet storage pool deficiency . Pursuit of the mechanism of mammalian vesicle formation and trafficking, impaired in HPS, relies upon investigation of these mouse models as well as studies of protein complexes involved in yeast vacuole formation.

Traffic, 2002 Dec, 3(12), 922 - 9
A new yeast endosomal SNARE related to mammalian syntaxin 8; Lewis MJ et al.; We report the identification of a yeast SNARE that has escaped notice because of an apparent error in the genome sequence and because it is functionally redundant . It is encoded by an extended version of ORF YAL014c, and since its SNARE motif is related to mammalian syntaxin 8 we term the gene SYN8 . Syn8p is in endosomes . Co-precipitation indicates a set of complexes containing Pep12p, Vti1p, either Syn8p or Tlg1p and either Snc1p or Ykt6p . Analysis of growth and trafficking defects demonstrates that in the absence of Tlg1p, Syn8p is required for Pep12p function . Conversely, when Tlg1p is present, Syn8p can be removed without loss of Pep12p function, or induction of any other obvious trafficking defect . Syn8p thus appears to be a functional homolog of mammalian syntaxin 8, but Tlg1p can, amongst other roles, provide an equivalent function.

J Appl Microbiol, 2002, 93(6), 1020 - 5
Extracellular enzymatic activity profiles in yeast and yeast-like strains isolated from tropical environments; Buzzini P et al.; AIMS: The objective of this study was to investigate the extracellular enzymatic activity (EEA) profile of yeasts isolated from tropical environments of the Brazilian rain forest . This screening survey could constitute the first approach in selecting yeast strains of environmental origin potentially exploitable as enzyme producers . METHODS AND RESULTS: In this study, 348 yeast (193 ascomycetes and 155 basidiomycetes) and 46 yeast-like strains (Aureobasidium pullulans) were screened for their EEA profile . The spread occurrence of extracellular amylases, esterases, lipases, proteases, pectinases and chitinases appeared to be a strain-related character . CONCLUSIONS: Yeasts isolated from tropical environments could represent a promising source of EEA . Selected strains showed maximum levels of EEA under acidic or neutral conditions . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the potential for yeasts isolated from extreme environments as sources of industrially relevant enzymes for biotechnological purposes.

Planta Med, 2002 Nov, 68(11), 1039 - 41
Differential activation of protein kinase C isoforms by euxanthone, revealed by an in vivo yeast phenotypic assay; Saraiva L et al.; The protein kinase C (PKC) modulatory effects of euxanthone, isolated from the wood of Cratoxylum maingayi, on isoforms alpha, betaI, delta, eta and zeta were characterised using an alternative in vivo yeast phenotypic assay . The present study shows that euxanthone can activate isoforms alpha, betaI, delta, eta and zeta, being more effective on PKC-betaI, -delta, -eta and -zeta than the established PKC activators used (the phorbol ester PMA and arachidonic acid for PKC-zeta) . Furthermore, euxanthone presents differences on its potency towards individual PKC isoforms, showing a remarkable selectivity for PKC-zeta . These results can help to clarify the molecular basis of the euxanthone-mediated effects.

Mol Cell Biol, 2002 Dec, 22(24), 8774 - 86
The novel SLIK histone acetyltransferase complex functions in the yeast retrograde response pathway; Pray-Grant MG et al.; The SAGA complex is a conserved histone acetyltransferase-coactivator that regulates gene expression in Saccharomyces cerevisiae . SAGA contains a number of subunits known to function in transcription including Spt and Ada proteins, the Gcn5 acetyltransferase, a subset of TATA-binding-protein-associated factors (TAF(II)s), and Tra1 . Here we report the identification of SLIK (SAGA-like), a complex related in composition to SAGA . Notably SLIK uniquely contains the protein Rtg2, linking the function of SLIK to the retrograde response pathway . Yeast harboring mutations in both SAGA and SLIK complexes displays synthetic phenotypes more severe than those of yeast with mutation of either complex alone . We present data indicating that distinct forms of the SAGA complex may regulate specific subsets of genes and that SAGA and SLIK have multiple partly overlapping activities, which play a critical role in transcription by RNA polymerase II.

Mol Cell Biol, 2002 Dec, 22(24), 8415 - 25
A sequence element downstream of the yeast HTB1 gene contributes to mRNA 3' processing and cell cycle regulation; Campbell SG et al.; Histone mRNAs accumulate in the S phase and are rapidly degraded as cells progress into the G(2) phase of the cell cycle . In Saccharomyces cerevisiae, fusion of the 3' untranslated region and downstream sequences of the yeast histone gene HTB1 to a neomycin phosphotransferase open reading frame is sufficient to confer cell cycle regulation on the resulting chimera gene (neo-HTB1) . We have identified a sequence element, designated the distal downstream element (DDE), that influences both the 3'-end cleavage site selection and the cell cycle regulation of the neo-HTB1 mRNA . Mutations in the DDE, which is located approximately 110 nucleotides downstream of the HTB1 gene, lead to a delay in the accumulation of the neo-HTB1 mRNA in the S phase and a lack of mRNA turnover in the G(2) phase . The DDE is transcribed as part of the primary transcript and binds a protein factor(s) . Maximum binding is observed in the S phase of the cell cycle, and mutations that affect the turnover of the HTB1 mRNA alter the binding activity . While located in the same general region, mutations that affect 3'-end cleavage site selection act independently from those that alter the cell cycle regulation.

J Biol Chem, 2003 Jan 31, 278(5), 3489 - 96 Epub 2002 Nov 22.
Translocation of the C terminus of a tail-anchored protein across the endoplasmic reticulum membrane in yeast mutants defective in signal peptide-driven translocation; Yabal M et al.; C-tail-anchored proteins are defined by an N-terminal cytosolic domain followed by a transmembrane anchor close to the C terminus . Their extreme C-terminal polar residues are translocated across membranes by poorly understood post-translational mechanism(s) . Here we have used the yeast system to study translocation of the C terminus of a tagged form of mammalian cytochrome b(5), carrying an N-glycosylation site in its C-terminal domain (b(5)-Nglyc) . Utilization of this site was adopted as a rigorous criterion for translocation across the ER membrane of yeast wild-type and mutant cells . The C terminus of b(5)-Nglyc was rapidly glycosylated in mutants where Sec61p was defective and incapable of translocating carboxypeptidase Y, a well known substrate for post-translational translocation . Likewise, inactivation of several other components of the translocon machinery had no effect on b(5)-Nglyc translocation . The kinetics of translocation were faster for b(5)-Nglyc than for a signal peptide-containing reporter . Depletion of the cellular ATP pool to a level that retarded Sec61p-dependent post-translational translocation still allowed translocation of b(5)-Nglyc . Similarly, only low ATP concentrations (below 1 microm), in addition to cytosolic protein(s), were required for in vitro translocation of b(5)-Nglyc into mammalian microsomes . Thus, translocation of tail-anchored b(5)-Nglyc proceeds by a mechanism different from that of signal peptide-driven post-translational translocation.

J Biol Chem, 2003 Feb 14, 278(7), 5009 - 20 Epub 2002 Nov 20.
Vps51 is part of the yeast Vps fifty-three tethering complex essential for retrograde traffic from the early endosome and Cvt vesicle completion; Reggiori F et al.; Autophagy, pexophagy, and the Cvt pathway are processes that deliver hydrolytic enzymes and substrates to the yeast vacuole/lysosome via double-membrane cytosolic vesicles . Whereas these pathways operate under different nutritional conditions, they all employ common machinery with only a few specific factors assisting in the choice of the delivery program and the membrane source for the sequestering vesicle . We found that the YKR020w gene product is essential for Cvt vesicle formation but not for pexophagy or induction of autophagy . Autophagosomes in the ykr020wdelta mutant, however, have a reduced size . We demonstrate that Ykr020 is a subunit of the Vps fifty-three tethering complex, composed of Vps52, Vps53, and Vps54, which is required for retrograde traffic from the early endosome back to the late Golgi, and for this reason we named it Vps51 . This complex participates in a fusion event together with Tlg1 and Tlg2, two SNAREs also shown to be necessary for Cvt vesicle assembly . In particular, those factors are essential to correctly target the prApe1-Cvt19-Cvt9 complex to the preautophagosomal structure, the site of Cvt vesicle formation.

J Invertebr Pathol, 2002 Oct, 81(2), 70 - 7
Beauveria bassiana yeast phase on agar medium and its pathogenicity against Diatraea saccharalis (Lepidoptera: Crambidae) and Tetranychus urticae (Acari: Tetranychidae); Alves SB et al.; Beauveria bassiana colonizes insect hosts initially through a yeast phase, which is common in some artificial liquid cultures, but not reported on artificial solid media . We describe a yeast-like phase for B . bassiana isolate 447 (ATCC 20872) on MacConkey agar and its virulence toward Diatraea saccharalis and Tetranychus urticae . The yeast-like cells of B . bassiana developed by budding from germinating conidia after 24-h incubation . Cells were typically 5-10 microm and fungal colonies were initially circular and mucoid, but later were covered with mycelia and conidia . Ability to produce yeast-like cells on MacConkey medium was relatively common among different B . bassiana isolates, but growth rate and timing of yeast-like cell production also varied . Metarhizium anisopliae and Paecilomyces spp . isolates did not grow as yeast-like cells on MacConkey medium . Yeast-like cells of B . bassiana 447 were more virulent against D . saccharalis than conidia when 10(7)cells/ml were used . At 10(8)cells/ml, the estimated mean survival time was 5.4 days for the yeast suspension and 7.7 days for the conidial suspension, perhaps due to faster germination . The LC(50) was also lower for yeast than conidial suspensions . Yeast-like cells and conidia had similar virulence against T . urticae; the average mortalities with yeast-like cells and conidia were, respectively, 42.8 and 45.0%, with 10(7)cells/ml, and 77.8 and 74.4%, with 10(8)cells/ml . The estimated mean survival times were 3.6 and 3.9 for yeast and conidial suspensions, respectively . The bioassay results demonstrate the yeast-like structures produced on MacConkey agar are effective as inoculum for B . bassiana applications against arthropod pests, and possibly superior to conidia against some species . Obtaining well-defined yeast phase cultures of entomopathogenic hyphomycetes may be an important step in studies of the biology and nutrition, pathogenesis, and the genetic manipulation of these fungi.

Biol Reprod, 2002 Dec, 67(6), 1936 - 42
Sperm PP1gamma2 is regulated by a homologue of the yeast protein phosphatase binding protein sds22; Huang Z et al.; Serine/threonine phosphatase PP1gamma2 is a testis-specific protein phosphatase isoform in spermatozoa . This enzyme appears to play a key role in motility initiation and stimulation . Catalytic activity of PP1gamma2 is higher in immotile compared with motile spermatozoa . Inhibition of PP1gamma2 activity causes both motility initiation and motility stimulation . Protein phosphatases, in general, are regulated by their binding proteins . The objective of this article is to understand the mechanisms by which PP1gamma2 is regulated, first by identifying its regulatory proteins . We had previously shown that a portion of bovine sperm PP1gamma2 is present in the cytosolic fraction of sperm sonicates . We purified PP1gamma2 from soluble bovine sperm extracts by immunoaffinity chromatography . Gel electrophoresis of the purified enzyme showed that it was complexed to a protein 43 M(r) x 10(-3) in size . Microsequencing revealed that this protein is a mammalian homologue of sds22, which is a yeast PP1 binding protein . Phosphatase activity measurements showed that PP1gamma2 complexed to sds22 is catalytically inactive . The complex cannot be activated by limited proteolysis . The complex is unable to bind to microcystin sepharose . This suggests that sds22 may block the microcystin binding site in PP1gamma2 . A proportion of PP1gamma2 in sperm extracts, which is presumably not complexed to sds22, is catalytically active . Fluorescence immunocytochemistry was used to determine the intrasperm localization of PP1gamma2 and sds22 . Both proteins are present in the tail . They are also present in distinct locations in the head . Our data suggest that PP1gamma2 binding to sds22 inhibits its catalytic activity . Mechanisms regulating sds22 binding to PP1gamma2 are likely to be important in understanding the biochemical basis underlying development and regulation of sperm function.

J Biotechnol, 2003 Feb 13, 100(3), 239 - 50
Applying the Taguchi robust design to the optimization of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers' yeast; Houng JY et al.; This study examined the characteristics and operational parameters of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers' yeast in order to produce S-4-chloro-3-hydroxybutyric acid ethyl ester . Eight operational variables were also optimized using the Taguchi method with consideration of the freshness of yeast cells as a noise factor . An L(18) orthogonal array was used to design the experiments . The reaction yield and the product's optical purity were considered as two product quality variables . A desirability function was applied to combine these two qualities as a single objective function . Additionally, the signal-to-noise (SN) ratio was used to estimate the variability in product quality . Optimization was undertaken not only to yield the best performance, but also to minimize the variation in quality . The confirmation experiments indicated that the reaction performance and the robustness of the product quality under the optimized conditions were higher than those obtained in other experiments in this study . Our results further demonstrate that the product's optical purity could be increased to >95% by adjusting the operational level of the main factors.

DNA Cell Biol, 2002 Oct, 21(10), 737 - 42
HBV C promoter Sp1 binding sequence functionally substitutes for the yeast ARS1 ABF1 binding site; Yan P et al.; Transcriptional factors have been implicated in eukaryotic DNA replication . We have studied the potential function of a viral promoter sequence in DNA replication . The hepatitis B virus (HBV) pregenomic promoter is regulated by two enhancers and cis-elements . The G-C rich region between 1734-1754 nt, which contains two SP1 binding sites, is necessary for transcription origin and HBV replication . We found that the Abf1-binding B3 element in yeast ARS1 can be functionally replaced by the viral Sp1-binding DNA sequence, which activates transcription from the HBV C promoter . Further, yeast RAP1 bound to the viral Sp1 binding sites in vitro . These results suggest that RAP1 binds to the Sp1 binding sites and stimulates yeast DNA replication.

Steroids, 2002 Dec, 67(13-14), 1109 - 19
Alternative pathways of sterol synthesis in yeast . Use of C(27) sterol tracers to study aberrant double-bond migrations and evaluate their relative importance; Ruan B et al.; Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations . We studied sterols generated in the absence of the delta(8)-delta(7) isomerase (Erg2p) or delta(5) desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5 alpha-cholest-8-en-3beta-ol, 8-dehydrocholesterol (delta(5,8) sterol), or isodehydrocholesterol (delta(6,8) sterol), together with the corresponding 3 alpha-3H isotopomer . Nine different incubations gave altogether 16 sterol metabolites, including seven delta(22E) sterols formed by action of the yeast C-22 desaturase (Erg5p) . These products were separated by silver-ion high performance liquid chromatography (Ag(+)-HPLC) and identified by gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag(+)-HPLC . When delta(8)-delta(7) isomerization was blocked, exogenous delta(8) sterol underwent desaturation to delta(5,8), delta(6,8), and delta(8,14) sterols . Formation of delta(5,8) sterol was strongly favored over delta(6,8) sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis . The delta(5,8) sterol was metabolically almost inert except for delta(22) desaturation, whereas the delta(6,8) sterol was readily converted to delta(5,7), delta(5,7,9(11)), and delta(7,9(11)) sterols . The combined results indicate aberrant metabolic pathways similar to those in mammalian systems . However, delta(5,7) sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of delta(5,8) and delta(5,7,9(11)) sterols in wild-type yeast compared to Smith-Lemli-Opitz individuals.

Biochem Soc Trans, 2002 Nov, 30(Pt 6), 1080 - 2
Yeast desaturases; Martin CE et al.; The Saccharomyces OLE1 gene encodes the intrinsic membrane-bound Delta-9 fatty acid desaturase . OLE1 expression is regulated at the levels of transcription and mRNA stability by nutrient fatty acids and molecular oxygen . Its transcription is controlled through two distinct promoter elements, the fatty acid response element (FAR) region, and a downstream low-oxygen response element (LORE) that dramatically amplifies FAR-activated expression under hypoxic or cobalt-stimulated growth conditions . Transcription activation through both elements is repressed by unsaturated fatty acids . The half-life of the OLE1 mRNA is also dramatically reduced upon exposure to unsaturated fatty acids . OLE1 expression is governed by two homologous membrane-bound proteins, Spt23p and Mga2p, which activate OLE1 expression through N-terminal polypeptides that are released from the membrane through a ubiquitin-mediated mechanism that involves processing by the 23 S proteosome . Although proteolytic processing of Spt23p can be repressed by polyunsaturated fatty acids, Mga2p processing in normoxic cells appears to be regulated by a different mechanism . Mga2p is essential, however, for the induction of the high levels of expression that are triggered by hypoxia through the LORE promoter element . Surprisingly, Mga2p also plays a critical role in controlling OLE1 mRNA stability, suggesting that there may be a functional linkage between OLE1 transcription and the regulation of OLE1 mRNA stability.

Z Naturforsch {C}, 2002 Sep-Oct, 57(9-10), 960 - 1
A modified procedure for isolation of yeast mitochondrial DNA; Nedeva T et al.; A modified, rapid and inexpensive method for preparation of mitochondrial DNA (mtDNA), suitable for molecular analysis is proposed . It comprises batch cultivation of Saccharomyces cerevisiae strain NBIMCC 583 on a simple nutrient medium at 28 degrees C; permeabialization of cells from late exponential growth phase with cetyltrimethylamonnium bromide, mechanical disintegration of the cell wall; preparation of a mitochondrial fraction and subsequent isolation and purification of mtDNA . The amount and the purity of the obtained mtDNA have been checked and its application for molecular analysis proven . The main advantages of the proposed procedure for isolation of mtDNA are introduction of simple nutrient medium, replacement of the enzymatic lysis of the cell wall by the cheaper mechanical one, avoidance of ultracentrifugation steps and use of harmful chemical substances.

Biochem J, 2002 Dec 1, 368(Pt 2), 433 - 46
Cell population modelling of yeast glycolytic oscillations; Henson MA et al.; We investigated a cell-population modelling technique in which the population is constructed from an ensemble of individual cell models . The average value or the number distribution of any intracellular property captured by the individual cell model can be calculated by simulation of a sufficient number of individual cells . The proposed method is applied to a simple model of yeast glycolytic oscillations where synchronization of the cell population is mediated by the action of an excreted metabolite . We show that smooth one-dimensional distributions can be obtained with ensembles comprising 1000 individual cells . Random variations in the state and/or structure of individual cells are shown to produce complex dynamic behaviours which cannot be adequately captured by small ensembles.

Immunogenetics, 2002 Nov, 54(8), 562 - 9 Epub 2002 Oct 02.
The model B6(dom1) minor histocompatibility antigen is encoded by a mouse homolog of the yeast STT3 gene; McBride K et al.; The B6(dom1) minor histocompatibility antigen (MiHA) is a model antigen, since it is both the epitome of an immunodominant epitope and an ideal target for adoptive cancer immunotherapy . Based on DNA sequencing and MS/MS analyses, we report that B6(dom1) corresponds to amino acids 770-778 (KAPDNRETL) of a protein we propose to call SIMP (source of immunodominant MHC-associated peptides) that is encoded by a mouse homolog of the yeast STT3gene . STT3, a member of the oligosaccharyltransferase complex, is essential for cell proliferation . Phenotypic and genotypic analyses among eight strains of mice revealed a precise correlation between susceptibility or resistance to B6(dom1)-specific cytotoxic T lymphocytes (CTLs) and the presence of a Glu vs Asp amino acid at position 776 of the SIMP protein, respectively . Strikingly, while the difference in the amino acid sequence 770-778 encoded by the two SIMP alleles represents a very conservative substitution, these allelic peptides were not crossreactive at the CTL level, and both peptides were immunodominant when presented to mice homozygous for the opposite allele . In addition, we have cloned a human ortholog of SIMP whose predicted protein shares 97% amino acid identity with mouse SIMP . These results strengthen the concept that MHC class-I-associated MiHAs originate as a consequence of rare polymorphisms among highly conserved genes . Furthermore, the notion that a peptide differing from a self analog by a single methylene group can be immunodominant has implications regarding our understanding of the mechanisms of immunodominance.

Science, 2002 Nov 15, 298(5597), 1412 - 4
Requirement of Hos2 histone deacetylase for gene activity in yeast; Wang A et al.; Histone deacetylases, typified by class I Rpd3 in the yeast Saccharomyces cerevisiae, have historically been associated with gene repression . We now demonstrate that Hos2, another member of the class I family, binds to the coding regions of genes primarily during gene activation, when it specifically deacetylates the lysines in H3 and H4 histone tails . Moreover, Hos2 is preferentially associated with genes of high activity genome-wide . We also show that Hos2 and an associated factor, Set3, are necessary for efficient transcription . Therefore, our data indicate that, in contrast to other class I histone deacetylases, Hos2 is directly required for gene activation.

Proc Natl Acad Sci U S A, 2002 Nov 26, 99(24), 15381 - 6 Epub 2002 Nov 13.
SWI/SNF-dependent long-range remodeling of yeast HIS3 chromatin; Kim Y et al.; Current models for the role of the SWISNF chromatin remodeling complex in gene regulation are focused on promoters, where the most obvious changes in chromatin structure occur . Here we present evidence that the SWISNF complex is involved in the remodeling of the chromatin structure of an entire gene in vivo . We compared the native chromatin structures of a small yeast plasmid containing the HIS3 gene purified from uninduced and induced cells . Relative to uninduced chromatin, induced chromatin displayed a large reduction in negative supercoiling, a large reduction in sedimentation rate, and increased accessibility to restriction enzymes with sites located both near and far from the HIS3 promoter . These observations indicate that the entire plasmid was remodeled as a result of induction . Loss of supercoiling required the presence of the SWISNF remodeling complex and the activator Gcn4p in vivo . The TATA boxes were not required, suggesting that remodeling was not the result of transcription . The induction-dependent loss of negative supercoiling was not apparent in cells, indicating that the supercoils were lost preferentially from induced chromatin during purification . Thus, induced HIS3 chromatin has a highly labile structure that is revealed as a result of purification . It is concluded that induction of HIS3 creates a domain of labile chromatin structure that extends far beyond the promoter to include the entire gene . We propose that the SWISNF complex is recruited to the HIS3 promoter by Gcn4p and then directs remodeling of a chromatin domain, with important implications for transcription.

Int J Antimicrob Agents, 2002 Nov, 20(5), 375 - 9
Ciclopiroxolamine: in vitro antifungal activity against clinical yeast isolates; Carrillo-Munoz AJ et al.; The in vitro susceptibility of 225 clinical isolates of yeasts to ciclopiroxolamine (CPO) was compared with that of clotrimazole, econazole, ketoconazole, miconazole, tioconazole, fluconazole, itraconazole and nystatin using a standardized agar diffusion method (NeoSensitabs) . Two hundred and eight strains of yeasts comprising 16 species of Candida and 22 strains belonging to other yeast genera were tested . One strain (0.4%) was resistant, four strains (1.8%) of intermediate susceptibility and 220 strains (97.3%) susceptible to CPO . More strains were susceptible to CPO than to the other antifungals studied . Susceptibility patterns of antifungal agents were not linked to species . The in vitro antifungal susceptibility profile of CPO was better than topical azole derivatives or fluconazole and itraconazole against a wide variety of clinically important yeasts.

BMC Bioinformatics . 2002 Nov 13;3(1):35.
FunSpec: a web-based cluster interpreter for yeast; Robinson MD et al.; BACKGROUND: For effective exposition of biological information, especially with regard to analysis of large-scale data types, researchers need immediate access to multiple categorical knowledge bases and need summary information presented to them on collections of genes, as opposed to the typical one gene at a time . RESULTS: We present here a web-based tool (FunSpec) for statistical evaluation of groups of genes and proteins (e.g . co-regulated genes, protein complexes, genetic interactors) with respect to existing annotations (e.g . functional roles, biochemical properties, localization) . FunSpec is available online at CONCLUSION: FunSpec is helpful for interpretation of any data type that generates groups of related genes and proteins, such as gene expression clustering and protein complexes, and is useful for predictive methods employing "guilt-by-association."

Cell Cycle, 2002 Mar-Apr, 1(2), 132 - 6
Improved flow cytometric analysis of the budding yeast cell cycle; Haase SB et al.; The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation . Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression . However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry . Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast . Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.

Mol Biol Cell, 2002 Nov, 13(11), 4074 - 87
Cofilin, but not profilin, is required for myosin-I-induced actin polymerization and the endocytic uptake in yeast; Idrissi FZ et al.; Mutations in the budding yeast myosins-I (MYO3 and MYO5) cause defects in the actin cytoskeleton and in the endocytic uptake . Robust evidence also indicates that these proteins induce Arp2/3-dependent actin polymerization . Consistently, we have recently demonstrated, using fluorescence microscopy, that Myo5p is able to induce cytosol-dependent actin polymerization on the surface of Sepharose beads . Strikingly, we now observed that, at short incubation times, Myo5p induced the formation of actin foci that resembled the yeast cortical actin patches, a plasma membrane-associated structure that might be involved in the endocytic uptake . Analysis of the machinery required for the formation of the Myo5p-induced actin patches in vitro demonstrated that the Arp2/3 complex was necessary but not sufficient in the assay . In addition, we found that cofilin was directly involved in the process . Strikingly though, the cofilin requirement seemed to be independent of its ability to disassemble actin filaments and profilin, a protein that closely cooperates with cofilin to maintain a rapid actin filament turnover, was not needed in the assay . In agreement with these observations, we found that like the Arp2/3 complex and the myosins-I, cofilin was essential for the endocytic uptake in vivo, whereas profilin was dispensable.

Mol Biol Cell, 2002 Nov, 13(11), 3901 - 14
Ammonia pulses and metabolic oscillations guide yeast colony development; Palkova Z et al.; On solid substrate, growing yeast colonies alternately acidify and alkalinize the medium . Using morphological, cytochemical, genetic, and DNA microarray approaches, we characterized six temporal steps in the "acid-to-alkali" colony transition . This transition is connected with the production of volatile ammonia acting as starvation signal between colonies . We present evidence that the three membrane proteins Ato1p, Ato2p, and Ato3p, members of the YaaH family, are involved in ammonia production in Saccharomyces cerevisiae colonies . The acid-to-alkali transition is connected with decrease of mitochondrial oxidative catabolism and by peroxisome activation, which in parallel with activation of biosynthetic pathways contribute to decrease the general stress level in colonies . These metabolic features characterize a novel survival strategy used by yeast under starvation conditions prevalent in nature.

J Biol Chem, 2003 Jan 31, 278(5), 3339 - 46 Epub 2002 Nov 11.
Whole genome expression profiles of yeast RNA polymerase II core subunit, Rpb4, in stress and nonstress conditions; Pillai B et al.; Organisms respond to environmental stress by adopting changes in gene expression at the transcriptional level . Rpb4, a nonessential subunit of the core RNA polymerase II has been proposed to play a role in non-stress-specific transcription and in the regulation of stress response in yeast . We find that in addition to the temperature sensitivity of the null mutant of Rpb4, diploid null mutants are also compromised in sporulation and show morphological changes associated with nitrogen starvation . Using whole genome expression analysis, we report here the effects of Rpb4 on expression of genes during normal growth and following heat shock and nutritional starvation . Our analysis shows that Rpb4 affects expression of a small yet significant fraction of the genome in both stress and normal conditions . We found that genes involved in galactose metabolism were dependent on the presence of Rpb4 irrespective of the environmental condition . Rpb4 was also found to affect the expression of several other genes specifically in conditions of nutritional starvation . The general defect in the absence of Rpb4 is in the expression of metabolic genes, especially those involved in carbon metabolism and energy generation . We report that various stresses are affected by RPB4 and that on overexpression the stress-specific activators can partially rescue the corresponding defects.

Genome Biol . 2002 Oct 10;3(11):RESEARCH0059 . Epub 2002 Oct 10.
Exploring the conditional coregulation of yeast gene expression through fuzzy k-means clustering; Gasch AP et al.; BACKGROUND: Organisms simplify the orchestration of gene expression by coregulating genes whose products function together in the cell . Many proteins serve different roles depending on the demands of the organism, and therefore the corresponding genes are often coexpressed with different groups of genes under different situations . This poses a challenge in analyzing whole-genome expression data, because many genes will be similarly expressed to multiple, distinct groups of genes . Because most commonly used analytical methods cannot appropriately represent these relationships, the connections between conditionally coregulated genes are often missed . RESULTS: We used a heuristically modified version of fuzzy k-means clustering to identify overlapping clusters of yeast genes based on published gene-expression data following the response of yeast cells to environmental changes . We have validated the method by identifying groups of functionally related and coregulated genes, and in the process we have uncovered new correlations between yeast genes and between the experimental conditions based on similarities in gene-expression patterns . To investigate the regulation of gene expression, we correlated the clusters with known transcription factor binding sites present in the genes' promoters . These results give insights into the mechanism of the regulation of gene expression in yeast cells responding to environmental changes . CONCLUSIONS: Fuzzy k-means clustering is a useful analytical tool for extracting biological insights from gene-expression data . Our analysis presented here suggests that a prevalent theme in the regulation of yeast gene expression is the condition-specific coregulation of overlapping sets of genes.

Microbiology, 2002 Nov, 148(Pt 11), 3725 - 35
YlBMH1 encodes a 14-3-3 protein that promotes filamentous growth in the dimorphic yeast Yarrowia lipolytica; Hurtado CA et al.; Most pathogenic fungi have the ability to alternate between a unicellular yeast form and different filamentous forms (hyphae and pseudohyphae) . This attribute is generally regarded as an important virulence factor and has also attracted attention because of its implications in the study of eukaryotic cell differentiation . To identify genes that are involved in the regulation of these events, chemical mutagenesis of the dimorphic yeast Yarrowia lipolytica was performed and morphological mutants that were unable to form hyphal cells were isolated . Screening of a Y . lipolytica genomic DNA library for genes able to complement this defect led to the isolation of YlBMH1, a gene encoding a 14-3-3 protein and whose transcription levels are increased during the yeast-to-hypha transition . Remarkably, overexpression of YlBMH1 was able to enhance pseudohyphae formation in a strain lacking functional YlRAC1 but caused no visible effects in deltamhy1 and deltabem1 cells, thus suggesting that YlBMH1 is involved in the regulation of both hyphal and pseudohyphal growth in Y . lipolytica . The identification of YlBMH2, a gene encoding a second 14-3-3 protein (YlBmh2p) that contains a 19 aa insertion absent in all other members of the 14-3-3 family, is also reported . Differently from YlBMH1, the transcription levels of YlBMH2 do not show any apparent variation during the induction of hyphal growth, and its overexpression has no effects on cells lacking functional MHY1, YlRAC1 or YlBEM1 . Taken together, these observations suggest that, in spite of their high conservation, YlBmh1p and YlBmh2p have different cellular functions.

Biochemistry, 2002 Nov 19, 41(46), 13681 - 9
Tyr266 in the sixth transmembrane domain of the yeast alpha-factor receptor plays key roles in receptor activation and ligand specificity; Lee BK et al.; To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapeptide ligand, alpha-factor (WHWLQLKPGQPMY), a variety of alpha-factor analogues were used in conjunction with site-directed mutagenesis of a targeted portion of Ste2p transmembrane domain six . Alanine substitution of residues in the 262-270 region of Ste2p did not affect pheromone binding or signal transduction, except for the Y266A mutant, which did not transduce signal yet exhibited only a small decrease in alpha-factor binding affinity . Substitutions with Ser, Leu, or Lys at Y266 also generated signaling-defective receptors . In contrast, Phe or Trp substitution at Y266 retained receptor function, suggesting that aromaticity at this position was critical . When coexpressed with WT receptor, the Y266A receptor exhibited a strong dominant-negative phenotype, indicating that this mutant bound G protein . A partial tryptic digest revealed that, in the presence of agonist, a different digestion profile for Y266A receptor was generated in comparison to that for WT receptor . The difference in trypsin-sensitive sites and their negative dominance indicated that the Y266A receptor was not able to switch into an "activated" conformation upon ligand binding . In comparison to WT Ste2p, the mutantY266A receptor showed increased binding affinity for N-terminal, alanine-substituted alpha-factor analogues (residues 1-4) and the antagonist {desW(1),desH(2)}alpha-factor . A substantial decrease in affinity was observed for alpha-factor analogues with Ala substitutions from residues 5-13 . The results suggest that Y266 is part of the binding pocket that recognizes the N-terminal portion of alpha-factor and is involved in the transformation of Ste2p into an activated state upon agonist binding.

EMBO J, 2002 Nov 15, 21(22), 6015 - 24
Spindle-kinetochore attachment requires the combined action of Kin I-like Klp5/6 and Alp14/Dis1-MAPs in fission yeast; Garcia MA et al.; Fission yeast Klp5 and Klp6 belong to the microtubule-destabilizing Kin I family . In klp5 mutants, spindle checkpoint proteins Mad2 and Bub1 are recruited to mitotic kinetochores for a prolonged duration, indicating that these kinetochores are unattached . Further analysis shows that there are kinetochores to which only Bub1, but not Mad2, localizes . These kinetochores are likely to have been captured, yet lack tension . Thus Klp5 and Klp6 play a role in a spindle- kinetochore interaction at dual steps, capture and generation of tension . The TOG/XMAP215 family, Alp14 and Dis1 are known to stabilize microtubules and be required for the bivalent attachment of the kinetochore to the spindle . Despite apparent opposing activities towards microtubule stability, Klp5/Klp6 and Alp14/Dis1 share an essential function, as either dis1klp or alp14klp mutants are synthetically lethal, like alp14dis1 . Defective phenotypes are similar to each other, characteristic of attachment defects and chromosome mis-segregation . Furthermore Alp14 is of significance for kinetochore localization of Klp5 . We propose that Klp5/Klp6 and Alp14/Dis1 play a collaborative role in bipolar spindle formation during prometaphase through producing spindle dynamism.

J Biol Chem, 2003 Jan 17, 278(3), 1603 - 11 Epub 2002 Nov 07.
The yeast mitochondrial degradosome . Its composition, interplay between RNA helicase and RNase activities and the role in mitochondrial RNA metabolism; Dziembowski A et al.; The yeast mitochondrial degradosome (mtEXO) is an NTP-dependent exoribonuclease involved in mitochondrial RNA metabolism . Previous purifications suggested that it was composed of three subunits . Our results suggest that the degradosome is composed of only two large subunits: an RNase and a RNA helicase encoded by nuclear genes DSS1 and SUV3, respectively, and that it co-purifies with mitochondrial ribosomes . We have found that the purified degradosome has RNA helicase activity that precedes and is essential for exoribonuclease activity of this complex . The degradosome RNase activity is necessary for mitochondrial biogenesis but in vitro the degradosome without RNase activity is still able to unwind RNA . In yeast strains lacking degradosome components there is a strong accumulation of mitochondrial mRNA and rRNA precursors not processed at 3'- and 5'-ends . The observed accumulation of precursors is probably the result of lack of degradation rather than direct inhibition of processing . We suggest that the degradosome is a central part of a mitochondrial RNA surveillance system responsible for degradation of aberrant and unprocessed RNAs.

Gene, 2002 Oct 2, 298(2), 121 - 7
The human homolog of yeast SEP1 is a novel candidate tumor suppressor gene in osteogenic sarcoma; Zhang K et al.; The hSEP1 gene is the human homolog of yeast SEP1 . Yeast SEP1 is a multifunctional gene that regulates a variety of nuclear and cytoplasmic functions including homologous recombination, meiosis, telomere maintenance, RNA metabolism and microtubule assembly . The function of hSEP1 is not known . We show loss or reduced expression of hSEP1 messenger RNA (mRNA) in three of four primary osteogenic sarcoma (OGS)-derived cell lines and in eight of nine OGS biopsy specimen . In addition, we find a heterozygous missense mutation (Valine(1484)>Alanine) at a conserved amino acid in the primary OGS-derived cell line U2OS . Importantly, we identified a homozygous missense mutation involving a CG-dinucleotide leading to a change in a conserved amino acid, aspartic acid(1137) >asparagine, in the primary OGS-derived cell line, TE85 . hSEP1 mRNA expression was nearly undetectable in TE85 and low in U2OS cell lines . None of these mutations were identified in 20 normal samples consisting of bone, cartilage and fibroblast . The hSEP1 gene is located in chromosome 3 at 3q25-26.1 between markers D3S1309 and D3S1569 . An adjacent locus defined by the polymorphic markers D3S1212 and D3S1245 has previously been reported to undergo loss of heterozygosity (LOH) at a >70% frequency in OGS and claimed to harbor an important tumor suppressor gene in osteosarcoma . The homozygous mutation in the hSEP1 mRNA in TE85 cell line suggest that this gene itself is subject to LOH . Taken together, these results suggest that hSEP1 acts as a tumor suppressor gene in OGS.

J Biol Chem, 2003 Jan 17, 278(3), 1751 - 7 Epub 2002 Nov 06.
A novel germ line-specific gene of the phosducin-like protein (PhLP) family . A meiotic function conserved from yeast to mice; Lopez P et al.; We identified a new member of the phosducin-like (PhLP) protein family that is predominantly, if not exclusively, expressed in male and female germ cells . In situ analysis on testis sections and analysis of purified spermatogenic cell fractions evidenced a stage-specific expression with high levels of RNA and protein in pachytene spermatocytes and round spermatids . Three mRNA species were detected, which correspond to different polyadenylation sites and vary in abundance during germ cell maturation . Only low levels of RNA were detected in whole ovary extracts, but expression of the protein became detectable within hours after hormonal induction of superovulation . The gene (Mgcphlp) is located on mouse chromosome 5 in the immediate vicinity of the Clock locus . The predicted amino acid sequence shows extensive similarities not only with the known mammalian PhLP proteins but also with the yeast phosducin-like protein Plp2, required for the production and growth of haploid cells . Expression of the murine protein was found to complement the defect of a yeast plp2 Delta mutant . We propose that MgcPhLP/Plp2 proteins exert a function in germ cell maturation that is conserved from yeast to mammals.

J Biol Chem, 2003 Jan 17, 278(3), 1924 - 31 Epub 2002 Nov 06.
Telomeric DNA ends are essential for the localization of Ku at telomeres in fission yeast; Miyoshi T et al.; The Ku70-Ku80 heterodimer is a conserved protein complex essential for the non-homologous end-joining pathway . Ku proteins are also involved in telomere maintenance, although their precise roles remain to be elucidated . In fission yeast, pku70(+), the gene encoding the Ku70 homologue, has been reported . Here we report the identification and characterization of pku80(+), the gene encoding Ku80 . Both pku70(+) and pku80(+) are essential for efficient non-homologous end-joining . We also found that the pku70 and pku80 mutants are sensitive to methyl methanesulfonate and hydroxyurea, suggesting their roles in the S phase . The pku80 mutant shows telomere shortening and tandem amplification of a subtelomeric sequence but no defects in the telomere position effect, as was previously reported for the pku70 mutant . By using the chromatin immunoprecipitation assay, we demonstrated that Pku70 and Pku80 physically interact with telomeric repeats and subtelomeric sequences . Interestingly, this telomere association of Pku proteins is independent of Taz1, a telomeric DNA-binding protein . We also showed that the Pku proteins do not associate with ectopically integrated telomeric repeats in the internal region of circular chromosomes . These results indicate that the physical end of DNA is necessary for the localization of Pku80 at telomeres.

J Biol Chem, 2003 Jan 17, 278(3), 1618 - 25 Epub 2002 Nov 06.
Okazaki fragment maturation in yeast . I . Distribution of functions between FEN1 AND DNA2; Ayyagari R et al.; In the presence of proliferating cell nuclear antigen, yeast DNA polymerase delta (Pol delta) replicated DNA at a rate of 40-60 nt/s . When downstream double-stranded DNA was encountered, Pol delta paused, but most replication complexes proceeded to carry out strand-displacement synthesis at a rate of 1.5 nt/s . In the presence of the flap endonuclease FEN1 (Rad27), the complex carried out nick translation (1.7 nt/s) . The Dna2 nuclease/helicase alone did not efficiently promote nick translation, nor did it affect nick translation with FEN1 . Maturation in the presence of DNA ligase was studied with various downstream primers . Downstream DNA primers, RNA primers, and small 5'-flaps were efficiently matured by Pol delta and FEN1, and Dna2 did not stimulate maturation . However, maturation of long 5'-flaps to which replication protein A can bind required both DNA2 and FEN1 . The maturation kinetics were optimal with a slight molar excess over DNA of Pol delta, FEN1, and proliferating cell nuclear antigen . A large molar excess of DNA ligase substantially enhanced the rate of maturation and shortened the nick-translation patch (nucleotides excised past the RNA/DNA junction before ligation) to 4-6 nt from 8-12 nt with equimolar ligase . These results suggest that FEN1, but not DNA ligase, is a stable component of the maturation complex.

J Biol Chem, 2003 Jan 17, 278(3), 1626 - 33 Epub 2002 Nov 06.
Okazaki fragment maturation in yeast . II . Cooperation between the polymerase and 3'-5'-exonuclease activities of Pol delta in the creation of a ligatable nick; Jin YH et al.; To address the different functions of Pol delta and FEN1 (Rad27) in Okazaki fragment maturation, exonuclease-deficient polymerase Pol delta-01 and Pol delta-5DV (corresponding to alleles pol3-01-(D321A, E323A) and pol3-5DV-(D520V), respectively) were purified and characterized in this process . In the presence of the replication clamp PCNA, both wild-type and exo(-) Pol delta carried out strand displacement synthesis with similar rates; however, initiation of strand displacement synthesis was much more efficient with Pol delta-exo(-) . When Pol delta-exo(-) encountered a downstream primer, it paused with 3-5 nucleotides of the primer displaced, whereas the wild type carried out precise gap filling . Consequently, in the absence of FEN1, Pol delta exonuclease activity was essential for closure of simple gaps by DNA ligase . Compared with wild type, Okazaki fragment maturation with Pol delta-exo(-) proceeded with an increased duration of nick translation prior to ligation . Maturation was efficient in the absence of Dna2 and required Dna2 only when FEN1 activity was compromised . In agreement with these results, the proposed generation of double strand breaks in pol3-exo(-) rad27 mutants was suppressed by the overexpression of DNA2 . Further genetic studies showed that pol3-exo(-) rad27 double mutants were sensitive to alkylation damage consistent with an in vivo defect in gap filling by exonuclease-deficient Pol delta.

Eur J Biochem, 2002 Nov, 269(22), 5440 - 50
Bax-induced cell death in yeast depends on mitochondrial lipid oxidation; Priault M et al.; The oxidant function of pro-apoptotic protein Bax was investigated through heterologous expression in yeast . Direct measurements of fatty acid content show that Bax-expression induces oxidation of mitochondrial lipids . This effect is prevented by the coexpression of Bcl-xL . The oxidation actually could be followed on isolated mitochondria as respiration-induced peroxidation of polyunsaturated cis-parinaric acid and on whole cells as the increase in the amount of thiobarbituric acid-reactive products . Treatments that increase the unsaturation ratio of lipids, making them more sensitive to oxidation, increase kinetics of Bax-induced death . Conversely, inhibitors of lipid oxidation and treatments that decrease the unsaturation ratio of fatty acids decrease kinetics of Bax-induced death . Taken together, these results show that Bax-induced mitochondrial lipid oxidation is relevant to Bax-induced cell death . Conversely, lipid oxidation is poorly related to the massive Bax-induced superoxide and hydrogen peroxide accumulation, which occurs at the same time, as chemical or enzymatic scavenging of ROS does not prevent lipid oxidation nor has any effects on kinetics of Bax-induced cell death . Whatever the origin of mitochondrial lipid oxidation, these data show that it represents a major step in the cascade of events leading to Bax-induced cell death . These results are discussed in the light of the role of lipid oxidation both in mammalian apoptosis and in other forms of cell death in other organisms.

Acta Pharmacol Sin, 2002 Nov, 23(11), 1013 - 7
Inhibition of human phosphodiesterase 4A expressed in yeast cell GL62 by theophylline, rolipram, and acetamide-45; Wang K et al.; AIM: To study the inductive expression of human phosphodiesterase 4A (hPDE4A) in yeast cell GL62 and investigate the inhibitory effects of theophylline, rolipram, and acetamide-45 on PDE4A activity of the expressed product in yeast cell GL62 . METHODS: Yeast cell GL62 were transfected with human PDE4A gene cloned in the expression plasmid p138NB . Expression was induced by adding CuSO4 to a final concentration of 150 micromol/L, and the expressed product was extracted . The activity of PDE4A was detected by HPLC . RESULTS: Yeast cell GL62 expressed a certain protein at CuSO4 150 micromol/L, the size of the expressed product was between 62 kDa and 83 kDa, the activity of PDE4A of the expressed product at 3 h was in maximum (188 23) micromol/g/min, and the Km was (17.7 2.6) micromol/L . Theophylline, rolipram, and acetamide-45 could inhibit the activity of PDE4A extracted from yeast cell GL62 . The IC50 (95 % confidence limits) of theophylline, rolipram, and acetamide-45 were 1642 (989-2727), 4.58 (3.45-6.08), and 275 (170-444) micromol/L respectively . CONCLUSION: PDE4A expressed in yeast cell GL62 is biologically active . Theophylline, rolipram, and acetamide-45 can inhibit the PDE4A activity . The expressed product in yeast cell GL62 may be used in the research work of PDE4 and its inhibitors.

Mol Cell, 2002 Oct, 10(4), 883 - 94
The yeast capping enzyme represses RNA polymerase II transcription; Myers LC et al.; Using a highly pure transcription system derived from Saccharomyces cerevisiae, we have purified an activity in yeast whole-cell extracts that represses RNA polymerase II transcription . Mechanistic studies suggest that this repressor specifically targets transcriptional reinitiation . The two polypeptides that constitute the repressor have been identified as Ceg1p and Cet1p, the two subunits of the yeast pre-mRNA capping enzyme . A purified recombinant capping enzyme is able to reconstitute repressor activity . Cet1p is necessary for and capable of this repression . Transcriptional run-on experiments indicate that the capping enzyme also serves as a repressor in vivo . Efficient pre-mRNA capping relies on interactions between the capping enzyme and transcription apparatus . Repression by the capping enzyme suggests a bidirectional flow of information between capping and transcription.

Curr Biol, 2002 Oct 29, 12(21), 1828 - 32
Responses of four yeast genes to changes in the transcriptional machinery are determined by their promoters; Cheng JX et al.; Many yeast genes are distinguished by their specific requirements for different components of the transcriptional machinery . Here we examine four genes that fall into two classes as defined by their dependence on specific components of the transcriptional machinery . We describe a series of hybrid constructs, each of which bears activator binding sites that are associated with a promoter other than that with which they are usually affiliated . We examine expression of these reporters in strains bearing three modifications of the transcriptional machinery . Our results indicate that, in each of these cases, the promoter (and not the activator) determines which components of the transcriptional machinery are required . These and additional results, including those of others, clarify how disparate activators can work at many different promoters.

Biosci Rep, 2002 Feb, 22(1), 59 - 79
Yeast as a model to study apoptosis?
Fleury C, Pampin M, Tarze A, Mignotte B.
Programmed cell death (PCD) serves as a major mechanism for the precise regulation of cell numbers, and as a defense mechanism to remove unwanted and potentially dangerous cells . Despite the striking heterogeneity of cell death induction pathways, the execution of the death program is often associated with characteristic morphological and biochemical changes termed apoptosis . Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of programmed cell death . This crucial position of mitochondria in programmed cell death control is not due to a simple loss of function (deficit in energy supplying), but rather to an active process in the regulation of effector mechanisms . The large diversity of regulators of apoptosis in mammals and their numerous interactions complicate the analysis of their individual functions . Yeast, eukaryotic but unicellular organism, lack the main regulators of apoptosis (caspases, Bcl-2 family members, ...) found in mammals . This absence render them a powerful tool for heterologous expression, functional studies, and even cloning of new regulators of apoptosis . Great advances have thus been made in our understanding of the molecular mechanisms of Bcl-2 family members interactions with themselves and other cellular proteins, specially thanks to the two hybrid system and the easy manipulation of yeast (molecular biology and genetics) . This review will focus on the use of yeast as a tool to identify new regulators and study function of mammalian apoptosis regulators.

Protoplasma, 2002 Oct, 220(1-2), 69 - 78
Differential localisation of GFP fusions to cytoskeleton-binding proteins in animal, plant, and yeast cells . Green-fluorescent protein; Timmers AC et al.; The structure and functioning of the cytoskeleton is controlled and regulated by cytoskeleton-associated proteins . Fused to the green-fluorescent protein (GFP), these proteins can be used as tools to monitor changes in the organisation of the cytoskeleton in living cells and tissues in different organisms . Since the localisation of a specific cytoskeleton protein may indicate a particular function for the associated cytoskeletal element, studies of cytoskeleton-binding proteins fused to GFP may provide insight into the organisation and functioning of the cytoskeleton . In this article, we focused on two animal proteins, human T-plastin and bovine tau, and studied the distribution of their respective GFP fusions in animal COS cells, plant epidermal cells (Allium cepa), and yeast cells (Saccharomyces cerevisiae) . Plastin-GFP localised preferentially to membrane ruffles, lamellipodia and focal adhesion points in COS cells, to the actin filament cytoskeleton within cytoplasmic strands in onion epidermal cells, and to cortical actin patches in yeast cells . Thus, in these 3 very different types of cells plastin-GFP associated with mobile structures in which there are high rates of actin turnover . Chemical fixation was found to drastically alter the distribution of plastin-GFP . Tau-GFP bound to microtubules in COS cells and onion epidermal cells but failed to bind to yeast microtubules . Thus, animal and plant microtubules appear to have a common tau binding site which is absent in yeast . We conclude that the study of the distribution patterns of microtubule- and actin-filament-binding proteins fused to GFP in heterologous systems should be a valuable tool in furthering our knowledge about cytoskeleton function in eukaryotic cells.

FEBS Lett, 2002 Nov 6, 531(2), 339 - 42
HspBP1, a homologue of the yeast Fes1 and Sls1 proteins, is an Hsc70 nucleotide exchange factor; Kabani M et al.; The yeast FES1 and SLS1 genes encode conserved nucleotide exchange factors that act on the cytoplasmic and endoplasmic reticulum luminal Hsp70s, Ssa1p and BiP, respectively . We report here that mammalian HspBP1 is homologous to Fes1p and that HspBP1 promotes nucleotide dissociation from both Ssa1p and mammalian Hsc70 . In contrast, Fes1p inefficiently strips nucleotide from mammalian Hsc70, and unlike HspBP1 does not inhibit chaperone-mediated protein refolding in vitro . Together, our data indicate that HspBP1 is a member of this new class of nucleotide exchange factors that exhibit varying degrees of compartment and species specificity.

FEBS Lett, 2002 Nov 6, 531(2), 168 - 72
Apoptosis-specific protein (ASP 45 kDa) is distinct from human Apg5, the homologue of the yeast autophagic gene apg5; Yung HW et al.; We have examined whether the apoptosis-specific protein p45ASP and human Apg5 are identical proteins . Like p45ASP, myc-hApg5 cross-reacted with a c-Jun antibody and approximately 50% of myc-hApg5 was bound to a Triton X-100-insoluble fraction in HeLa cells . However, soluble myc-hApg5 was degraded during apoptosis induced by staurosporine or TNFalpha/cycloheximide whilst expression of soluble p45ASP was stabilised . Furthermore, myc-hApg5 degradation was blocked by the caspase inhibitor Boc-Asp(OMe)FMK whilst p45ASP expression was eliminated . Moreover, myc-hApg5 ( approximately 32 kDa) never assumed the size of p45ASP (45 kDa) . It is therefore likely that p45ASP and human Apg5 are distinct proteins although they do share some common characteristics.

Trends Biochem Sci, 2002 Nov, 27(11), 559 - 63
Analysis of membrane protein interactions using yeast-based technologies; Stagljar I et al.; Proteins associated with membranes total approximately a third of all proteins in a typical eukaryotic cell . However, the analysis of interactions between membrane proteins is difficult because of the hydrophobic nature of these proteins, and conventional biochemical and genetic assays are often of limited use . We summarize here recent yeast-based interaction technologies that can be applied to membrane proteins.

Genet Eng (N Y), 2002, 24, 191 - 213
Import of nuclear encoded RNAs into yeast and human mitochondria: experimental approaches and possible biomedical applications; Entelis N et al.; Mitochondria import from the cytoplasm the vast majority of proteins and some RNAs . Although there exists extended knowledge concerning the mechanisms of protein import, the import of RNA is poorly understood . It was almost exclusively studied on the model of tRNA import, in several protozoans, plants and yeast . Mammalian mitochondria, which do not import tRNAs naturally, are hypothesized to import other small RNA molecules from the cytoplasm . We studied tRNA import in the yeast system, both in vitro and in vivo, and applied similar approaches to study 5S rRNA import into human mitochondria . Despite the obvious divergence of RNA import systems suggested for different species, we find that in yeast and human cells this pathway involves similar mechanisms exploiting cytosolic proteins to target the RNA to the organelle and requiring the integrity of pre-protein import apparatus . The import pathway might be of interest from a biomedical point of view, to target into mitochondria RNAs that could suppress pathological mutations in mitochondrial DNA . Yeast represents a good model to elaborate such a gene therapy approach . We have described here the various approaches and protocols to study RNA import into mitochondria of yeast and human cells in vitro and in vivo.

Anal Biochem, 2002 Oct 15, 309(2), 279 - 92
Application of kinetic-based biospecific affinity chromatographic systems to ATP-dependent enzymes: studies with yeast hexokinase; Mulcahy P et al.; This study is concerned with the development of kinetic-based bioaffinity chromatographic systems for purification of ATP-dependent kinases, with a particular focus on the allosteric yeast hexokinase enzyme (EC 2.7.1.1) . Synthesis and characterization of highly substituted N(6)-linked and S(6)-linked immobilized ATP derivatives are described using a rapid solid-phase modular approach . Evaluation of the new immobilized ATP derivatives has been carried out using model chromatographic studies with yeast hexokinase, employing specific substrate analogues (N-acetyl-D-glucosamine and suramin) to promote biospecific adsorption, in the presence and absence of citrate (a so-called allosteric activator of hexokinase activity) . In this paper, successful bioaffinity chromatography systems were developed for yeast hexokinase and, as a result, interesting binding and catalytic properties of the enzyme were highlighted and explored . The overall results confirm the potential for extrapolation of the kinetic locking-on tactic, a general kinetic-based bioaffinity approach already developed for the NAD(P)(+)-dependent dehydrogenases, to ATP/ADP-dependent enzymes . However, in view of the enhancement of the intrinsic ATPase activity of hexokinase with glucosamine derivatives, and the coincidental hydrolysis of immobilized ATP to immobilized ADP, future developments necessary to support adaptation of the approach to ATP-dependent enzymes are discussed.

EMBO J, 2002 Nov 1, 21(21), 5745 - 55
Plo1(+) regulates gene transcription at the M-G(1) interval during the fission yeast mitotic cell cycle; Anderson M et al.; The regulation of gene expression plays an important part in cell cycle controls . We describe the molecular machinery that co-ordinates gene transcription at the M-G(1) interval during the fission yeast mitotic cell cycle . A sequence is identified in the cdc15(+) promoter that we call a PCB (pombe cell cycle box), which confers M-G(1)-specific transcription . Sequences similar to the PCB are present in the promoters of seven other genes, spo12(+), cdc19(+), fin1(+), sid2(+), ppb1(+), mid1(+)/dmf1(+) and plo1(+), which we find to be transcribed at M-G(1) . A transcription factor complex is identified that binds to the PCB sequence, which we name PBF, for PCB-binding factor . Finally, we show that PBF binding activity and consequent gene transcription are regulated by the Plo1p protein kinase, thus invoking a potential auto-feedback loop mechanism that regulates mitotic gene transcription and passage through septation and cytokinesis.

J Biol Chem, 2003 Jan 10, 278(2), 1273 - 80 Epub 2002 Oct 30.
A dominant allele of PDR1 alters transition metal resistance in yeast; Tuttle MS et al.; A yeast mutant was found to have defective growth on low iron medium despite a normal high affinity iron transport system . The phenotype results from a gain of function mutation in PDR1, which encodes a transcription factor that acts as a regulator of pleiotropic drug resistance in Saccharomyces cerevisiae . The mutant allele, PDR1(R821H), was found to result in increased expression of at least 19 genes, three of which are ATP-binding cassette (ABC) transporters . Expression of at least six genes was required to show the low iron growth defect . Wild type cells transformed with the PDR1(R821H) allele or a PDR1 dominant allele (PDR1-3) showed the low iron growth defect as well as increased resistance to drugs such as cycloheximide and oligomycin . Transformation of PDR1(R821H) into Deltaccc1 cells, which were previously shown to have increased sensitivity to high iron medium because of defective vacuolar iron storage (Li, L., Chen, O . S., Ward, D . M., and Kaplan, J . (2001) J . Biol . Chem . 276, 29515-29519), conferred resistance to high iron medium . Cells expressing PDR1(R821H) also showed increased resistance to copper and manganese because of increased metal export . These results suggest that expression of PDR1-regulated genes affects both efflux and storage of transition metals.

Mol Microbiol, 2002 Nov, 46(3), 845 - 54
Rst1 and Rst2 are required for the a/alpha diploid cell type in yeast; Gelli A; In the budding yeast Saccharomyces cerevisiae, the preservation of the mating competent haploid (a or alpha) and the mating incompetent diploid (a/alpha) is necessary to prevent aneuploidy . Once haploid cells respond to pheromone, the mating-specific signal transduction pathway is activated, and the MAP kinase Fus3 phosphorylates two specific repressor proteins Rst1 and Rst2 (also known as Dig1 and Dig2) to promote Ste12-dependent transcription of mating-specific genes . In contrast, diploid cells cannot mate because genes that encode components of the mating pathway are repressed through the combined action of the Mata1-Matalpha2 and Matalpha2-Mcm1 repressors . Surprisingly, repression of Ste12 by Rst1 and Rst2 is essential for diploid sterility . Homozygous deletion of both RST1 and RST2 (rst-) causes a/alpha diploid cells constitutively to express a-specific genes and mate preferentially as a-cells . This phenotype is sensitive to Ste12 dosage, as removal of one copy of STE12 completely reduces the ectopic activation of a-specific genes . The Matalpha2-Mcm1 complex, which normally represses a-specific genes, is defective in rst- diploids because Matalpha2 is destabilized in rst- diploids, possibly as a consequence of its relocalization from the nucleus to the cytoplasm . This study finds that Rst1 and Rst2 are necessary for the a/alpha diploid cell type . Rst1 and Rst2 are required in order to prevent the amplification of a robust Ste12 transcriptional programme that appears to over-ride Matalpha2-dependent repression of haploid and a-specific genes.

Mol Microbiol, 2002 Nov, 46(3), 781 - 9
Regulation of the yeast Rlm1 transcription factor by the Mpk1 cell wall integrity MAP kinase; Jung US et al.; The Mpk1 MAP kinase of the Saccharomyces cerevisiae cell wall integrity signalling pathway phosphorylates and activates the Rlm1 transcription factor in response to cell wall stress . Rlm1 is related to mammalian MEF2 isoforms, and shares a similar DNA-binding specificity . Signalling through Rlm1 regulates the expression of at least 25 genes, most of which have been implicated in cell wall biogenesis . We report here the transcriptional induction by agents of cell wall stress of a set of lacZ reporter plasmids derived from several Rlm1-responsive genes . Analysis of substitution mutations at putative Mpk1 phosphorylation sites within Rlm1 revealed that Ser427 and Thr439 are important for its stress-induced transcriptional activation of these reporter plasmids . Assessment of Rlm1 activation potency when fused to a heterologous DNA-binding domain showed that the identified seryl and threonyl residues are necessary for the Rlm1 transcriptional activation function independently of its DNA binding . We also demonstrate that a MAP kinase docking site, shown recently to mediate activation of MEF2A and MEF2C, is conserved in Rlm1 and is required for its ability to mediate transcriptional activation in response to agents that induce cell wall stress . Finally, intracellular localization analyses show that Rlm1 resides in the nucleus regardless of its activation and phosphorylation status . Together these observations support the inference that Mpk1 regulates the Rlm1 transcriptional activation function by phosphorylation of Ser427 and Thr439.

Mol Microbiol, 2002 Nov, 46(3), 745 - 8
The glycosylphosphatidylinositol (GPI) signal sequence of human placental alkaline phosphatase is not recognized by human Gpi8p in the context of the yeast GPI anchoring machinery; Meyer U et al.; Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins involves the action of a GPI trans-amidase, which replaces the C-terminal GPI signal sequence (GPI-SS) of the primary translation product with a preformed GPI lipid . The transamidation depends on a complex of four proteins, Gaa1p, Gpi8p, Gpi16p and Gpi17p . Although the GPI anchoring pathway is conserved throughout the eukaryotic kingdom, it has been reported recently that the GPI-SS of human placental alkaline phosphatase (hPLAP) is not recognized by the yeast transamidase, but is recognized in yeast that contain the human Gpi8p homologue . This finding suggests that Gpi8p is intimately involved in the recognition of GPI precursor proteins and may also be responsible for the subtle taxon-specific differences in transamidase specificity that sometimes prevent the efficient GPI anchoring of heterologously expressed GPI proteins . Here, we confirm that the GPI signal sequence of hPLAP is indeed not recognized by the yeast GPI-anchoring machinery . However, in our hands, GPI attachment cannot be restored by the co-expression of human Gpi8p in yeast cells under any circumstances.

J Biol Chem, 2003 Jan 10, 278(2), 843 - 52 Epub 2002 Oct 29.
The Cdc42 binding and scaffolding activities of the fission yeast adaptor protein Scd2; Endo M et al.; The small GTP-binding protein Cdc42, the guanine nucleotide exchange factor Scd1, the p21-activated kinase Shk1, and the adaptor protein Scd2 are involved in the Cdc42-dependent signaling cascade in fission yeast . In the present study, we analyzed the Cdc42 binding and scaffolding activities of Scd2 by co-precipitation assays . We found that two SH3-containing regions, amino acid residues 1-87 (CB1 (Cdc42-binding region 1)) and 110-266 (CB2), of Scd2 can bind to the GTP-bound form of Cdc42 . CB2 is cryptic because of the intramolecular binding between the SH3 domain in CB2 (SH3(C)) and the PX domain and binds to Cdc42 only when the Scd2 PB1 domain binds to the PC motif-containing region (residues 760-872) of Scd1 . This CB2.Cdc42 association, which would stabilize the open configuration of Scd2, enables the SH3(C) domain to bind to the polyproline motif of Shk1 . We also found that the GTP-bound form of Cdc42 binds to the CRIB motif of Shk1 more strongly than to Scd2 . Thus, Scd2 functions as a scaffold to form a protein complex, and the GTP-bound Cdc42 might be transferred effectively from the upstream activator Scd1 to the downstream effector Shk1 via Scd2.

Cell, 2002 Oct 18, 111(2), 155 - 8
Nutrient-regulated protein kinases in budding yeast; Wilson WA et al.; The ability of cells to react appropriately to nutritional cues is of fundamental importance, and in budding yeast, a small number of intracellular protein kinases, PKA, Snf1p/AMP-activated kinase, TOR, Gcn2p, and the cyclin-dependent kinase Pho85p have key roles . A recently characterized enzyme, PAS kinase, may be a new member of this group of nutritional transducers.

Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 14670 - 5 Epub 2002 Oct 29.
The A14-A43 heterodimer subunit in yeast RNA pol I and their relationship to Rpb4-Rpb7 pol II subunits; Peyroche G et al.; A43, an essential subunit of yeast RNA polymerase I (pol I), interacts with Rrn3, a class I general transcription factor required for rDNA transcription . The pol I-Rrn3 complex is the only form of enzyme competent for promoter-dependent transcription initiation . In this paper, using biochemical and genetic approaches, we demonstrate that the A43 polypeptide forms a stable heterodimer with the A14 pol I subunit and interacts with the common ABC23 subunit, the yeast counterpart of the omega subunit of bacterial RNA polymerase . We show by immunoelectronic microscopy that A43, ABC23, and A14 colocalize in the three-dimensional structure of the pol I, and we demonstrate that the presence of A43 is required for the stabilization of both A14 and ABC23 within the pol I . Because the N-terminal half of A43 is clearly related to the pol II Rpb7 subunit, we propose that the A43-A14 pair is likely the pol I counterpart of the Rpb7-Rpb4 heterodimer, although A14 distinguishes from Rpb4 by specific sequence and structure features . This hypothesis, combined with our structural data, suggests a new localization of Rpb7-Rpb4 subunits in the three-dimensional structure of yeast pol II.

Bioorg Chem, 2002 Jun, 30(3), 188 - 98
Spectroscopic evidence for participation of the 1',4'-imino tautomer of thiamin diphosphate in catalysis by yeast pyruvate decarboxylase; Jordan F et al.; The 1',4'-iminopyrimidine tautomeric form of the coenzyme thiamin diphosphate (ThDP), implicated in catalysis on the basis of the conformation of enzyme-bound ThDP, has been observed by both ultraviolet absorption and circular dichroism spectroscopy . On yeast pyruvate decarboxylase, the unusual tautomer is observed in an active center variant in which catalysis in the post-decarboxylation regime of the reaction is compromised . In a model system consisting of N1-methyl-4-aminopyrimidinium or N1-methyl-N4-n-butylpyrimidinium salts, on treatment with either NaOH in water, or DBU in DMSO there is an intermediate formed with lambda(max) near 310 nm, and this intermediate reverts back to the starting salt on acidification . Proton NMR chemical shifts are consistent with the intermediate representing the 1-methyl-4-imino tautomer . On the enzyme, the intermediate could be observed by rapid-scan stopped flow with UV detection when reacting holoenzyme of the E477Q active center variant with pyruvate, and by circular dichroism even in the absence of pyruvate . This represents the first direct observation of the imino tautomeric form of ThDP both on the enzyme and in models, although some years ago, this laboratory had already reported some pertinent acid-base properties for its formation {Jordan, F., and Mariam, Y . H . (1978) J . Am . Chem . Soc.100, 2534-2541} . The work also represents the first instance in which a rare tautomer implicated in catalysis is identified and suggests that such tautomeric catalysis may be more common in biology than hitherto recognized.

RNA, 2002 Oct, 8(10), 1348 - 60
An active precursor in assembly of yeast nuclear ribonuclease P; Srisawat C et al.; The RNA-protein subunit assembly of nuclear RNase P was investigated by specific isolation and characterization of the precursor and mature forms of RNase P using an RNA affinity ligand . Pre-RNase P was as active in pre-tRNA cleavage as mature RNase P, although it contained only seven of the nine proteins found in mature RNase P . Pop3p and Rpr2p were not required for maturation of the RPR1 RNA subunit and virtually absent from pre-RNase P, implying that they are dispensable for pre-tRNA substrate recognition and cleavage . The RNase P subunit assembly is likely to occur in the nucleolus, where both precursor and mature forms of RNase P RNA are primarily localized . The results provide insight into assembly of nuclear RNase P, and suggest pre-tRNA substrate recognition is largely determined by the RNA subunit.

Physiol Chem Phys Med NMR, 2002, 34(1), 43 - 60
Fluorescence quenching of dimeric and monomeric forms of yeast hexokinase (PII): effect of substrate binding steady-state and time-resolved fluorescence studies; Maity H et al.; Fluorescence quenching studies on the PII isoenzyme of yeast hexokinase have been performed using charged as well as polar uncharged quenchers . In both 'open' (i.e . in the absence of glucose) and 'closed' (i.e . in the presence of glucose) forms of the enzyme, bimolecular quenching rate constant (kq) for acrylamide is significantly larger than that of KI, indicating that all the tryptophans are not fully exposed to the solvent . Overall accessibility of tryptophans towards KI was greater in the presence of glucose than in the absence of glucose . At high ionic strength, the value of bimolecular quenching rate constant (kq) for KI did not change suggesting that the average environment of the accessible tryptophan residue(s) is almost neutral . Quenching by KI is dynamic in nature . Accessibility of tryptophans towards acrylamide at concentration > or = 0.2 M was more in the 'open' form of the enzyme than that observed in the 'closed' form whereas at concentration < or = 0.2 M no significant difference in the extent of quenching was observed . It is reasonable to conclude that glucose induced conformational change leads some tryptophan residue(s) to be more exposed and at the same time some tryptophan residue(s) in the hydrophobic region become more buried . Dimeric and monomeric forms of the enzyme behave similarly towards the quenching by acrylamide . In the unfolded state, the accessibility of tryptophans was considerably higher for both the quenchers . Temperature dependent study and the fluorescence lifetime data indicate that the mechanism of quenching by acrylamide is primarily dynamic in nature.

Yeast, 2002 Nov, 19(15), 1295 - 8
Simultaneous determination of potassium and rubidium content in yeast; Mulet JM et al.; Rubidium is widely used as a potassium analogue in transport studies in yeast and other organisms . As rubidium (potassium) uptake is modulated by the internal potassium concentration, it is often necessary to determine both Rb(+) and K(+) concentrations in the same cell extract . Current methods based on atomic absorption/emission spectroscopy require separate analysis for each cation . Alternatively, unsafe radioactive isotopes can be used . Here we report a convenient, non-radioactive, HPLC/conductivity-based method that allows a complete analysis of both cations with a single injection from a cell extract . The increase in Rb(+) uptake during K(+) starvation in yeast is easily demonstrated with this method .

Yeast, 2002 Nov, 19(15), 1285 - 93
Overexpressed yeast mitochondrial putative RNA helicase Mss116 partially restores proper mtRNA metabolism in strains lacking the Suv3 mtRNA helicase; Minczuk M et al.; RNA helicase, encoded by the Saccharomyces cerevisiae nuclear gene SUV3, is a subunit of the mitochondrial (mt) degradosome: an enzyme complex that takes part in turnover of mtRNAs . Deletion of the SUV3 gene leads to a variety of disturbances in mtRNA metabolism and results in respiratory incompetence of yeast cells . Here we show that the nuclear gene MSS116, which codes for a mitochondrial putative RNA helicase necessary for splicing of several mt introns, can suppress the lack of the SUV3 gene . Overexpression of the Mss116 putative helicase from a multicopy plasmid present in the SUV3-deleted strains partially restores respiratory competence, brings the steady-state levels of COB and ATP6/8 mRNA back almost to normal and lowers the accumulation of 21S rRNA and ATP6/8 RNA precursors to the wild-type levels . To the best of our knowledge, this is the first reported case of a substitution of one RNA helicase by another, belonging to a different class of RNA helicases .

J Biol Chem, 2003 Jan 10, 278(2), 784 - 90 Epub 2002 Oct 24.
Translesion synthesis past acrolein-derived DNA adduct, gamma -hydroxypropanodeoxyguanosine, by yeast and human DNA polymerase eta; Minko IG et al.; gamma-Hydroxy-1,N(2)-propano-2'deoxyguanosine (gamma-HOPdG) is a major deoxyguanosine adduct derived from acrolein, a known mutagen . In vitro, this adduct has previously been shown to pose a severe block to translesion synthesis by a number of polymerases (pol) . Here we show that both yeast and human pol eta can incorporate a C opposite gamma-HOPdG at approximately 190- and approximately 100-fold lower efficiency relative to the control deoxyguanosine and extend from a C paired with the adduct at approximately 8- and approximately 19-fold lower efficiency . Although DNA synthesis past gamma-HOPdG by yeast pol eta was relatively accurate, the human enzyme misincorporated nucleotides opposite the lesion with frequencies of approximately 10(-1) to 10(-2) . Because gamma-HOPdG can adopt both ring closed and ring opened conformations, comparative replicative bypass studies were also performed with two model adducts, propanodeoxyguanosine and reduced gamma-HOPdG . For both yeast and human pol eta, the ring open reduced gamma-HOPdG adduct was less blocking than gamma-HOPdG, whereas the ring closed propanodeoxyguanosine adduct was a very strong block . Replication of DNAs containing gamma-HOPdG in wild type and xeroderma pigmentosum variant cells revealed a somewhat decreased mutation frequency in xeroderma pigmentosum variant cells . Collectively, the data suggest that pol eta might potentially contribute to both error-free and mutagenic bypass of gamma-HOPdG.

J Biol Chem, 2003 Jan 10, 278(2), 791 - 9 Epub 2002 Oct 24.
Biochemical and genetic evidence for the involvement of yeast Ypt6-GTPase in protein retrieval to different Golgi compartments; Luo Z et al.; Yeast Ypt6p, the homologue of the mammalian Rab6 GTPase, is not essential for cell viability . Based on previous studies with ypt6 deletion mutants, a regulatory role of the GTPase either in protein retrieval to the trans-Golgi network or in forward transport between the endoplasmic reticulum (ER) and early Golgi compartments was proposed . To assess better the primary role(s) of Ypt6p, temperature-sensitive ypt6 mutants were generated and analyzed biochemically and genetically . Defects in N-glycosylation of proteins passing the Golgi and of Golgi-resident glycosyltransferases as well as protein sorting defects in the trans-Golgi were recorded shortly after functional loss of Ypt6p . ER-to-Golgi transport and protein secretion were delayed but not interrupted . Mis-sorting of the vesicular SNARE Sec22p to the late Golgi was also observed . Combination of the ypt6-2 mutant allele with a number of mutants in forward and retrograde transport between ER, Golgi, and endosomes led to synthetic negative growth defects . The results obtained indicate that Ypt6p acts in endosome-to-Golgi, in intra-Golgi retrograde transport, and possibly also in Golgi-to-ER trafficking.

Genetics, 2002 Oct, 162(2), 677 - 88
A genomics-based screen for yeast mutants with an altered recombination/end-joining repair ratio; Wilson TE; We recently described a yeast assay suitable for genetic screening in which simple religation nonhomologous end-joining (NHEJ) and single-strand annealing (SSA) compete for repair of an I-SceI-created double-strand break . Here, the required allele has been introduced into an array of 4781 MATa deletion mutants and each strain screened individually . Two mutants (rad52 and srs2) showed a clear increase in the NHEJ/SSA ratio due to preferential impairment of SSA, but no mutant increased the absolute frequency of NHEJ significantly above the wild-type level . Seven mutants showed a decreased NHEJ/SSA ratio due to frank loss of NHEJ, which corresponded to all known structural/catalytic NHEJ components (yku70, yku80, dnl4, lif1, rad50, mre11, and xrs2); no new mutants in this category were identified . A clearly separable and surprisingly large set of 16 other mutants showed partial defects in NHEJ . Further examination of these revealed that NEJ1 can entirely account for the mating-type regulation of NHEJ, but that this regulatory role was distinct from the postdiauxic/stationary-phase induction of NHEJ that was deficient in other mutants (especially doa1, fyv6, and mck1) . These results are discussed in the context of the minimal set of required proteins and regulatory inputs for NHEJ.

Genetics, 2002 Oct, 162(2), 567 - 78
Mutant membrane protein of the budding yeast spindle pole body is targeted to the endoplasmic reticulum degradation pathway; McBratney S et al.; Mutation of either the yeast MPS2 or the NDC1 gene leads to identical spindle pole body (SPB) duplication defects: The newly formed SPB is improperly inserted into the nuclear envelope (NE), preventing the cell from forming a bipolar mitotic spindle . We have previously shown that both MPS2 and NDC1 encode integral membrane proteins localized at the SPB . Here we show that CUE1, previously known to have a role in coupling ubiquitin conjugation to ER degradation, is an unusual dosage suppressor of mutations in MPS2 and NDC1 . Cue1p has been shown to recruit the soluble ubiquitin-conjugating enzyme, Ubc7p, to the cytoplasmic face of the ER membrane where it can ubiquitinate its substrates and target them for degradation by the proteasome . Both mps2-1 and ndc1-1 are also suppressed by disruption of UBC7 or its partner, UBC6 . The Mps2-1p mutant protein level is markedly reduced compared to wild-type Mps2p, and deletion of CUE1 restores the level of Mps2-1p to nearly wild-type levels . Our data indicate that Mps2p may be targeted for degradation by the ER quality control pathway.

Cancer Cell, 2002 Oct, 2(4), 267 - 73
Cancer therapeutics in yeast; Bjornsti MA; The budding yeast Saccharomyces cerevisiae is a genetically tractable model system with which to establish the cellular target of a given agent and investigate mechanisms of drug action.

Biotechniques . 2002 Oct;33(4):788, 790, 792 passim.
Yeast tRNA as carrier in the isolation of microscale RNA for global amplification and expression profiling; Wang QT et al.; The characterization of global gene expression patterns of microscale samples is important in many areas of biological and clinical research . The choice of carrier is critical for the efficient isolation and successful amplification of RNA at the nanogram level . Here we show that recovery of nanograms of RNA is significantly higher when carrier linear polyacrylamide is supplemented with carrier tRNA . Reverse transcription and in vitro transcription reactions remain efficient and specific in the presence of carrier tRNA . Finally, comparison of GeneChip array hybridization patterns demonstrates that the presence of carrier tRNA does not cause detectable distortion in global amplification . Taken together, tRNA is a superior carrier for the isolation and global amplification of microscale RNA.

Biofizika, 2002 Sep-Oct, 47(5), 852 - 7
{Fluorescent photobleaching of endogenous protoporphyrin IX in yeast cells}; Strakhovskaia MG et al.; Yeast Saccharomyces cerevisiae accumulate in the presence of chelator 2,2'-dipyridyl, two fractions of photosensitizer protoporphyrin IX, which fluoresce with maxima at 625 and 635 nm . The two fractions were found to differ from each other in the dynamics and character of fluorescence photobleaching . In contrast to the protoporphyrin IX that fluoresces with a maximum at 625 nm, the protoporphyrin IX fraction that fluoresces with a maximum at 635 nm is more photolable; in addition, upon photobleaching of its fluorescence, a new maximum at 675 nm appears.

J Biol Chem, 2002 Dec 20, 277(51), 49554 - 61 Epub 2002 Oct 18.
A lack of SUMO conjugation affects cNLS-dependent nuclear protein import in yeast; Stade K et al.; Yeast SUMO (Smt3) and its mammalian ortholog SUMO-1 are ubiquitin-like proteins that can reversibly be conjugated to other proteins . Among the substrates for SUMO modification in vertebrates are RanGAP1 and RanBP2/Nup358, two proteins previously implicated in nucleocytoplasmic transport . Sumoylated RanGAP1 binds to the nuclear pore complex via RanBP2/Nup358, a giant nucleoporin, which was recently reported to act as a SUMO E3 ligase on some nuclear substrates . However, no direct evidence for a role of the SUMO system in nuclear transport has been obtained so far . By the use of conditional yeast mutants, we examined nuclear protein import in vivo . We show here that cNLS-dependent protein import is impaired in mutants with defective Ulp1 and Uba2, two enzymes involved in the SUMO conjugation reaction . In contrast, other transport pathways such as rgNLS-mediated protein import and mRNA export are not affected . Furthermore, we find that the yeast importin-alpha subunit Srp1 accumulates in the nucleus of ulp1 and uba2 strains but not the importin-beta subunit Kap95, indicating that a lack of Srp1 export might impair cNLS import . In summary, our results provide evidence that SUMO modification in yeast, as has been suspected for vertebrates, plays an important role in nucleocytoplasmic trafficking.

J Biol Chem, 2002 Dec 20, 277(51), 49538 - 44 Epub 2002 Oct 18.
Sphingolipids are required for the stable membrane association of glycosylphosphatidylinositol-anchored proteins in yeast; Watanabe R et al.; Ongoing sphingolipid synthesis is specifically required in vivo for the endoplasmic reticulum (ER) to Golgi transport of glycosylphosphatidylinositol (GPI)-anchored proteins . However, the sphingolipid intermediates that are required for transport nor their role(s) have been identified . Using stereoisomers of dihydrosphingosine, together with specific inhibitors and a mutant defective for sphingolipid synthesis, we now show that ceramides and/or inositol sphingolipids are indispensable for GPI-anchored protein transport . Furthermore, in the absence of sphingolipid synthesis, a significant fraction of GPI-anchored proteins is no longer associated tightly with the ER membrane . The loose membrane association is neither because of the lack of a GPI-anchor nor because of prolonged ER retention of GPI-anchored proteins . These results indicate that ceramides and/or inositol sphingolipids are required to stabilize the association of GPI-anchored proteins with membranes . They could act either by direct involvement as membrane components or as substrates for the remodeling of GPI lipid moieties.

Biochim Biophys Acta, 2002 Oct 11, 1578(1-3), 51 - 8
The yeast gene COQ5 is differentially regulated by Mig1p, Rtg3p and Hap2p; Hagerman RA et al.; Ubiquinone (CoQ) is an important component of the electron transport chain and serves to regenerate cellular anti-oxidants . In Saccharomyces cerevisiae, expression of the COQ5 gene, encoding for a C-methyltransferase involved in CoQ synthesis, is transcriptionally regulated by carbon source . We have identified three transcription factors involved in this regulation . Mig1p repressed COQ5 expression on dextrose, while Rtg1p/Rtg3p heterodimers up-regulated COQ5 expression on oleic acid . Hap2p modulated the response to oleic acid but did not have an effect in other nonfermentable carbon sources such as glycerol . These results suggest that the regulation of COQ5 gene expression by carbon source is multifactorial and involves the interaction of various transcription factors.

Eur J Biochem, 2002 Nov, 269(21), 5203 - 14
Tag-mediated isolation of yeast mitochondrial ribosome and mass spectrometric identification of its new components; Gan X et al.; Mitochondrial ribosomal proteins (mrps) of the budding yeast, Saccharomyces cerevisiae, have been extensively characterized genetically and biochemically . However, the list of the genes encoding individual mrps is still not complete and quite a few of the mrps are only predicted from their similarity to bacterial ribosomal proteins . We have constructed a yeast strain in which one of the small subunit proteins, termed Mrp4, was tagged with S-peptide and used for affinity purification of mitochondrial ribosome . Mass spectrometric analysis of the isolated proteins detected most of the small subunit mrps which were previously identified or predicted and about half of the large subunit mrps . In addition, several proteins of unknown function were identified . To confirm their identity further, we added tags to these proteins and analyzed their localization in subcellular fractions . Thus, we have newly established Ymr158w (MrpS8), Ypl013c (MrpS16), Ymr188c (MrpS17) and Ygr165w (MrpS35) as small subunit mrps and Img1, Img2, Ydr116c (MrpL1), Ynl177c (MrpL22), Ynr022c (MrpL50) and Ypr100w (MrpL51) as large subunit mrps.

Genes Cells, 2002 Nov, 7(11), 1113 - 24
Cut1/separase C-terminus affects spindle pole body positioning in interphase of fission yeast: pointed nuclear formation; Nakamura T et al.; BACKGROUND: The separase-securin complex is required for anaphase . Separase activated by securin destruction cleaves the cohesin subunit Scc1/Rad21 enriched in kinetochores . Fission yeast Cut1/separase resides in interphase cytoplasm and mobilizes to the spindle and the spindle pole bodies (SPBs) in mitosis, while Cut2/securin remains in the nucleus from interphase to metaphase, and temporarily locates at the short spindle . RESULTS: We here report a novel SPB-led dynamic nuclear movement in fission yeast, when the Cut1 C-terminal fragment is over-expressed . The tip of the pointed nucleus contained both SPB and centromeric DNA, and rapidly moved along the bundled cytoplasmic microtubules . The same pointed nucleus was produced when the human separase C-fragment was over-expressed . The pointed nuclear formation did not require the protease site of separase, but required the conserved C-terminus and a microtubule- and kinetochore-binding protein Mtc1/Alp14, a homologue of frog XMAP215 and budding yeast Stu2 . The movement-inducing C-fragment should be cytoplasmic, as the pointed nucleus was abolished when the fragment contained the NLS (nuclear localization signal) . CONCLUSIONS: Overproduced separase C-fragment abolishes correct SPB-positioning in interphase . Resulting pointed nuclear formation (alternatively called 'pigtail movement') requires cytoplasmic microtubules and Mtc1/Alp14.

FEBS Lett, 2002 Oct 23, 530(1-3), 174 - 80
Yeast Mon1p/Aut12p functions in vacuolar fusion of autophagosomes and cvt-vesicles; Meiling-Wesse K et al.; Here we identify Mon1p as being essential for the cvt-pathway and autophagy . Thus, mon1Delta cells are impaired in proaminopeptidase I maturation and homozygous diploid mon1Delta cells do not sporulate . Quantitative autophagy measurements suggest a complete autophagy block . The autophagosomal marker protein GFP-Aut7p accumulates in mon1Delta cells at punctate structures outside the vacuole . Furthermore, proaminopeptidase I accumulates in mon1Delta cells in a proteinase-protected form . Our data demonstrate that mon1Delta cells are defective in the fusion of cvt-vesicles and autophagosomes with the vacuole . Consistent with this, GFP-Mon1p localizes to the cytosol and to punctate structures within the cytosol.

Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14041 - 6 Epub 2002 Oct 16.
Crystal structure of the DNA-binding domain from Ndt80, a transcriptional activator required for meiosis in yeast; Montano SP et al.; Ndt80 is a transcriptional activator required for meiosis in the yeast Saccharomyces cerevisiae . Here, we report the crystal structure at 2.3 A resolution of the DNA-binding domain of Ndt80 experimentally phased by using the anomalous and isomorphous signal from a single ordered Se atom per molecule of 272-aa residues . The structure reveals a single approximately 32-kDa domain with a distinct fold comprising a beta-sandwich core elaborated with seven additional beta-sheets and three short alpha-helices . Inspired by the structure, we have performed a mutational analysis and defined a DNA-binding motif in this domain . The DNA-binding domain of Ndt80 is homologous to a number of proteins from higher eukaryotes, and the residues that we have shown are required for DNA binding by Ndt80 are highly conserved among this group of proteins . These results suggest that Ndt80 is the defining member of a previously uncharacterized family of transcription factors, including the human protein (C11orf9), which has been shown to be highly expressed in invasive or metastatic tumor cells.

Curr Opin Cell Biol, 2002 Aug, 14(4), 454 - 62
Insights from yeast endosomes; Pelham HR; The endosomal system of yeast is simpler than that of animal cells, but as it is mapped more similarities are emerging . A key role for ubiquitin in sorting proteins to and into multivesicular bodies has been demonstrated . The finding that Phox homology domains recognise phosphatidylinositol 3-phosphate explains how sorting nexins are recruited to endosomes, where they mediate the retrieval of membrane proteins from the endocytic pathway.

Gene, 2002 Aug 21, 296(1-2), 1 - 9
Detection of Arabidopsis thaliana AtRAD1 cDNA variants and assessment of function by expression in a yeast rad1 mutant; Vonarx EJ et al.; The Saccharomyces cerevisiae RAD1 and human XPF genes encode a subunit of a nucleotide excision repair endonuclease that also is implicated in some forms of homologous recombination . An Arabidopsis thaliana gene (AtRAD1) encoding the orthologous plant protein has been identified recently . Here we report the isolation of three structurally distinct AtRAD1 cDNAs from A . thaliana leaf tissue RNA . One of the isolates (AtRAD1-1) corresponds to the cDNA previously shown to encode the full-length AtRad1 protein, whereas the other two (AtRAD1-2, AtRAD1-3) differ slightly in size due to variations at the 5' end of exon 6 or the 3' end of exon 7, respectively . The sequence differences argue that these cDNAs were probably templated by mRNAs generated via alternative splicing . Diagnostic polymerase chain reaction pointed to the presence of the AtRAD1-1 and AtRAD1-2 but not AtRAD1-3 transcripts in bud and root tissue, and to a fourth transcript (AtRAD1-4), having both alterations identified in AtRAD1-2 and AtRAD1-3, in root tissue . However, the low frequency of detection of AtRAD1-3 and AtRAD1-4 makes the significance of these tissue-specific patterns unclear . The predicted AtRad1-2, AtRad1-3 and AtRad1-4 proteins lack part of the region likely required for endonuclease complex formation . Expression of AtRAD1-2 and AtRAD1-3 in a yeast rad1 mutant did not complement the sensitivity to ultraviolet radiation or the recombination defect associated with the rad1 mutation . These results suggest that alternative splicing may modulate the levels of functional AtRad1 protein.

J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Nov 15, 780(1), 161 - 9
Quantitation of yeast ceramides using high-performance liquid chromatography-evaporative light-scattering detection; Zhou Q et al.; A high-performance liquid chromatograph equipped with an evaporative light scattering detector (ELSD) (HPLC-ELSD) was used to assay the ceramides in yeast cells . The HPLC-ELSD method employed a cyanopropyl bonded column (CN column) that effectively separated the main interfering substance ergosterol without any derivatization process; most other interfering substances were also removed . The method can be applied for routine assay of ceramide content in yeast.

Nat Genet, 2002 Nov, 32(3), 378 - 83 Epub 2002 Oct 15.
Sir2p and Sas2p opposingly regulate acetylation of yeast histone H4 lysine16 and spreading of heterochromatin; Suka N et al.; The Sir3 protein helps form telomeric heterochromatin by interacting with hypoacetylated histone H4 lysine 16 (H4-Lys16) . The molecular nature of the heterochromatin boundary is still unknown . Here we show that the MYST-like acetyltransferase Sas2p is required for the acetylation (Ac) of H4-Lys16 in euchromatin . In a sas2Delta strain or a phenocopy Lys16Arg mutant, Sir3p spreads from roughly 3 kb to roughly 15 kb, causing hypoacetylation and repression of adjacent chromatin . We also found that disruption of Sir3p binding in a deacetylase-deficient Sir 2Delta strain can be suppressed by sas2Delta . These data indicate that opposing effects of Sir2p and Sas2p on acetylation of H4-Lys16 maintain the boundary at telomeric heterochromatin.

J Exp Bot, 2002 Nov, 53(378), 2273 - 5
Functional expression of Acetabularia acetabulum vacuolar H(+)-pyrophosphatase in a yeast VMA3-deficient strain; Ikeda M et al.; The function of the translation product of cDNA for Acetabularia vacuolar H(+)-pyrophosphatase was examined using the Saccharomyces cerevisiae VMA3-deficient strain . The open reading frame of Acetabularia H(+)-pyrophosphatase was revealed to encode 751 amino acids (721 or 751 amino acids in a previous paper) . The acidification of the vacuole was observed by fluorescence microscopy when the cDNA was constructed in pYES2 . Immunoblot analysis also supported the localization of the translation product in the vacuolar-membrane-enriched fraction.

J Biol Chem, 2002 Dec 20, 277(51), 50015 - 21 Epub 2002 Oct 11.
Comprehensive expression analysis of time-dependent genetic responses in yeast cells to low temperature; Sahara T et al.; We performed genome-wide expression analysis to determine genetic responses in Saccharomyces cerevisiae to a low temperature environment using a cDNA microarray . Approximately 25% of the genes in the yeast genome were found to be involved in the response of yeast to low temperature . This finding of a large number of genes being involved in the response to low temperature enabled us to give a functional interpretation to the genetic responses to the stimulus . Functional and clustering analyses of temporal changes in gene expression revealed that global states of the expressions of up-regulated genes could be characterized as having three phases (the early, middle, and late phases) . In each phase, genes related to rRNA synthesis, ribosomal proteins, or several stress responses are time-dependently up-regulated, respectively . Through these phases, yeast cells may improve reduced efficiency of translation and enhance cell protection mechanisms to survive under a low temperature condition . Furthermore, these time-dependent regulations of these genes would be controlled by the cAMP-protein kinase A pathway . The results of our study provide a global description of transcriptional response for adaptation to low temperature in yeast cells.

J Biol Chem, 2002 Dec 20, 277(51), 49352 - 9 Epub 2002 Oct 11.
Erf4p and Erf2p form an endoplasmic reticulum-associated complex involved in the plasma membrane localization of yeast Ras proteins; Zhao L et al.; Ras oncogene proteins are plasma membrane-associated signal transducers that are found in all eukaryotes . Posttranslational addition of lipid to a carboxyl-terminal CaaX box (where "C" represents a cysteine, "a" is generally an aliphatic residue, and X can be any amino acid) is required to target Ras proteins to the cytosolic surface of the plasma membrane . The pathway by which Ras translocates from the endoplasmic reticulum to the plasma membrane is currently not clear . We have performed a genetic screen to identify components of the Ras plasma membrane localization pathway . Mutations in two genes, ERF2 and ERF4/SHR5, have been shown to affect the palmitoylation and subcellular localization of Ras proteins . In this report, we show that Erf4p is localized on the endoplasmic reticulum as a peripheral membrane protein in a complex with Erf2p, an integral membrane protein that was identified from the same genetic screen . Erf2p has been shown to be required for the plasma membrane localization of GFP-Ras2p via a pathway distinct from the classical secretory pathway (X . Dong and R . J . Deschenes, manuscript in preparation) . We show here that Erf4p, like Erf2p, is involved in the plasma membrane localization of Ras2p . Erf2p and Erf4p represent components of a previously uncharacterized subcellular transport pathway involved in the plasma membrane targeting of Ras proteins.

Mutat Res, 2002 Oct 31, 508(1-2), 137 - 45
Benzo{a}pyrene diol epoxide-deoxyguanosine adducts are accurately bypassed by yeast DNA polymerase zeta in vitro; Simhadri S et al.; The possible role of bypass DNA polymerase zeta in mutagenic translesion synthesis past benzo{a}pyrene (BP) 7,8-diol-9,10-epoxide (DE) N(2)-deoxyguanosine (dG) adducts has been examined . We prepared 59-mer DNA templates containing dG adducts derived from trans opening of enantiomers of BP DE-2, in which the 7-hydroxyl group and epoxide oxygen are trans . The 10S-BP DE-dG and 10R-BP DE-dG adducts derive from the (+)- and (-)-DE-2 enantiomers, respectively . The adducted dG is located at a site identified as a G-->T mutational hotspot in random mutagenesis studies of (+)-BP DE-2 in Chinese hamster V-79 cells . Yeast pol zeta (complex of Gst-Rev3p and Rev7p) formed extension products (total of all lengths) of 71, 74 and 88% of a primer annealed to the 10S-BP DE-dG, 10R-BP DE-dG and non-adducted 59-mer templates, respectively . However, only 18 and 19% of the primer was extended to the full-length product on 10S-BP DE-dG and 10R-BP DE-dG adducted templates compared to 55% of the primer on the non-adducted template . A major 34-mer product corresponding to primer elongation up to and including the base before the adduct indicated that nucleotide incorporation opposite both adducts was strongly blocked . Full-length products were isolated from gels and subjected to PCR amplification and cloning . Sequence analysis of more than 300 clones of these full-length products on each template showed that only the correct dCMP was incorporated opposite both the adducted and non-adducted G-hotspot in the template . This corresponds to a probability of mutation lower than 0.3%, the limit of detection, and demonstrates the remarkable fidelity of yeast pol zeta in translesion synthesis past these BP DB-dG lesions in vitro.

Biotechnol Bioeng, 2002 Dec 20, 80(6), 610 - 21
Enzyme recovery during gas/liquid two-phase flow microfiltration of enzyme/yeast mixtures; Mercier-Bonin M et al.; The effect of a gas/liquid two-phase flow on the recovery of an enzyme was evaluated and compared with standard crossflow operation when confronted with the microfiltration of a high-fouling yeast suspension . Ceramic tubular and flat sheet membranes were used . At constant feed concentration (permeate recycling) and transmembrane pressure, the results obtained with the tubular membrane were dependent on the two-phase flow pattern . In comparison with single-phase flow performances at the same liquid velocity, the enzyme transmission was maintained at a high level with a bubble flow pattern but it decreased by 70% with a slug flow, whatever the flow rate ratio . Identical results were obtained with flat sheet membranes: for the highest flow rate ratio, the enzyme transmission was reduced by 70% even though the permeate flux was improved by 240% . During diafiltration experiments with the tubular membrane, it was found that a bubble flow pattern led to a 13% higher enzyme recovery compared to single-phase flow conditions, whereas with a slug flow the enzyme recovery was strongly reduced . With bubble flow conditions, energy consumption was minimal, confirming that this flow pattern was the most suitable for enzyme recovery .

J Biol Chem, 2002 Dec 13, 277(50), 48484 - 9 Epub 2002 Oct 10.
Su e of the yeast F1Fo-ATP synthase forms homodimers; Brunner S et al.; The yeast F(1)F(o)-ATP synthase forms a dimeric complex in the mitochondrial inner membrane . Dimerization of two F(1)F(o) monomeric complexes involves the physical association of two membrane-embedded F(o) sectors and in a manner, which is dependent on the F(o) subunit, Su e . Sequence analysis of Su e protein family members indicated the presence of a conserved coiled-coil motif . As this motif is often the basis for protein homodimerization events, it was hypothesized that Su e forms homodimers in the inner membrane and that formation of Su e dimers between two neighboring F(o) complexes would facilitate dimerization of the F(1)F(o)-ATP synthase complex (Arnold, I., Pfeiffer, K., Neupert, W., Stuart, R . A., and Schagger, H . (1998) EMBO J . 17, 7170-7178) . Using a histidine-tagged derivative of yeast Su e, Su e-His(12), combined with cross-linking and affinity purification approaches, we have directly demonstrated the ability of the yeast Su e protein to form homodimers . Functionality of the Su e-His(12) derivative was confirmed by its ability to assemble into the ATP synthase complex and to support its dimerization in the Deltasu e null mutant yeast cells . The close association of two neighboring Su e proteins was also demonstrated using cross-linking with Cu(2+), which binds and cross-links a unique Cys residue in neighboring Su e proteins . Finally, we propose a model for the molecular basis of the homodimerization of the Su e proteins.

Mol Cell Proteomics, 2002 Aug, 1(8), 561 - 6
Roles for the two-hybrid system in exploration of the yeast protein interactome; Ito T et al.; Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast . The yeast protein interactome analysis was achieved first by using the yeast two-hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes . While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome . Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal . Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network . Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction . Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research.

J Cell Sci, 2002 Nov 15, 115(Pt 22), 4375 - 85
An evolutionarily conserved fission yeast protein, Ned1, implicated in normal nuclear morphology and chromosome stability, interacts with Dis3, Pim1/RCC1 and an essential nucleoporin; Tange Y et al.; We identified a novel fission yeast gene, ned1(+), with pleiotropic mutations that have a high incidence of chromosome missegregation, aberrantly shaped nuclei, overdeveloped endoplasmic reticulum-like membranes, and increased sensitivity to a microtubule destabilizing agent . Ned1 protein, which was phosphorylated in a growth-related manner, interacted in a yeast two-hybrid system with Dis3 as well as with Pim1/RCC1 (nucleotide exchange factor for Ran) . Ned1 also interacted with an essential nucleoporin, a probable homologue of mammalian Nup98/96 . The ned1 gene displayed a variety of genetic interactions with factors involved in nuclear transport and chromosome segregation, including the crm1 (exportin), spi1 (small GTPase Ran), pim1, and dis genes . A substitution mutation that affected the two-hybrid interaction with Dis3 increased chromosome instability, suggesting the functional importance of the interaction . Overproduction of Ned1 protein induced formation of an abnormal microtubule bundle within the nucleus, apparently independently of the spindle pole body, but dependent on pim1(+) activity . The ned1(+) gene belongs to an evolutionarily conserved gene family, which includes the mouse Lpin genes, one of whose mutations is responsible for lipodystrophy.

Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14183 - 8 Epub 2002 Oct 08.
Cell surface polarization during yeast mating; Bagnat M et al.; Exposure to mating pheromone in haploid Saccharomyces cerevisiae cells results in the arrest of the cell cycle, expression of mating-specific genes, and polarized growth toward the mating partner . Proteins involved in signaling, polarization, cell adhesion, and fusion are localized to the tip of the mating cell (shmoo) where fusion will eventually occur . The mechanisms ensuring the correct targeting and retention of these proteins are poorly understood . Here we show that in pheromone-treated cells, a reorganization of the plasma membrane involving lipid rafts results in the retention of proteins at the tip of the mating projection, segregated from the rest of the membrane . Sphingolipid and ergosterol biosynthetic mutants fail to polarize proteins to the tip of the shmoo and are deficient in mating . Our results show that membrane microdomain clustering at the mating projection is involved in the generation and maintenance of polarity during mating.

EMBO J, 2002 Oct 15, 21(20), 5498 - 507
Hmo1, an HMG-box protein, belongs to the yeast ribosomal DNA transcription system; Gadal O et al.; Hmo1 is one of seven HMG-box proteins of Saccharo myces cerevisiae . Null mutants have a limited effect on growth . Hmo1 overexpression suppresses rpa49-Delta mutants lacking Rpa49, a non-essential but conserved subunit of RNA polymerase I corresponding to the animal RNA polymerase I factor PAF53 . This overexpression strongly increases de novo rRNA synthesis . rpa49-Delta hmo1-Delta double mutants are lethal, and this lethality is bypassed when RNA polymerase II synthesizes rRNA . Hmo1 co-localizes with Fob1, a known rDNA-binding protein, defining a narrow territory adjacent to the nucleoplasm that could delineate the rDNA nucleolar domain . These data identify Hmo1 as a genuine RNA polymerase I factor acting synergistically with Rpa49 . As an HMG-box protein, Hmo1 is remotely related to animal UBF factors . hmo1-Delta and rpa49-Delta are lethal with top3-Delta DNA topoisomerase (type I) mutants and are suppressed in mutants lacking the Sgs1 DNA helicase . They are not affected by top1-Delta defective in Top1, the other eukaryotic type I topoisomerase . Conversely, rpa34-Delta mutants lacking Rpa34, a non-essential subunit associated with Rpa49, are lethal in top1-Delta but not in top3-Delta.

Genome Biol . 2002 Sep 20;3(10):REVIEWS1028 . Epub 2002 Sep 20.
A genomic approach to studying cell-size homeostasis in yeast; Millar JB; Using a complete set of budding-yeast mutants bearing deletions of all known open reading frames, a recent study has revealed multiple overlapping pathways that coordinately regulate cell-cycle progression with ribosome biogenesis and translation efficiency, providing new insights into the mechanisms governing cell-size homeostasis in eukaryotes..

Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14298 - 302 Epub 2002 Oct 07.
Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals; Ishida S et al.; Cisplatin is a chemotherapeutic drug used to treat a variety of cancers . Both intrinsic and acquired resistance to cisplatin, as well as toxicity, limit its effectiveness . Molecular mechanisms that underlie cisplatin resistance are poorly understood . Here we demonstrate that deletion of the yeast CTR1 gene, which encodes a high-affinity copper transporter, results in increased cisplatin resistance and reduced intracellular accumulation of cisplatin . Copper, which causes degradation and internalization of Ctr1 protein (Ctr1p), enhances survival of wild-type yeast cells exposed to cisplatin and reduces cellular accumulation of the drug . Cisplatin also causes degradation and delocalization of Ctr1p and interferes with copper uptake in wild-type yeast cells . Mouse cell lines lacking one or both mouse Ctr1 (mCtr1) alleles exhibit increased cisplatin resistance and decreased cisplatin accumulation in parallel with mCtr1 gene dosage . We propose that cisplatin uptake is mediated by the copper transporter Ctr1p in yeast and mammals . The link between Ctr1p and cisplatin transport may explain some cases of cisplatin resistance in humans and suggests ways of modulating sensitivity and toxicity to this important anticancer drug.

J Cell Biol, 2002 Oct 14, 159(1), 23 - 8 Epub 2002 Oct 07.
The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase; Roth AF et al.; Protein palmitoylation has been long appreciated for its role in tethering proteins to membranes, yet the enzymes responsible for this modification have eluded identification . Here, experiments in vivo and in vitro demonstrate that Akr1p, a polytopic membrane protein containing a DHHC cysteine-rich domain (CRD), is a palmitoyl transferase (PTase) . In vivo, we find that the casein kinase Yck2p is palmitoylated and that Akr1p function is required for this modification . Akr1p, purified to near homogeneity from yeast membranes, catalyzes Yck2p palmitoylation in vitro, indicating that Akr1p is itself a PTase . Palmitoylation is stimulated by added ATP . Furthermore, during the reaction, Akr1p is itself palmitoylated, suggesting a role for a palmitoyl-Akr1p intermediate in the overall reaction mechanism . Mutations introduced into the Akr1p DHHC-CRD eliminate both the trans- and autopalmitoylation activities, indicating a central participation of this conserved sequence in the enzymatic reaction . Finally, our results indicate that palmitoylation within the yeast cell is controlled by multiple PTase specificities . The conserved DHHC-CRD sequence, we propose, is the signature feature of an evolutionarily widespread PTase family.

Plant Mol Biol, 2002 Oct, 50(3), 405 - 13
Tobacco Nia2 cDNA functionally complements a Hansenula polymorpha yeast mutant lacking nitrate reductase . A new expression system for the study of plant proteins involved in nitrate assimilation; Perdomo G et al.; An integrative expression vector based on promoter and terminator transcriptional sequences from the Hansenula polymorpha nitrate reductase gene (YNR1) has been developed to express nitrate assimilation plant genes in the nitrate assimilatory yeast H . polymorpha . Using this vector a plant nitrate reductase cDNA (tobacco Nia2) was expressed for the first time in a nitrate assimilatory yeast . The heterologous nitrate reductase produced retained its biochemical and physiological properties such as its NADH-dependent nitrate reductase activity, and allowed growth in nitrate containing media in a strain lacking endogenous nitrate reductase activity . In the transgenic strain, maximum tobacco nitrate reductase activity was about 70% of that presented in the wild-type . On the other hand, the disappearance of nitrate reductase activity correlated with that of the enzyme protein in response to the addition of ammonium to the medium and took place more rapidly in the transgenic strain than in the wild-type . Nitrate reductase activity of the recombinant strain assayed in the presence of Mg2+ was about 30% of that observed when assayed with EDTA . This result, together with a decreased growth rate in nitrate, suggests that tobacco nitrate reductase could be partially inactivated in H . polymorpha by phosphorylation and binding of 14-3-3-like proteins . These results show that H . polymorpha is a useful yeast heterologous expression system for studying plant proteins involved in nitrate assimilation.

Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13669 - 74 Epub 2002 Oct 04.
Yeast Tdp1 and Rad1-Rad10 function as redundant pathways for repairing Top1 replicative damage; Vance JR et al.; When a replication fork collides with a DNA topoisomerase I (Top1) cleavage complex, the covalently bound enzyme must be removed from the DNA 3' end before recombination-dependent replication restart . Here we report that the tyrosyl-DNA phosphodiesterase Tdp1 and the structure-specific endonuclease Rad1-Rad10 function as primary alternative pathways of Top1 repair in Saccharomyces cerevisiae . Thus, tdp1 rad1 cells (including the catalytic point mutant rad1-D869A) not only are highly sensitive to the Top1 poison camptothecin but also exhibit a TOP1-dependent growth delay . Extensive genetic analysis revealed that both Tdp1 and Rad1-Rad10 repair proceed through recombination that equally depends on RAD52, RAD51, and RAD50 . The Rad1-Rad10 pathway further particularly depends on RAD59 and SRS2 but is independent of other nucleotide excision repair genes . Although this pattern is consistent with Rad1-Rad10 removing Top1 in a manner similar to its removal of nonhomologous tails during gene conversion, these differ in that Top1 removal does not require Msh2-Msh3 . Finally, we show that yeast lacking the Rad1-Rad10-related proteins Mus81-Mms4 display a unique pattern of camptothecin sensitivity and suggest a concerted model for the action of these endonucleases.

Genes Dev, 2002 Oct 1, 16(19), 2479 - 84
Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus; Pasero P et al.; How eukaryotes specify their replication origins is an important unanswered question . Here, we analyze the replicative organization of yeast rDNA, which consists of approximately 150 identical repeats, each containing a potential origin . Using DNA combing and single-molecule imaging, we show that functional rDNA origins are clustered and interspersed with large domains where initiation is silenced . This repression is largely mediated by the Sir2p histone-deacetylase . Increased origin firing in sir2 Delta mutants leads to the accumulation of circular rDNA species, a major determinant of yeast aging . We conclude that rDNA replication is regulated epigenetically and that Sir2p may promote genome stability and longevity by suppressing replication-dependent rDNA recombination.

Genome Res, 2002 Oct, 12(10), 1533 - 9
Molecular characterization of a chromosomal rearrangement involved in the adaptive evolution of yeast strains; Perez-Ortin JE et al.; Wine yeast strains show a high level of chromosome length polymorphism . This polymorphism is mainly generated by illegitimate recombination mediated by Ty transposons or subtelomeric repeated sequences . We have found, however, that the SSU1-R allele, which confers sulfite resistance to yeast cells, is the product of a reciprocal translocation between chromosomes VIII and XVI due to unequal crossing-over mediated by microhomology between very short sequences on the 5' upstream regions of the SSU1 and ECM34 genes . We also show that this translocation is only present in wine yeast strains, suggesting that the use for millennia of sulfite as a preservative in wine production could have favored its selection . This is the first time that a gross chromosomal rearrangement is shown to be involved in the adaptive evolution of Saccharomyces cerevisiae.

Trends Biochem Sci, 2002 Oct, 27(10), 514 - 20
Evolution of protein kinase signaling from yeast to man; Manning G et al.; Protein phosphorylation controls many cellular processes, especially those involved in intercellular communication and coordination of complex functions . To explore the evolution of protein phosphorylation, we compared the protein kinase complements ('kinomes') of budding yeast, worm and fly, with known human kinases . We classify kinases into putative orthologous groups with conserved functions and discuss kinase families and pathways that are unique, expanded or lost in each lineage . Fly and human share several kinase families involved in immunity, neurobiology, cell cycle and morphogenesis that are absent from worm, suggesting that these functions might have evolved after the divergence of nematodes from the main metazoan lineage.

Trends Pharmacol Sci, 2002 Oct, 23(10), 445 - 7
New genome-wide methods bring more power to yeast as a model organism; Game JC; Recent developments in genetic research using Saccharomyces cerevisiae have provided rapid new methods for identifying the genes that control cellular responses to treatment with a wide variety of agents, including chemicals and radiation . This significantly increases the power of yeast as a model system for studying human cellular responses to these agents, and for identifying human genes that are important in DNA repair and cancer.

BMC Mol Biol . 2002 Oct 07;3(1):15.
Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum; Agaphonov MO et al.; BACKGROUND: Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway . Recombinant proteins can also be degraded by the vacuolar protease complex . Human urokinase type plasminogen activator (uPA) is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown . RESULTS: We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation . The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum . Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate . Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree . Retention of uPA in the endoplasmic reticulum stimulates its aggregation . CONCLUSIONS: The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum . Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

Nucleic Acids Res, 2002 Oct 1, 30(19), 4222 - 31
Deletions in the S1 domain of Rrp5p cause processing at a novel site in ITS1 of yeast pre-rRNA that depends on Rex4p; Eppens NA et al.; Rrp5p is the only protein so far known to be required for the processing of yeast pre-rRNA at both the early sites A0, A1 and A2 leading to 18S rRNA and at site A3, the first step specific for the pathway leading to 5.8S/25S rRNA . Previous in vivo mutational analysis of Rrp5p demonstrated that the first 8 of its 12 S1 RNA-binding motifs are involved in the formation of the 'short' form of 5.8S rRNA (5.8S(S)), which is the predominant species under normal conditions . We have constructed two strains in which the genomic RRP5 gene has been replaced by an rrp5 deletion mutant lacking either S1 motifs 3-5 (rrp5-Delta3) or 5-8 (rrp5-Delta4) . The first mutant synthesizes almost exclusively 5.8S(L) rRNA, whereas the second one still produces a considerable amount of the 5.8S(S) species . Nevertheless, both mutations were found to block cleavage at site A3 completely . Instead, a novel processing event occurs at a site in a conserved stem-loop structure located between sites A2 and A3, which we have named A4 . A synthetic lethality screen using the rrp5-Delta3 and rrp-Delta4 mutations identified the REX4 gene, which encodes a non-essential protein belonging to a class of related yeast proteins that includes several known 3'-->5' exonucleases . Inactivation of the REX4 gene in rrp5-Delta3 or rrp-Delta4 cells abolished cleavage at A4, restored cleavage at A3 and returned the 5.8S(S):5.8S(L) ratio to the wild-type value . The sl phenotype of the rrp5Delta/rex4(-) double mutants appears to be due to a severe disturbance in ribosomal subunit assembly, rather than pre-rRNA processing . The data provide direct evidence for a crucial role of the multiple S1 motifs of Rrp5p in ensuring the correct assembly and action of the processing complex responsible for cleavage at site A3 . Furthermore, they clearly implicate Rex4p in both pre-rRNA processing and ribosome assembly, even though this protein is not essential for yeast.

Nucleic Acids Res, 2002 Oct 1, 30(19), 4199 - 207
Transcription initiation in vivo without classical transactivators: DNA kinks flanking the core promoter of the housekeeping yeast adenylate kinase gene, AKY2, position nucleosomes and constitutively activate transcription; Angermayr M et al.; The housekeeping gene of the major adenylate kinase in Saccharomyces cerevisiae (AKY2, ADK1) is constitutively transcribed at a moderate level . The promoter has been dissected in order to define elements that effect constitutive transcription . Initiation of mRNA synthesis at the AKY2 promoter is shown to be mediated by a non-canonic core promoter, (TA)(6) . Nucleotide sequences 5' of this element only marginally affect transcription suggesting that promoter activation can dispense with transactivators and essentially involves basal transcription . We show that the core promoter of AKY2 is constitutively kept free of nucleosomes . Analyses of permutated AKY2 promoter DNA revealed the presence of bent DNA . DNA structure analysis by computer and by mutation identified two kinks flanking an interstitial stretch of 65 bp of moderately bent core promoter DNA . Kinked DNA is likely incompatible with packaging into nucleosomes and responsible for positioning nucleosomes at the flanks allowing unimpeded access of the basal transcription machinery to the core promoter . The data show that in yeast, constitutive gene expression can dispense with classical transcriptional activator proteins, if two prerequisites are met: (i) the core promoter is kept free of nucleosomes; this can be due to structural properties of the DNA as an alternative to chromatin remodeling factors; and (ii) the core promoter is pre-bent to allow a high rate of basal transcription initiation.

Acta Microbiol Pol, 2002, 51(2), 115 - 20
Isolation of alcohol tolerant, osmotolerant and thermotolerant yeast strains and improvement of their alcohol tolerance by UV mutagenesis; Unaldi MN et al.; In this study, the yeast strains were isolated from grapes by serial dilution technique to determine their alcohol-, sugar- and thermotolerance . 34 wild type yeast strains were isolated and alcohol-, sugar- and thermotolerance of these strains were determined . The maximum alcohol tolerance was found to be 9% (v/v) in yeast strain which is named Y2 . Thermotolerance behavior of 6 strains were investigated . The strains were treated with UV light with intervals of 20, 30, 40 and 50 seconds . Selected resistant colonies were investigated for alcohol tolerance . It was found that alcohol tolerance increased from 9% (v/v) to 12% (v/v) on Y2 strain.

Cell Mol Life Sci, 2002 Aug, 59(8), 1241 - 5
Differential display analysis of gene expression in yeast; Ivanova AV et al.; RNA differential display (DD) is a powerful and straightforward method that employs random reverse-transcription polymerase chain reaction amplification of mRNA species with electrophoresis for comparative analysis of two or more transcriptomes . The small yeast genome represents a convenient model for studying basic functions of the eukaryotic genome and simultaneously provides valuable information towards further refinement of this technique . Several examples discussed below illustrate how DD coupled with classical yeast genetic approaches may be used for studying transcriptionally regulated genetic systems.

Food Nutr Bull, 2002 Sep, 23(3 Suppl), 61 - 5
Uptake rate measurement of some amino acids on normal and treated yeast cells to xenobiotics using 14C labelled amino acid; Tanudjojo N et al.; Benzo(alpha)pyrene (BP) and 7,12--dimethylbenz(alpha) anthracene (DMBA) are potent carcinogens for mammals, which are able to affect the normal metabolic processes . The influence both of BP and DMBA to the transport rate of individual 14C labeled amino acids (14C-lysine; 14C-valine; 14C-leucine or 14C-tyrosine) in yeast Saccharomyces cerevisiae strain A3 were studied by introducing about one microCi (37 kBq) of individual 14C labeled amino acid into 30 ml liquid ethanol media that contained BP (0.001% v/v) or DMBA (0.001% v/v), then followed by inoculating a known concentration of yeast suspension in such a manner to get the initial optical density (OD) of new cultures were about 0.10 . Uptake rates were determined at certain intervals after inoculation, using a liquid scintillation counter . The results show that BP had the tendency to increase the uptake rate while DMBA showed a reversed effect on the use of amino acids . It was also found that tyrosine was absorbed faster than valine as well as leucine and this was different with the result reported by the former investigators.

Curr Biol, 2002 Oct 1, 12(19), 1652 - 60
cis-acting DNA from fission yeast centromeres mediates histone H3 methylation and recruitment of silencing factors and cohesin to an ectopic site; Partridge JF et al.; BACKGROUND: Metazoan centromeres are generally composed of large repetitive DNA structures packaged in heterochromatin . Similarly, fission yeast centromeres contain large inverted repeats and two distinct silenced domains that are both required for centromere function . The central domain is flanked by outer repetitive elements coated in histone H3 methylated on lysine 9 and bound by conserved heterochromatin proteins . This centromeric heterochromatin is required for cohesion between sister centromeres . Defective heterochromatin causes premature sister chromatid separation and chromosome missegregation . The role of cis-acting DNA sequences in the formation of centromeric heterochromatin has not been established . RESULTS: A deletion strategy was used to identify centromeric sequences that allow heterochromatin formation in fission yeast . Fragments from the outer repeats are sufficient to cause silencing of an adjacent gene when inserted at a euchromatic chromosomal locus . This silencing is accompanied by the local de novo methylation of histone H3 on lysine 9, recruitment of known heterochromatin components, Swi6 and Chp1, and the provision of a new strong cohesin binding site . In addition, we demonstrate that the chromodomain of Chp1 binds to MeK9-H3 and that Chp1 itself is required for methylation of histone H3 on lysine 9 . CONCLUSIONS: A short sequence, reiterated at fission yeast centromeres, can direct silent chromatin assembly and cohesin recruitment in a dominant manner . The heterochromatin formed at the euchromatic locus is indistinguishable from that found at endogenous centromeres . Recruitment of Rad21-cohesin underscores the link between heterochromatin and chromatid cohesion and indicates that these centromeric elements act independently of kinetochore activity to recruit cohesin.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1887 - 92
Udeniomyces pannonicus sp . nov., a ballistoconidium-forming yeast isolated from leaves of plants in Hungary; Niwata Y et al.; Fifteen ballistoconidium-forming yeasts, isolated from the leaves of plants in Hungary, showed morphological, physiological and biochemical characteristics similar to those of Udeniomyces pyricola . The identical sequences of internal transcribed spacer regions for selected strains (HY-16T, HY-29, HY-111 and HY-186) indicated that they should be classified as one species . Although a representative strain, HY-16T, showed a closer relationship to Itersonilia perplexans than to known Udeniomyces species in phylogenetic trees constructed using 18S rDNA and the D1/D2 region of the 26S rDNA sequence, this species was placed in the genus Udeniomyces on the basis of its morphological and chemotaxonomic characteristics . Udeniomycespannonicus sp . nov . (type strain HY-16T = JCM 11145T = NCAIM Y 01556T = CBS 9123T) is proposed.

J Biol Chem, 2002 Dec 13, 277(50), 48627 - 34 Epub 2002 Sep 30.
An essential function of yeast cyclin-dependent kinase Cdc28 maintains chromosome stability; Kitazono AA et al.; Multiple surveillance pathways maintain genomic integrity in yeast during mitosis . Although the cyclin-dependent kinase Cdc28 is a well established regulator of mitotic progression, evidence for a direct role in mitotic surveillance has been lacking . We have now implicated a conserved sequence in the Cdc28 carboxyl terminus in maintaining chromosome stability through mitosis . Six temperature-sensitive mutants were isolated via random mutagenesis of 13 carboxyl-terminal residues . These mutants identify a Cdc28 domain necessary for proper mitotic arrest in the face of kinetochore defects or microtubule inhibitors . These chromosome stability-defective cdc28(CST) mutants inappropriately continue mitosis when the mitotic spindle is disrupted at 23 degrees C, display high rates of spontaneous chromosome loss at 30 degrees C, and suffer catastrophic aneuploidy at 35 degrees C . A dosage suppression screen identified Cak1, a kinase known to phosphorylate and activate Cdc28, as a specific high copy suppressor of cdc28(CST) temperature sensitivity and chromosome instability . Suppression is independent of the kinase activity of Cak1, suggesting that Cak1 may bind to the carboxyl terminus to serve a non-catalytic role in assembly and/or stabilization of active Cdc28 complexes . Significantly, these studies implicate Cdc28 and Cak1 in an essential surveillance function required to maintain genetic stability through mitosis.

J Biochem (Tokyo), 2002 Oct, 132(4), 649 - 54
A yeast mitochondrial ATPase inhibitor interacts with three proteins that are easy to dissociate from the mitochondrial inner membrane; Ichikawa N et al.; A mitochondrial ATPase inhibitor is a 7.4 kDa protein that regulates the catalytic activity of ATP synthase (F(1)F(o)-ATPase) . In the present study, we examined the binding sites of the inhibitor on the mitochondrial membrane using chemical cross-linkers, disuccinimidyl suberate (DSS) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) . Most of the inhibitors were recovered from the inner membrane fraction of mitochondria, indicating that the inhibitor binds to the membrane . Seven different cross-linked products that reacted with the antibody against the inhibitor were detected . The apparent molecular masses of the products were 61, 58, 47, 41, 28, 27, and 26 kDa . The 61 and 58 kDa products were attributed to the inhibitor+alpha and inhibitor+beta adducts on immunoblotting . The proteins cross-linked to the inhibitor in the 28, 27, and 26 kDa products were distinguished from subunit 4 (23 kDa), oligomycin sensitivity conferring protein (21 kDa), and subunit d (20 kDa) of F(1)F(o)-ATPase by analysis of the cross-linked products of mutant mitochondria in which the three proteins were replaced by hemagglutinin-tagged versions . The 28, 27, and 26 kDa products could be gradually dissociated from the mitochondrial membrane by increasing the salt concentration . These results shows that the endogenous inhibitor binds not only to the catalytic part of the enzyme, but also to the 19-21 kDa proteins that loosely associate with the mitochondrial inner membrane.

Hypertens Res, 2002 Jul, 25(4), 647 - 52
Screening the proteins that interact with calpain in a human heart cDNA library using a yeast two-hybrid system; Jiang LQ et al.; Calpain, a cytosolic cysteine protease, requires calcium ions for activity . It has been reported that calpain is involved in the degradation of myofibrillar and neurofilament proteins, and the activation of phosphorylase b kinase and protein kinase C . More recently, calpain was shown to participate in apoptosis . In order to understand the calpain-related signal transduction pathway and its changes during hypertrophy, and especially in hypertension, we screened a human heart cDNA library to find proteins that interact with calpain . 1) Using PCR we amplified the full-length, domain II, domain III and domain IV cDNA of calpain (calcium-activated neutral protease, CANP) I large subunit respectively . 2) Then the fragments were cloned into pGBKT7 vector, resulting in 4 bait expression constructs (pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III, and pG BKT7-CANP IV) . 3) After 4 bait vectors were transformed into AH109 by the lithium acetate-mediated method, AH109/pGBKT7-CANP, AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III, and AH109/pGBKT7-CANP IV were obtained, respectively . 4) After the human heart cDNA library was sequentially transformed into AH109/ pGBKT7-CANP, 1000-1200 positive clones were grown on SD/Trp-Leu-Ade-His- . Only 150 positive clones were obtained through a colony-lift filter assay to detect beta-galactosidase activity . 5) Total 105 clones among above 150 positive clones were eliminated through that the duplicate, pseudopositive and autoactive detection, respectively . 6) Finally, sequencing eliminated clones with a wrong open reading frame (ORF) . Eight clones were cancelled with wrong ORF . The remaining 37 positive clones were analyzed using BLAST software available on the Internet and classified as follows: 1 . enzymes or proteins related to signal transduction in the cell; 2 . contraction proteins 3 . matrix proteins 4 . unknown proteins . 7) In order to determine which domain of the calpain I large subunit was involved in the interaction with these real clones, the 37 clones were transformed into AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III or AH109/pGBKT7-CANP IV . Among these 37 clones, 29 clones could interact with domain II, 5 clones could interact with domain III and 6 clones could interact with domain IV . Thus, we successfully constructed 4 bait expression vectors, pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III and pGBKT7-CANP IV, and obtained 37 real positive clones that interacted with the calpain I large subunit by screening a human heart cDNA library using pGBKT7-CANP as bait . Among them, 29 clones could interact with domain II of the calpain I large subunit, where the active site of calpain is located . Additional studies will be needed to clarify the calpain-related signal transduction pathway in greater detail.

J Biol Chem, 2002 Dec 13, 277(50), 48002 - 8 Epub 2002 Sep 27.
Protein phosphatase-1 binding to scd5p is important for regulation of actin organization and endocytosis in yeast; Chang JS et al.; SCD5, an essential gene, encodes a protein important for endocytosis and actin organization in yeast . Previous two-hybrid screens showed that Scd5p interacts with Glc7p, a yeast Ser/Thr-specific protein phosphatase-1 (PP1) that participates in a variety of cellular processes . PP1 substrate specificity in vivo is regulated by association with different regulatory or targeting subunits, many of which have a consensus PP1-binding site ((V/I)XF, with a basic residue at the -1 or -2 position) . Scd5p contains two of these potential PP1-binding motifs: KVDF (amino acids 240-243) and KKVRF (amino acids 272-276) . Deletion analysis mapped the PP1-binding domain to a region of Scd5p containing these motifs . Therefore, the consequence of mutating these two potential PP1-binding sites was examined . Although mutation of KVDF had no effect, alteration of KKVRF dramatically reduced Scd5p interaction with Glc7p and resulted in temperature-sensitive growth . Furthermore, this mutation caused defects in fluid phase and receptor-mediated endocytosis and actin organization . Overexpression of GLC7 suppressed the temperature-sensitive growth of the KKVRF mutant and partially rescued the actin organization phenotype . These results provide evidence that Scd5p is a PP1 targeting subunit for regulation of actin organization and endocytosis or that Scd5p is a PP1 substrate, which regulates the function of Scd5p in these processes.

Nat Biotechnol, 2002 Oct, 20(10), 991 - 7
Analyzing yeast protein-protein interaction data obtained from different sources; Bader GD et al.; High-throughput methods for detecting protein interactions, such as mass spectrometry and yeast two-hybrid assays, continue to produce vast amounts of data that may be exploited to infer protein function and regulation . As this article went to press, the pool of all published interaction information on Saccharomyces cerevisiae was 15,143 interactions among 4,825 proteins, and power-law scaling supports an estimate of 20,000 specific protein interactions . To investigate the biases, overlaps, and complementarities among these data, we have carried out an analysis of two high-throughput mass spectrometry (HMS)-based protein interaction data sets from budding yeast, comparing them to each other and to other interaction data sets . Our analysis reveals 198 interactions among 222 proteins common to both data sets, many of which reflect large multiprotein complexes . It also indicates that a "spoke" model that directly pairs bait proteins with associated proteins is roughly threefold more accurate than a "matrix" model that connects all proteins . In addition, we identify a large, previously unsuspected nucleolar complex of 148 proteins, including 39 proteins of unknown function . Our results indicate that existing large-scale protein interaction data sets are nonsaturating and that integrating many different experimental data sets yields a clearer biological view than any single method alone.

Hum Mol Genet, 2002 Oct 1, 11(21), 2635 - 43
A non-essential function for yeast frataxin in iron-sulfur cluster assembly; Duby G et al.; Friedreich's ataxia is caused by a deficit in frataxin, a small mitochondrial protein of unknown function that has been conserved during evolution . Previous studies have pointed out a role for frataxin in mitochondrial iron-sulfur (Fe-S) metabolism . Here, we have analyzed the incorporation of Fe-S clusters into yeast ferredoxin imported into isolated energized mitochondria from cells grown in the presence of glycerol, an obligatory respiratory carbon source . Similar amounts of apo-ferredoxin precursor were imported into mitochondria and processed in wild-type and yfh1-deleted (delta YF111) strains . However, the incorporation of Fe-S clusters into apo-ferredoxin was significantly reduced in delta YFH1 mitochondria . The newly assembled ferredoxin was stable, excluding the possibility that the decreased incorporation was a result of increased oxidative damage . When delta YFH1 cells were grown in raffinose medium, the formation of holo-ferredoxin was low, as a consequence of the decrease in ferredoxin precursor import into mitochondria . However, the decrease in the conversion rate of apo- into holo-ferredoxin was in the same range as for glycerol-grown cells, indicating that the extent of the defect in Fe-S protein assembly is similar under different physiological conditions . These data show that frataxin is not essential for Fe-S protein assembly, but improves the efficiency of the process . The large variations observed in the activity of Fe-S cluster proteins under different physiological conditions result from secondary defects in the physiology of delta YFH1 cells.

Genes Cells, 2002 Oct, 7(10), 1043 - 57
Mouse GSPT2, but not GSPT1, can substitute for yeast eRF3 in vivo; Le Goff C et al.; BACKGROUND: The termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs), eRF1 and eRF3 . In mammals two genes encoding eRF3 structural homologues were identified and named GSPT1 and GSPT2 . RESULTS: In the present study, we demonstrate that mouse mGSPT2 but not mGSPT1 could functionally substitute the essential yeast gene SUP35 . However, we show that the complementation property of mGSPT1 protein is modified when NH2-tagged by GST . Since mGSPT1 and mGSPT2 differ mainly in their N-terminal regions, we developed a series of N-terminal deleted constructs and tested them for complementation in yeast . We found that at least amino acids spanning 84-120 of mGSPT1 prevent the complementation of sup35 mutation . The fact that chimeras between mGSPT1, mGSPT2 and yeast Sup35 complement the disruption of the SUP35 gene indicates that the N-terminal region of mGSPT1 is not sufficient by itself to prevent complementation . Complementation of the mutant with a double disruption of SUP35 and SUP45 genes is obtained when mGSPT2 and human eRF1 are co-expressed but not by co-expression of mGSPT1 and human eRF1 . CONCLUSIONS: Our results strongly suggest that the two proteins (mGSPT1 and mGSPT2) are different . We hypothesize that the full length mGSPT1 does not have the properties expected for eRF3.

Genes Cells, 2002 Oct, 7(10), 1009 - 19
Calcineurin is implicated in the regulation of the septation initiation network in fission yeast; Lu Y et al.; BACKGROUND: In fission yeast, calcineurin has been implicated in cytokinesis because calcineurin-deleted cells form multiple septa and cell separation is impeded . However, this mechanism remains unclear . RESULTS: We screened for mutations that confer synthetic lethality with calcineurin deletion and isolated a mutant, its 10-1/cdc7-i10, a novel allele of the cdc7+ gene involved in the septation initiation network (SIN) . The mutation created a termination codon, resulting in the truncation of Cdc7 by 162 amino acids, which is not localized in the spindle pole body . Following treatment with the immune suppressive drug FK506, cdc7-i10 and the original cdc7-24 mutant cells showed highly elongated multinuclear morphology with few visible septa, closely resembling the phenotype at the restrictive temperature . Other SIN mutants, cdc11, spg1, sid2 and mob1 showed similar phenotypes following FK506 treatment . Consistent with this, expression of the constitutively active calcineurin suppressed the growth defects and septum initiation deficiency of these SIN mutants at the restrictive temperature . Moreover, electron microscopy revealed that calcineurin-deleted cells had very thick multiple septa which were partially and ectopically formed . CONCLUSION: These results suggest that calcineurin is involved in the regulation of the SIN pathway, and is required for the proper formation and maturation of the septum in fission yeast.

Biosci Biotechnol Biochem, 2002 Aug, 66(8), 1744 - 7
Iron absorption in rats increased by yeast glucan; Mai TT et al.; The effects of brewer's yeast cell walls and two of its components, glucan and mannan, on the absorption of 59Fe by anemic rats were investigated . After administration of the label, the percentage of 59Fe taken up into the blood of group given glucan was generally similar to that of a group given yeast cell walls, both values were higher than in controls . The incorporation of 59Fe into the small intestines was higher in the group given glucan than in the controls or a group given a glucan-mannan mixture . Glucan is the main substance in yeast cell walls that increases iron absorption.

Anal Chem, 2002 Sep 15, 74(18), 4602 - 10
Identification of selenium-containing glutathione S-conjugates in a yeast extract by two-dimensional liquid chromatography with inductively coupled plasma MS and nanoelectrospray MS/MS detection; Lindemann T et al.; An approach for the identification of unknown selenium-containing biomolecules was developed, enabling the identification of selenodiglutathione (GS-Se-SG) and the mixed selenotrisulfide of glutathione and cysteinylglycine (GS-Se-SCG) in aqueous yeast extracts . The method consists of two-dimensional liquid chromatography, inductively coupled plasma mass spectrometry (ICPMS) and nanoelectrospray tandem mass spectrometry . Analytes were separated by size-exclusion chromatography followed by preconcentration and separation on a porous graphitic carbon HPLC column . The HPLC effluent was monitored for selenium by ICPMS, and two selenium-containing fractions were isolated and analyzed by nanoelectrospray MS . The nanoelectrospray technique has a low sample consumption of approximately 80 nL/min, enabling a preconcentration of the sample to a few microliters . Mass spectra of the two fractions showed the characteristic Se isotopic pattern centered at m/z 693.1 and 564.0 for the {M + H}+ 80Se ions . MS/MS spectra of adjacent parent ions confirmed the presence of Se . The two selenium species were identified as GS-Se-SG and GS-Se-SCG by collision induced dissociation (CID) . The accurately measured masses of the most abundant 691 and 693 u parent ions are in good agreement (differences = 3 ppm) with the theoretical masses . To our knowledge, this is the first identification of GS-Se-SG and GS-Se-SCG in biological matrixes by MS/MS.

J Biol Chem, 2002 Nov 22, 277(47), 45099 - 107 Epub 2002 Sep 23.
Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1; Bitterman KJ et al.; The Saccharomyces cerevisiae Sir2 protein is an NAD(+)-dependent histone deacetylase that plays a critical role in transcriptional silencing, genome stability, and longevity . A human homologue of Sir2, SIRT1, regulates the activity of the p53 tumor suppressor and inhibits apoptosis . The Sir2 deacetylation reaction generates two products: O-acetyl-ADP-ribose and nicotinamide, a precursor of nicotinic acid and a form of niacin/vitamin B(3) . We show here that nicotinamide strongly inhibits yeast silencing, increases rDNA recombination, and shortens replicative life span to that of a sir2 mutant . Nicotinamide abolishes silencing and leads to an eventual delocalization of Sir2 even in G(1)-arrested cells, demonstrating that silent heterochromatin requires continual Sir2 activity . We show that physiological concentrations of nicotinamide noncompetitively inhibit both Sir2 and SIRT1 in vitro . The degree of inhibition by nicotinamide (IC(50) < 50 microm) is equal to or better than the most effective known synthetic inhibitors of this class of proteins . We propose a model whereby nicotinamide inhibits deacetylation by binding to a conserved pocket adjacent to NAD(+), thereby blocking NAD(+) hydrolysis . We discuss the possibility that nicotinamide is a physiologically relevant regulator of Sir2 enzymes.

Vaccine, 2002 Oct 4, 20(29-30), 3523 - 31
Yeast-expressed Puumala hantavirus nucleocapsid protein induces protection in a bank vole model; Dargeviciute A et al.; Hantaviruses are rodent-borne agents that cause severe human diseases . The coding sequences for the authentic and a His-tagged Puumala hantavirus (PUUV) nucleocapsid (N) protein were expressed in yeast (Saccharomyces cerevisiae) . N-specific monoclonal antibodies demonstrated native antigenicity of the two proteins . All bank voles vaccinated with the His-tagged N protein in Freund's adjuvant (n=12) were defined as completely protected against subsequent virus challenge, based on the absence of viral N protein, RNA and G2-specific antibodies . In the group vaccinated with the yeast-expressed authentic N protein in Freund's adjuvant, 2/6 animals were defined as completely protected and 4/6 as partially protected . Moreover, when animals were vaccinated with the His-tagged N protein in an adjuvant certified for human use (alum), all (n=8) were at least partially protected (six completely, two partially) . The general advantages of the yeast expression system make the described recombinant proteins promising candidate vaccines against hantavirus infection.

FEBS Lett, 2002 Sep 25, 528(1-3), 23 - 6
Programmed death in yeast as adaptation?
Skulachev VP.
During recent years, several pieces of indirect evidence of a programmed death in yeast have been published . Among them there are observations that some mammalian pro- or anti-apoptotic proteins induce or prevent the death of yeast; some toxic compounds kill yeast at lower concentrations if protein synthesis is operative; this death, as well as the death due to certain mutations, shows some apoptotic markers . In April 2002, the yeast programmed death concept received direct support . Madeo et al . {Madeo et al., Mol . Cell 9 (2002) 911-917} disclosed a caspase which is activated by H(2)O(2) or aging and is required for the protein-synthesis-dependent death of yeast . Thus, a specific apoptosis-mediating protein was identified for the first time in Saccharomyces cerevisiae . Independently, Severin and Hyman {Severin, F.F., Hyman, A.A., Curr . Biol . 12 (2002) R233-R235} discovered that death of yeast, induced by a high level of a pheromone, is programmed . In particular, the death was found to be prevented by cycloheximide and cyclosporin A . It required mitochondrial DNA, cytochrome c and the pheromone-initiated protein kinase cascade . When haploids of opposite mating types were mixed, some cells died, the inhibitory pattern being the same as in the case of the killing by pheromone . Inhibition of mating proved to be favorable for death . Thus, pheromone not only activates mating but also eliminates yeast cells failing to mate . Such an effect should (i) stimulate switch of the yeast population from vegetative to sexual reproduction, and (ii) shorten the life span and, hence, accelerate changing of generations . As a result, the probability of appearance of new traits could be enhanced when ambient conditions turned for the worse.

Biol Trace Elem Res, 2002 Aug, 88(2), 193 - 9
A preliminary study of chromium distribution in chromium-rich brewer's yeast cell by NAA; Ding WJ et al.; The purpose of this study was to assess the chromium (Cr) distribution in chromium-rich brewer's yeast cell . The chromium concentrations in the cell wall and protoplast fractions of the chromium-rich yeast were determined by neutron activation analysis (NAA) . Moreover, the combined state of chromium and amino acid content in the Cr-rich brewer's yeasts was analyzed and measured . The experimental results indicate that the introduction of water-soluble chromium(III) salt as a component of the culture medium for yeasts results in a substantial amount of chromium absorbed through the cell wall by the yeast, among which 80.9% are accumulated in the protoplast . It implies that, under optimal conditions, yeasts are capable of accumulating large amounts of chromium and incorporating chromium into organic compounds.

Biol Trace Elem Res, 2002 Aug, 88(2), 185 - 91
Evaluation of methods for total selenium determination in yeast; Renard NE et al.; The selenium determination in biological materials by the classical fluorometric method (FM) is time-consuming and also hazardous, as it requires the destruction of the organic matrix samples with hot HNO3/HClO4 mixtures prior to analysis . Accordingly, commercial analytical laboratories are increasingly using faster instrumental methods; for sample digestion, avoid using HClO4 . Because of these procedural changes, the results obtained by commercial laboratories may be unreliable, especially for samples containing Se in organic forms . One such "difficult" substrate is Se yeast, which contains most of its Se as selenomethionine . To establish which methods for Se analysis and sample digestion are applicable, samples of Se yeast and of selenomethionine standards were sent to laboratories employing either flame atomic absorption spectrometry (FAAS), inductively coupled plasma-mass spectrometry (ICP-MS), or hydride generation atomic absorption spectrometry (HGAAS) . The results were compared with those obtained by FM and non-destructive instrumental neutron activation analysis (INAA) . ICP-MS, after microwave digestion of sample with HNO3/H2O2, produced results within 5% of the expected values, as did those obtained by FM and INAA . With FAAS, acceptable results were obtained after digestion with HNO3/HCl . With HGAAS, sample digestion with HNO3/H2O2 produced values that were systematically elevated by about 10% and exhibited standard deviations of > or = 10% . Thus, current methods of sample digestion are applicable for Se yeast analysis by ICP-MS and FAAS, but not by HGAAS.

Yeast, 2002 Oct, 19(14), 1261 - 76
Functional analysis of yeast gene families involved in metabolism of vitamins B1 and B6; Rodriguez-Navarro S et al.; In order to clarify their physiological functions, we have undertaken a characterization of the three-membered gene families SNZ1-3 and SNO1-3 . In media lacking vitamin B(6), SNZ1 and SNO1 were both required for growth in certain conditions, but neither SNZ2, SNZ3, SNO2 nor SNO3 were required . Copies 2 and 3 of the gene products have, in spite of their extremely close sequence similarity, slightly different functions in the cell . We have also found that copies 2 and 3 are activated by the lack of thiamine and that the Snz proteins physically interact with the thiamine biosynthesis Thi5 protein family . Whereas copy 1 is required for conditions in which B(6) is essential for growth, copies 2 and 3 seem more related with B(1) biosynthesis during the exponential phase .

J Biol Chem, 2002 Nov 22, 277(47), 45704 - 14 Epub 2002 Sep 20.
Import of yeast mitochondrial transcription factor (Mtf1p) via a nonconventional pathway; Biswas TK et al.; The yeast mitochondrial (mt) transcription factor Mtf1p is imported into the mitochondria from the cytoplasm without a conventional mt-targeting presequence . To understand its import the mt translocation of wild type and mutant Mtf1p constructs was investigated in vitro under various assay conditions . We report here that Mtf1p, unlike most mt matrix proteins hitherto studied, is translocated into the mitochondria independent of membrane potential, ATP hydrolysis, and membrane receptor . This unusual import of Mtf1p was also observed on ice (3 degrees C) . Sub-mitochondrial fractionation demonstrated that Mtf1p was translocated in vitro to one or more of the same mt sites as the endogenous protein that includes the matrix . To identify the mt-targeting sequence of Mtf1p, various N-terminal, C-terminal, or internally deleted Mtf1p derivatives were generated . The full-length and C-terminal deletions but not the N-terminal truncated Mtf1p were imported into mitochondria, indicating the importance of its N-terminal sequence for mt targeting . However, the internal deletion of Mtf1p revealed that the first 150-amino acid N-terminal sequence alone was not sufficient for mt targeting of Mtf1p, suggesting that an extended rather than a short N-terminal sequence is required for import . We favor a model in which Mtf1p adopts an import-competent conformation during translation . Consistent with this model are three findings: most of the protein sequence appears to be required for optimal import, urea denaturation eliminates its import competence, and the import-competent form of the protein is more resistant to tryptic hydrolysis than is the denatured protein . This represents a novel mechanism for mitochondrial protein import.

Mol Biol Evol, 2002 Oct, 19(10), 1760 - 8
Asymmetric functional divergence of duplicate genes in yeast; Wagner A; Most duplicate genes are eliminated from a genome shortly after duplication, but those that remain are an important source of biochemical diversity . Here, I present evidence from genome-scale protein-protein interaction data, microarray expression data, and large-scale gene knockout data that this diversification is often asymmetrical: one duplicate usually shows significantly more molecular or genetic interactions than the other . I propose a model that can explain this divergence pattern if asymmetrically diverging duplicate gene pairs show increased robustness to deleterious mutations.

J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Sep 25, 777(1-2), 167 - 78
Yeast reporter system for rapid determination of estrogenic activity; Jungbauer A et al.; An in vitro test system for the determination of estrogens, xeno- and phytoestrogens, based on the activation of human estrogen receptor-alpha, has been examined for ability in monitoring environmental estrogens . The system consists of an expression plasmid for the human estrogen receptor-alpha and a reporter plasmid containing the lacZ gene under the control of the vitellogenin hormone response element . These plasmids have been transformed into S . cerevisae . Cultivation of yeast in the presence of estrogenic substances leads to activation of the estrogen receptor and induces the expression of the reporter lacZ . beta-Galactosidase activity of the translated gene lacZ is a measure of the estrogenic activity of a compound . First, the selectivity of the system was compared to data available in the literature . Then the sensitivity of the system was checked . The detection limit is 0.1 ng 17-beta estradiol or an equivalent activity per liter, if a sample can be concentrated 1000-fold . The system has been further characterized by selected compounds with known and unknown estrogenic activity.

J Oral Pathol Med, 2002 Oct, 31(9), 534 - 8
Detection of TP53 mutation in ameloblastoma by the use of a yeast functional assay; Shibata T et al.; BACKGROUND: Mutations in TP53 have been observed in a variety of tumors including oral lesions, though there are no reports in ameloblastomas . The purpose of the study was to examine the TP53 status of ameloblastomas using newly developed yeast functional assay whose accuracy and sensitivity has been proven to be higher than those of the previous DNA structure-based methods such as single strand conformation polymorphism (SSCP) analysis . METHODS: TP53 status was analyzed by yeast functional assay and DNA sequencing in 12 cases of ameloblastoma which were diagnosed histologically and represented the clinical features of a benign tumor . After the extraction of RNA from the frozen tissue samples without microdissection, reverse transcription (RT)-PCR was carried out and these samples were used . The assay can detect mutations of p53 mRNA between codons 67 and 347 by the DNA-binding activity of the protein and reveal them as red colonies . RESULTS: One case of 47-year-old male gave 17% red colonies of yeast and the other 11 cases gave 5.4% (mean; range, 3-8%) . To confirm this result, we obtained other nine samples from the case of 17% red colonies, which contained or did not contain tumor tissues, and analyzed them by the assay . Seven samples that were histologically negative for tumor cells gave 4.7% red colonies (mean; range, 1-7%) . Two samples that were histologically positive for tumor cells gave 20 or 46% of red colonies . The p53 plasmids were recovered from the red colonies of these three samples showing high red colony ratios and were subjected to sequencing analysis after purification . In all these samples, the same clonal mutation of TGT (Cys) 238 TAT (Tyr) was demonstrated . CONCLUSIONS: These results suggest that TP53 mutation may be involved in molecular pathogenesis in a subset of ameloblastomas, though it is infrequent.

Biochemistry, 2002 Oct 1, 41(39), 11914 - 20
Tail-anchored protein insertion into yeast ER requires a novel posttranslational mechanism which is independent of the SEC machinery; Steel GJ et al.; Tail-anchored or C-terminally-anchored proteins play many essential roles in eukaryotic cells . However, targeting and insertion of this class of membrane protein has remained elusive . In this study, we reconstitute insertion of tail-anchored proteins into microsomes derived from Saccharomyces cerevisiae . Using this approach, we are able to genetically manipulate the composition of the microsomes in order to address the question of which components of the endoplasmic reticulum (ER) are required for this process . We show that tail-anchored protein insertion is not dependent on the classical SEC translocation machinery but rather occurs via an ATP-dependent pathway involving at least one novel membrane protein factor . We further demonstrate that the specificity of this pathway is conserved between yeast and mammals.

Environ Sci Technol, 2002 Sep 15, 36(18), 3908 - 15
Effects of the pesticide thiuram: genome-wide screening of indicator genes by yeast DNA microarray; Kitagawa E et al.; Although there have been studies on the toxicity of the pesticide thiuram, the present study is the first one to attempt to integrate a whole genomic response using microarray technology . From the DNA microarray experiment it was found that exposure to thiuram led to alterations of gene expression in yeast cells and that many genes involved in detoxification and stress response were highly induced . The induced genes were classified according to the MIPS yeast database . The induction of genes concerned with folding and proteolysis reflects the protein denaturing and degradation effects of the thiuram treatment The induction of genes involved in redox and defense against reaction oxygen species also suggests that thiuram has other effects, such as oxidative stress . Genes classified for carbohydrate metabolism and energy were also highly induced, and these gene products may play the role of providing the energy for the detoxification mechanism . In addition, in view of the induction of some genes involved in DNA repair, thiuram potentially causes DNA damage . Therefore, as stated in previous reports, thiuram is a potential positive toxic chemical . On the other hand, YKL071W, YCR102C, YLR303W, and YLL057C were selected based on the result of a DNA microarray experiment and used for the promoter activity assay . Thiuram treatment affected the promoter of these genes, indicating that this technique could be used for the selection of biomarker candidates.

Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12853 - 8 Epub 2002 Sep 20.
Sphingoid base synthesis is required for oligomerization and cell surface stability of the yeast plasma membrane ATPase, Pma1; Wang Q et al.; The plasma membrane H(+)-ATPase, Pma1, is an essential and long-lived integral membrane protein . Previous work has demonstrated that the Pma1-D378N mutant is a substrate for endoplasmic reticulum (ER)-associated degradation and causes a dominant negative effect on cell growth by preventing ER export of wild-type Pma1 . We now show that Pma1-D378N is ubiquitylated, and it heterooligomerizes with wild-type Pma1, resulting in ubiquitylation and ER-associated degradation of wild-type Pma1 . In temperature-sensitive lcb1-100 cells, defective in sphingoid base synthesis, Pma1 fails to oligomerize . At 30 degrees C, lcb1-100 is a suppressor of pma1-D378N because wild-type Pma1 fails to heterooligomerize with Pma1-D378N; wild-type Pma1 moves to the cell surface, indicating that oligomerization is not required for delivery to the plasma membrane . Even in the absence of Pma1-D378N, wild-type Pma1 is ubiquitylated and it undergoes internalization from the cell surface and vacuolar degradation at 30 degrees C in lcb1-100 cells . At 37 degrees C in lcb1-100 cells, a more severe defect occurs in sphingoid base synthesis, and targeting of newly synthesized Pma1 to the plasma membrane is impaired . These data indicate requirements for sphingolipids at three discrete stages: Pma1 oligomerization at the ER, targeting to the plasma membrane, and stability at the cell surface.

J Cell Sci, 2002 Oct 15, 115(Pt 20), 3889 - 900
Retromer function in endosome-to-Golgi retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase; Burda P et al.; A direct role for phosphoinositides in vesicular trafficking has been demonstrated by the identification of the yeast VPS34 gene encoding the phosphatidylinositol 3-kinase responsible for the synthesis of phosphatidylinositol 3-phosphate (PtdIns3P) . Vps34p binds the protein kinase Vps15p, and it has recently been shown that Vps15p and Vps34p associate with Vps30p and Vps38p to form a multimeric complex, termed complex II . We observed that mutations in the VPS30 and VPS38 genes led to a selective sorting and maturation phenotype of the soluble vacuolar protease CPY . Localization studies revealed that the CPY receptor Vps10p and the Golgi-endoprotease Kex2p were mislocalized to vacuolar membranes in strains deficient for either Vps30p or Vps38p, respectively . Interestingly, we measured decreased PtdIns3P levels in Deltavps30 and Deltavps38 cells and observed redistribution of Vps5p and Vps17p to the cytoplasm in these mutants . Vps5p and Vps17p are subunits of the retromer complex that is required for endosome-to-Golgi retrograde transport . Both proteins contain the Phox homology (PX) domain, a recently identified phosphoinositide-binding motif . We demonstrate that the PX domains of Vps5p and Vps17p specifically bind to PtdIns3P in vitro and in vivo . On the basis of these and other observations, we propose that the PtdIns 3-kinase complex II directs the synthesis of a specific endosomal pool of PtdIns3P, which is required for recruitment/activation of the retromer complex, thereby ensuring efficient endosome-to-Golgi retrograde transport.

J Ind Microbiol Biotechnol, 2002 Sep, 29(3), 134 - 9
Yeast adapted to wine: nitrogen compounds released during induced autolysis in a model wine; Perrot L et al.; As important as the blend of base wines before bottling, one of the most important steps in the champagne-making process is the long ageing on lees . Two yeast strains of Saccharomyces cerevisiae MC001 and MC002, used in champagne wine production, were allowed to autolyse . After 8 days of autolysis, active dry yeasts adapted to wine released 1.7- to 1.8-fold more nitrogen compounds than nonadapted active dry yeast . The nitrogen content (total, proteins, peptides and amino) present in autolysates was measured for yeasts adapted to wine . The composition of free amino acids and amino acids constituting peptides showed no difference between the two strains of yeast used . Studies of intracellular proteolytic activity and release of peptides showed no correlation between these two phenomena . These results indicate that yeasts adapted to wine give results similar to those that occur in wine during ageing.

Mol Genet Genomics, 2002 Sep, 268(1), 70 - 80 Epub 2002 Jul 20.
High dosage of the small nucleolar RNA snR10 specifically suppresses defects of a yeast rrp5 mutant; Torchet C et al.; We have previously described a yeast strain in which cleavage at site A2 during processing of rRNA is absent and is functionally replaced by cleavage at site A3 . This strain expresses a variant of the essential RRP5 gene that results in the synthesis of two noncontiguous segments of the protein . We have used the slow-growth phenotype of this strain to screen for revertants . The gene for the small nucleolar RNA snR10 was isolated as a multicopy suppressor of this "bipartite" RRP5 allele . Suppression by snR10 efficiently rescues the slow-growth (sg) and temperature-sensitive (ts) phenotypes of the mutant strain and is specific for this small nucleolar RNA . Deletion derivatives of snR10 were constructed and tested for the ability to suppress the sg and ts phenotypes of the RRP5 mutant, as well as for complementation of the cold sensitivity of a delta snr10 strain . The results indicate that the suppression effect is more sensitive to snR10 mutations than is complementation . The high dosage of wild-type snR10 does not restore cleavage at A2, but improves the rate of pre-rRNA processing and significantly increases the level of active ribosomes in the suppressed strain . These effects probably account for the suppression of the sg and ts phenotypes of the rrp5 mutant strain.

Mol Genet Genomics, 2002 Sep, 268(1), 10 - 8 Epub 2002 Aug 13.
Identification of a role for actin in translational fidelity in yeast; Kandl KA et al.; Numerous studies have suggested a role for actin in translation, but the molecular details of this role are unknown . To elucidate the function(s) of actin in translation, we have studied 25 isogenic, conditional yeast actin mutants . Strikingly, analysis of these mutants indicates that none of those tested have conditional growth defects caused by reduced rates of protein synthesis; and analysis of latrunculin A-treated wild-type cells indicates that even complete disruption of the actin cytoskeleton has no significant effect on the rate of translation . However, analysis of the effect of the 25 actin mutations on fidelity and sensitivity to translation inhibitors identified two mutations ( act1-2 and act1-122) that cause a significant reduction in the fidelity of translation, as assayed by nonsense suppression, and several mutants that are sensitive to paromomycin, which affects translational fidelity . Translation elongation factor 1A (eEF1A) also has a role in fidelity, and in the presence of excess eEF1A four of the mutants ( act1-2, act1-20, act1-120, and act1-125) are even more sensitive to paromomycin, while one mutant ( act1-122) becomes less sensitive . Together, these findings suggest that actin may not be important for the rate of translation, but may have a critical role in ensuring translational fidelity.

Mol Cell Biol, 2002 Oct, 22(20), 7168 - 83
The mal2p protein is an essential component of the fission yeast centromere; Jin QW et al.; Precise segregation of chromosomes requires the activity of a specialized chromatin region, the centromere, that assembles the kinetochore complex to mediate the association with spindle microtubules . We show here that Mal2p, previously identified as a protein required for genome stability, is an essential component of the fission yeast centromere . Loss of functional Mal2p leads to extreme missegregation of chromosomes due to nondisjunction of sister chromatids and results in inviable cells . Mal2p associates specifically with the central region of the complex fission yeast centromere, where it is required for the specialized chromatin architecture as well as for transcriptional silencing of this region . Genetic evidence indicates that mal2(+) interacts with mis12(+), encoding another component of the inner centromere core complex . In addition, Mal2p is required for correct metaphase spindle length . Our data imply that the Mal2p protein is required to build up a functional fission yeast centromere.

Genetics, 2002 Sep, 162(1), 45 - 58
The Scw1 RNA-binding domain protein regulates septation and cell-wall structure in fission yeast; Karagiannis J et al.; Loss of the nonessential RNA-binding domain protein, Scw1, increases resistance to cell-wall-degrading enzymes in fission yeast . Surprisingly, scw1 null mutations also suppress the lethality of mutations (cdc11-136, cdc7-24, cdc14-118, sid1-239, sid2-250, sid3-106, sid4-A1, and mob1-1) at all levels of the sid pathway . This pathway forms part of the septation initiation network (SIN), which regulates the onset of septum formation and ensures the proper coupling of mitosis to cytokinesis . In contrast, scw1(-) mutations do not suppress ts alleles of the rng genes, cdc12 or cdc15 . These mutations also prevent the formation of a septum and in addition block assembly and/or function of the contractile acto-myosin ring . sid mutants exhibit a hyper-sensitivity to cell-wall-degrading enzymes that is suppressed by loss of Scw1 . Furthermore, scw1(-)-mediated rescue of sid mutants is abolished in the presence of calcofluor white, a compound that interferes with cell-wall synthesis . These data suggest that Scw1 acts in opposition to the SIN as a negative regulator of cell-wall/septum deposition . Unlike components of the SIN, Scw1 is predominantly a cytoplasmic protein and is not localized to the spindle pole body.

Gene, 2002 Jul 24, 295(1), 143 - 9
The Hypocrea jecorina gal10 (uridine 5'-diphosphate-glucose 4-epimerase-encoding) gene differs from yeast homologues in structure, genomic organization and expression; Seiboth B et al.; As part of a comprehensive study on lactose metabolism in Hypocrea jecorina (anamorph: Trichoderma reesei), a genomic clone of the gal10 gene encoding H . jecorina uridine 5'-diphosphate (UDP)-glucose 4-epimerase has been cloned and sequenced . It contains an open reading frame of 1548-base pair, interrupted by three introns, and encoding a 370-amino acids protein with similarity to pro- and eukaryotic UDP-glucose-4-epimerases . H . jecorina Gal10 does not contain the C-terminal mutarotase domain which is present in yeast Gal10 proteins but is able to functionally complement a corresponding Saccharomyces cerevisiae gal10 mutant . gal10 is not clustered with other H . jecorina gal genes (gal7, gene encoding galactose-1-phosphate uridylyltransferase and gal1, gene encoding galactokinase) . The genomic location of H . jecorina gal10 and gal7 was syntenic with that in Neurospora crassa and colinear over an area of 6 and 3.5-kilobase . gal10 is constitutively expressed, and--unlike H . jecorina gal7--not further stimulated by D-galactose or L-arabinose or its corresponding polyols.

J Virol, 2002 Oct, 76(20), 10485 - 96
Mitochondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells; Weber-Lotfi F et al.; Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection . ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon . We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria . Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side . Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains . No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.

Protein Sci, 2002 Oct, 11(10), 2471 - 8
A structure for the yeast prohibitin complex: Structure prediction and evidence from chemical crosslinking and mass spectrometry; Back JW et al.; The mitochondrial prohibitin complex consists of two subunits (PHB1 of 32 kD and PHB2 of 34 kD), assembled into a membrane-associated supercomplex of approximately 1 MD . A chaperone-like function in holding and assembling newly synthesized mitochondrial polypeptide chains has been proposed . To further elucidate the function of this complex, structural information is necessary . In this study we use chemical crosslinking, connecting lysine side chains, which are well scattered along the sequence . Crosslinked peptides from protease digested prohibitin complexes were identified with mass spectrometry . From these results, spatial restraints for possible protein conformation were obtained . Many interaction sites between PHB1 and PHB2 were found, whereas no homodimeric interactions were observed . Secondary and tertiary structural predictions were made using several algorithms and the models best fitting the spatial restraints were selected for further evaluation . From the structure predictions and the crosslink data we derived a structural building block of one PHB1 and one PHB2 subunit, strongly intertwined along most of their length . The size of the complex implies that approximately 14 of these building blocks are present . Each unit contains a putative transmembrane helix in PHB2 . Taken together with the unit building block we postulate a circular palisade-like arrangement of the building blocks projecting into the intermembrane space.

Tsitologiia, 2002, 44(6), 555 - 60
{Dark recovery of diploid yeast cells after simultaneous exposure to UV-irradiation and hyperthermia}; Tkhabisimova MD et al.; Quantitative regularities of dark recovery of wild-type diploid yeast cells of Saccharomyces cerevisiae simultaneously treated with UV-light (254 nm) and high temperatures (53-56 degrees C) were studied . Under this combined action, the constant of recovery, which defines the probability of elimination of the UV-radiation induced damage per unit of time, did not depend on the temperature of irradiation . It was shown that both the irreversible component of cell damage and the number of cells that died without division gradually increased as the temperature of exposure increased . It is concluded, on this basis, that the mechanism of synergistic interaction of UV-radiation and hyperthermia is related not to the inhibition of dark recovery itself, but to the increase in the shape of irreversibly damaged cells incapable of recovering from the induced damage.

J Biol Chem, 2002 Nov 22, 277(47), 45630 - 7 Epub 2002 Sep 13.
A physical and functional interaction between yeast Pol4 and Dnl4-Lif1 links DNA synthesis and ligation in nonhomologous end joining; Tseng HM et al.; Genetic studies have implicated the Saccharomyces cerevisiae POL4 gene product in the repair of DNA double-strand breaks by nonhomologous end joining . Here we show that Pol4 preferentially catalyzes DNA synthesis on small gaps formed by the alignment of linear duplex DNA molecules with complementary ends, a DNA substrate specificity that is compatible with its predicted role in the repair of DNA double-strand breaks . Pol4 also interacts directly with the Dnl4 subunit of the Dnl4-Lif1 complex via its N-terminal BRCT domain . This interaction stimulates the DNA synthesis activity of Pol4 and, to a lesser extent, the DNA joining activity of Dnl4-Lif1 . Notably, the joining of DNA substrates that require the combined action of Pol4 and Dnl4-Lif1 is much more efficient than the joining of similar DNA substrates that require only ligation . Thus, the physical and functional interactions between Pol4 and Dnl4-Lif1 provide a molecular mechanism for both the recruitment of Pol4 to in vivo DNA double-strand breaks and the coupling of the gap filling DNA synthesis and DNA joining reactions that complete the microhomology-mediated pathway of nonhomologous end joining.

J Cell Biol, 2002 Sep 16, 158(6), 1067 - 78 Epub 2002 Sep 16.
A yeast model system for functional analysis of beta-catenin signaling; Lee MS et al.; We have developed a novel Saccharomyces cerevisiae model system to dissect the molecular events of beta-catenin (beta-cat) signaling . Coexpression of mammalian beta-cat with TCF4 or LEF1 results in nuclear accumulation of these proteins and a functional complex that activates reporter gene transcription from constructs containing leukocyte enhancer factor (LEF)/T cell factor (TCF) response elements . Reporter transcription is constitutive, requires expression of both beta-cat and TCF4 or LEF1, and is not supported by mutated LEF/TCF binding elements or by TCF4 or LEF1 mutants . A cytoplasmic domain of E-cadherin or a functional fragment of adenomatous polyposis coli (APC) protein (APC-25) complexes with beta-cat, reduces beta-cat binding to TCF4, and leads to increased cytoplasmic localization of beta-cat and a reduction in reporter activation . Systematic mutation of putative nuclear export signal sequences in APC-25 decreases APC-25 binding to beta-cat and restores reporter gene transcription . Additional beta-cat signaling components, Axin and glycogen synthase kinase 3beta, form a multisubunit complex similar to that found in mammalian cells . Coexpression of the F-box protein beta-transducin repeat-containing protein reduces the stability of beta-cat and decreases reporter activation . Thus, we have reconstituted a functional beta-cat signal transduction pathway in yeast and show that beta-cat signaling can be regulated at multiple levels, including protein subcellular localization, protein complex formation, and protein stability.

EMBO J, 2002 Sep 16, 21(18), 4959 - 68
RPD3 is required for the inactivation of yeast ribosomal DNA genes in stationary phase; Sandmeier JJ et al.; rRNA transcription in Saccharomyces cerevisiae is performed by RNA polymerase I and regulated by changes in growth conditions . During log phase, approximately 50% of the ribosomal DNA (rDNA) genes in each cell are transcribed and maintained in an open, psoralen-accessible conformation . During stationary phase, the percentage of open rDNA genes is greatly reduced . In this study we found that the Rpd3 histone deacetylase was required to inactivate (close) individual rDNA genes as cells entered stationary phase . Even though approximately 50% of the rDNA genes remained open during stationary phase in rpd3Delta mutants, overall rRNA synthesis was still reduced . Using electron microscopy of Miller chromatin spreads, we found that the number of RNA polymerases transcribing each open gene in the rpd3Delta mutant was significantly reduced when cells grew past log phase . Bulk levels of histone H3 and H4 acetylation were reduced during stationary phase in an RPD3-dependent manner . However, histone H3 and H4 acetylation was not significantly altered at the rDNA locus in an rpd3Delta mutant . Rpd3 therefore regulates the number of open rDNA repeats.

EMBO J, 2002 Sep 16, 21(18), 4863 - 74
Fission yeast Mor2/Cps12, a protein similar to Drosophila Furry, is essential for cell morphogenesis and its mutation induces Wee1-dependent G(2) delay; Hirata D et al.; Fission yeast cells identify growing regions at the opposite ends of the cell, producing the rod-like shape . The positioning of the growth zone(s) and the polarized growth require CLIP170-like protein Tip1 and the Ndr kinase Orb6, respectively . Here, we show that the mor2/cps12 mutation disrupts the localization of F-actin at the cell ends, producing spherical cells and concomitantly inducing a G(2) delay at 36 degrees C . Mor2 is important for the localization of F-actin at the cell end(s) but not at the medial region, and is essential for the restriction of the growth zone(s) where Tip1 targets . Mor2 is homologous to the Drosophila Furry protein, which is required to maintain the integrity of cellular extensions, and is localized at both cell ends and the medial region of the cell in an actin-dependent fashion . Cellular localization of Mor2 and Orb6 was interdependent . The tyrosine kinase Wee1 is necessary for the G(2) delay and maintenance of viability of the mor2 mutant . These results indicate that Mor2 plays an essential role in cell morphogenesis in concert with Orb6, and the mutation activates the mechanism coordinating morphogenesis with cell cycle progression.

Arch Biochem Biophys, 2002 Oct 1, 406(1), 96 - 104
Yeast mitochondrial oxodicarboxylate transporters are important for growth on oleic acid; Tibbetts AS et al.; The yeast genes ODC1 and ODC2 encode members of the Saccharomyces cerevisiae family of mitochondrial transport proteins that transport oxodicarboxylates . In these studies, the ODC1 gene was identified as able, in low-copy, to rescue a yeast strain that is unable to grow on oleic acid but can grow on other nonfermentable carbon sources . ODC2 was shown to be a high-copy suppressor of this mutant . Odc1delta odc2delta double mutants are unable to grow on oleic acid at 36 degrees C . ODC1 mRNA and protein expression is elevated in oleic acid medium as compared to glucose or glycerol . The ODC1 promoter contains sequences required for the oleic acid response . However, regulation of ODC1 does not require the transcription factors Oaf1p and Pip2p, known to mediate oleic acid induction of other genes . These studies provide the first link between these mitochondrial transporters and peroxisomal beta-oxidation.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(5), 547 - 554
Studies on the Potential Phosphorylation Sites of the Yeast PHO2 Factor; Yang ZY et al.; We report here that PHO2 protein is also phosphorylated by an unidentified protein kinase . A Ser-230 to Ala mutation in the consensus sequence (SPIK) recognized by cc2/CDC28-related kinase in the PHO2 protein led to the complete loss of its ability to activate the transcription of PHO5 gene . Further work showed that Pro-231 to Ser mutation inactivated PHO2 protein as well, while Ser-230 to Asp mutation did not affect PHO2 activity . Since PHO2 Asp-230 mutant mimics Ser-230 phosphorylated PHO2, we postulate that only phosphorylated PHO2 protein could activate the transcription of the PHO5 gene . The results of in vitro phospho-labelling experiments showed that the whole cell extract of the YPH499 strain grown under low phosphate conditions phosphorylated GST (glutathione S-transferase)- PHO2 (wild type) fusion protein, but not the GST-PHO2 mutant (Pro-231 to Ser) protein in which the putative phosphorylation sequence was destroyed . We therefore propose that the PHO2 protein may also be phosphorylated in vitro at Ser-230, and the phosphorylation of this site may be necessary for its function in controlling PHO5 gene expression.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(5), 516 - 522
The Effect of Yeast Transcriptional Factor PHO2 on the Gene Expression of PHO5, HIS4 and HO; Yang J et al.; Yeast PHO2 protein plays a role in the expression of several different genes and acts as a multiple global activator . Here we report the comparison of the effect of PHO2 protein on the expression of PHO5, HIS4 and Ho genes . In the PHO2 defective yeast strain, PHO5 gene could not be depressed in low Pi and the expression of the HIS4 and HO genes was decrease to 25% and 40% of the normal level respectively . When the PHO2 gene carried by low copy shuttle vector was transformed into this strain, the expression of the three genes could be restored . Previously the PHO2 gene was mutated on its conspicuous regions with site-directed mutagenesis, deletion and linker insertion . Here the effects of these mutations on these genes were compared.

Biogerontology, 2002, 3(4), 243 - 56
Application of the yeast two-hybrid system in molecular gerontology; Knudsen CR et al.; Most -- if not all -- proteins are bound to interact with other proteins to exert their function, and thus the identification of the interaction partners of a protein is vital in proteomics . The yeast two-hybrid system is a popular and effective tool for studying protein-protein interactions . Although the advantages of the system are manifold, it also has certain drawbacks and limitations . The two-hybrid system has been shown to be extremely useful for placing a protein of unknown function within a functional context, thereby providing information about a putative role of the uncharacterised protein . This concept has also been successfully applied in molecular gerontology.

Plant Physiol, 1993 May, 102(1), 61 - 69
Wheat (Triticum aestivum L.) {gamma}-Gliadin Accumulates in Dense Protein Bodies within the Endoplasmic Reticulum of Yeast; Rosenberg N et al.; Following their sequestration into the endoplasmic reticulum (ER), wheat storage proteins may either be retained and packaged into protein bodies within this organelle or transported via the Golgi to vacuoles . We attempted to study the processes of transport and packaging of wheat storage proteins using the heterologous expression system of yeast . A wild-type wheat {gamma}-gliadin, expressed in the yeast cells, accumulated mostly within the ER and was deposited in protein bodies with similar density to natural protein bodies from wheat endosperm . This suggested that wheat storage proteins contain sufficient information to initiate the formation of protein bodies in the ER of a heterologous system . Only a small amount of the {gamma}-gliadin was transported to the yeast vacuoles . When a deletion mutant of the {gamma}-gliadin, lacking the entire N-terminal repetitive region, was expressed in the yeast cells, the mutant was unable to initiate the formation of protein bodies within the ER and was completely transported to the yeast vacuole . This strongly indicated that the information for packaging into dense protein bodies within the ER resides in the N-terminal repetitive region of the {gamma}-gliadin . The advantage of using yeast to identify the signals and mechanisms controlling the transport of wheat storage proteins and their deposition in protein bodies is discussed.

Traffic, 2002 Oct, 3(10), 730 - 9
An ER membrane protein, Sop4, facilitates ER export of the yeast plasma membrane {H+}ATPase, Pma1; Luo WJ et al.; We have analyzed the mechanism by which Sop4, a novel ER membrane protein, regulates quality control and intracellular transport of Pma1-7, a mutant plasma membrane ATPase . At the restrictive temperature, newly synthesized Pma1-7 is targeted for vacuolar degradation instead of being correctly delivered to the cell surface . Loss of Sop4 at least partially corrects vacuolar mislocalization, allowing Pma1-7 routing to the plasma membrane . Ste2-3 is a mutant pheromone receptor which, like Pma1-7, is defective in targeting to the cell surface, resulting in a mating defect . sop4delta suppresses the mating defect of ste2-3 cells as well as the growth defect of pma1-7 . Visualization of newly synthesized Pma1-7 in sop4delta cells by indirect immunofluorescence reveals delayed export from the ER . Similarly, ER export of wild-type Pma1 is delayed in the absence of Sop4 although intracellular transport of Gas1 and CPY is unaffected . These observations suggest a model in which a selective increase in ER residence time for Pma1-7 may allow it to achieve a more favorable conformation for subsequent delivery to the plasma membrane . In support of this model, newly synthesized Pma1-7 is also routed to the plasma membrane upon release from a general block of ER-to-Golgi transport in sec13-1 cells.

J Biol Chem, 2002 Nov 15, 277(46), 43792 - 8 Epub 2002 Sep 11.
A higher plant mitochondrial homologue of the yeast m-AAA protease . Molecular cloning, localization, and putative function; Kolodziejczak M et al.; Mitochondrial AAA metalloproteases play a fundamental role in mitochondrial biogenesis and function . They have been identified in yeast and animals but not yet in plants . This work describes the isolation and sequence analysis of the full-length cDNA from the pea (Pisum sativum) with significant homology to the yeast matrix AAA (m-AAA) protease . The product of this clone was imported into isolated pea mitochondria where it was processed to its mature form (PsFtsH) . We have shown that the central region of PsFtsH containing the chaperone domain is exposed to the matrix space . Furthermore, we have demonstrated that the pea protease can complement respiration deficiency in the yta10 and/or yta12 null yeast mutants, indicating that the plant protein can compensate for the loss of at least some of the important m-AAA functions in yeast . Based on biochemical experiments using isolated pea mitochondria, we propose that PsFtsH-like m-AAA is involved in the accumulation of the subunit 9 of the ATP synthase in the mitochondrial membrane.

Plant Physiol, 2002 Sep, 130(1), 210 - 20
Arabidopsis CYP98A3 mediating aromatic 3-hydroxylation . Developmental regulation of the gene, and expression in yeast; Nair RB et al.; The general phenylpropanoid pathways generate a wide array of aromatic secondary metabolites that range from monolignols, which are ubiquitous in all plants, to sinapine, which is confined to crucifer seeds . The biosynthesis of these compounds involves hydroxylated and methoxylated cinnamyl acid, aldehyde, or alcohol intermediates . Of the three enzymes originally proposed to hydroxylate the 4-, 3-, and 5-positions of the aromatic ring, cinnamate 4-hydroxylase (C4H), which converts trans-cinnamic acid to p-coumaric acid, is the best characterized and is also the archetypal plant P450 monooxygenase . Ferulic acid 5-hydroxylase (F5H), a P450 that catalyzes 5-hydroxylation, has also been studied, but the presumptive 3-hydroxylase converting p-coumarate to caffeate has been elusive . We have found that Arabidopsis CYP98A3, also a P450, could hydroxylate p-coumaric acid to caffeic acid in vivo when expressed in yeast (Saccharomyces cerevisiae) cells, albeit very slowly . CYP98A3 transcript was found in Arabidopsis stem and silique, resembling both C4H and F5H in this respect . CYP98A3 showed further resemblance to C4H in being highly active in root, but differed from F5H in this regard . In transgenic Arabidopsis, the promoters of CYP98A3 and C4H showed wound inducibility and a comparable developmental regulation throughout the life cycle, except in seeds, where the CYP98A3 promoter construct was inactive while remaining active in silique walls . Within stem and root tissue, the gene product and the promoter activity of CYP98A3 were most abundant in lignifying cells . Collectively, these studies show involvement of CYP98A3 in the general phenylpropanoid metabolism, and suggest a downstream function for CYP98A3 relative to the broader and upstream role of C4H.

Biochim Biophys Acta, 2002 Sep 20, 1565(1), 36 - 40
The yeast SR protein kinase Sky1p modulates salt tolerance, membrane potential and the Trk1,2 potassium transporter; Forment J et al.; Protein kinases dedicated to the phosphorylation of SR proteins have been implicated in the processing and nuclear export of mRNAs . Here we demonstrate in Saccharomyces cerevisiae their participation in cation homeostasis . A null mutant of the single yeast SR protein kinase Sky1p is viable but exhibits increased tolerance to diverse toxic cations such as Na(+), Li(+), spermine, tetramethylammonium, hygromycin B and Mn(2+) . This pleiotropic phenotype correlates with reduced accumulation of cations, suggesting a decrease in membrane electrical potential . Genetic analysis and Rb(+) uptake measurements indicate that Sky1p modulates Trk1,2, the high-affinity K(+) uptake system of yeast and a major determinant of membrane potential.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2002 Jun, 19(2), 284 - 6, 290
{Cloning of AE1-c-end cDNA and construction of its expression plasmid for yeast two-hybrid system}; Li H et al.; In this study, about 350 bp cDNA fragment was amplified by PCR . After being sequenced, the AE1-c-end gene fragment was cloned into EcoR I-Pst I site of pGADT7 to form AD ends in the yeast two-hybrid system . The recombinant plasmid was transformed into yeast AH109, and the expression in the yeast was observed . The results demonstrate that AE1-c-end was obtained . pGADT7-AE1-c-end has no toxic effect on the yeast . It can serve as a target gene of yeast two-hybrid system.

J Muscle Res Cell Motil, 2001, 22(8), 665 - 74
Functional studies of yeast actin mutants corresponding to human cardiomyopathy mutations; Wong WW et al.; The molecular mechanisms by which different mutations in actin lead to distinct cardiomyopathies are unknown . Here, actin mutants corresponding to alpha-cardiac actin mutations causing hypertrophic cardiomyopathy {(HCM) P164A and A331P} and dilated cardiomyopathy {(DCM) R312H and E361G} were expressed in yeast and purified for in vitro functional studies . While P164A appeared unaltered compared to wild-type (WT) actin, A331P function was impaired . A331P showed reduced stability in circular dichroism melting experiments; its monomer unfolding transition was 10 degrees C lower compared to WT actin . Additionally, in vitro filament formation was hampered, and yeast cell cultures were temperature sensitive, implying perturbations in actin-actin interactions . Filament instability of the A331P mutant actin could lead to actomyosin dysfunction observed in HCM . Yeast strains harboring the R312H mutation did not grow well in culture, suggesting that cell viability is compromised . The E361G substitution is located at an alpha-actinin binding region where the actin filament is anchored . The mutant actin, though unaltered in the in vitro motility and standard actomyosin functions, had a threefold reduction in alpha-actinin binding . This could result in impairment of force-transduction in muscle fibers, and a DCM phenotype.

Biol Chem, 2002 Jun, 383(6), 969 - 75
DNA binding properties of the yeast Msh2-Msh6 and Mlh1-Pms1 heterodimers; Drotschmann K et al.; We describe here our recent studies of the DNA binding properties of Msh2-Msh6 and Mlh1-Pms1, two protein complexes required to repair mismatches generated during DNA replication . Mismatched DNA binding by Msh2-Msh6 was probed by mutagenesis based on the crystal structure of the homologous bacterial MutS homodimer bound to DNA . The results suggest that several amino acid side chains inferred to interact with the DNA backbone near the mismatch are critical for repair activity . These contacts, which are different in Msh2 and Msh6, likely facilitate stacking and hydrogen bonding interactions between side chains in Msh6 and the mismatched base, thus stabilizing a kinked DNA conformation that permits subsequent repair steps coordinated by the Mlh1-Pms1 heterodimer . Mlh1-Pms1 also binds to DNA, but independently of a mismatch . Mlh1-Pms1 binds short DNA substrates with low affinity and with a slight preference for single-stranded DNA . It also binds longer duplex DNA molecules, but with a higher affinity indicative of cooperative binding . Indeed, imaging by atomic force microscopy reveals cooperative DNA binding and simultaneous interaction with two DNA duplexes . The novel DNA binding properties of Mlh1-Pms1 may be relevant to signal transduction during DNA mismatch repair and to recombination, meiosis and cellular responses to DNA damage.

Mol Biol Cell, 2002 Sep, 13(9), 3078 - 95
ADP-ribosylation factor (ARF) interaction is not sufficient for yeast GGA protein function or localization; Boman AL et al.; Golgi-localized gamma-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic . Human and yeast GGA proteins are only ~25% identical, but all GGA proteins have four similar domains based on function and sequence homology . GGA proteins are most conserved in the region that interacts with ARF proteins . To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs . To our surprise, yeast and human GGAs differ in their requirement for ARF interaction . We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs . In mammalian cells, this mutation disrupts the localization of human GGA proteins . Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs and VPS27 . Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking . Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi . Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function . Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo . Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

Mol Biol Cell, 2002 Sep, 13(9), 2990 - 3004
The roles of bud-site-selection proteins during haploid invasive growth in yeast; Cullen PJ et al.; In haploid strains of Saccharomyces cerevisiae, glucose depletion causes invasive growth, a foraging response that requires a change in budding pattern from axial to unipolar-distal . To begin to address how glucose influences budding pattern in the haploid cell, we examined the roles of bud-site-selection proteins in invasive growth . We found that proteins required for bipolar budding in diploid cells were required for haploid invasive growth . In particular, the Bud8p protein, which marks and directs bud emergence to the distal pole of diploid cells, was localized to the distal pole of haploid cells . In response to glucose limitation, Bud8p was required for the localization of the incipient bud site marker Bud2p to the distal pole . Three of the four known proteins required for axial budding, Bud3p, Bud4p, and Axl2p, were expressed and localized appropriately in glucose-limiting conditions . However, a fourth axial budding determinant, Axl1p, was absent in filamentous cells, and its abundance was controlled by glucose availability and the protein kinase Snf1p . In the bud8 mutant in glucose-limiting conditions, apical growth and bud site selection were uncoupled processes . Finally, we report that diploid cells starved for glucose also initiate the filamentous growth response.

J Biol Chem, 2002 Nov 15, 277(46), 44497 - 506 Epub 2002 Sep 06.
Trypanosoma cruzi H+-ATPase 1 (TcHA1) and 2 (TcHA2) genes complement yeast mutants defective in H+ pumps and encode plasma membrane P-type H+-ATPases with different enzymatic properties; Luo S et al.; Previous studies in Trypanosoma cruzi have shown that intracellular pH homeostasis requires ATP and is affected by H(+)-ATPase inhibitors, indicating a major role for ATP-driven proton pumps in intracellular pH control . In the present study, we report the cloning and sequencing of a pair of genes linked in tandem (TcHA1 and TcHA2) in T . cruzi which encode proteins with homology to fungal and plant P-type proton-pumping ATPases . The genes are expressed at the mRNA level in different developmental stages of T . cruzi: TcHA1 is expressed maximally in epimastigotes, whereas TcHA2 is expressed predominantly in trypomastigotes . The proteins predicted from the nucleotide sequence of the genes have 875 and 917 amino acids and molecular masses of 96.3 and 101.2 kDa, respectively . Full-length TcHA1 and an N-terminal truncated version of TcHA2 complemented a Saccharomyces cerevisiae strain deficient in P-type H(+)-ATPase activity, the proteins localized to the yeast plasma membrane, and ATP-driven proton pumping could be detected in proteoliposomes reconstituted from plasma membrane purified from transfected yeast . The reconstituted proton transport activity was reduced by inhibitors of P-type H(+)-ATPases . C-terminal truncation did not affect complementation of mutant yeast, suggesting the lack of C-terminal autoinhibitory domains in these proteins . ATPase activity in plasma membrane from TcHA1- and (N-terminal truncated) TcHA2-transfected yeast was inhibited to different extents by vanadate, whereas the latter yeast strain was more resistant to extremes of pH, suggesting that the native proteins may serve different functions at different stages in the T . cruzi life cycle.

Biochemistry, 2002 Sep 17, 41(37), 11301 - 7
Localization of subunits D, E, and G in the yeast V-ATPase complex using cysteine-mediated cross-linking to subunit B; Arata Y et al.; Using a combination of cysteine mutagenesis and covalent cross-linking, we have identified subunits in close proximity to specific sites within subunit B of the vacuolar (H(+))-ATPase (V-ATPase) of yeast . Unique cysteine residues were introduced into subunit B by site-directed mutagenesis, and the resultant V-ATPase complexes were reacted with the bifunctional, photoactivatable maleimide reagent 4-(N-maleimido)benzophenone (MBP) followed by irradiation . Cross-linked products were identified by Western blot using subunit-specific antibodies . Introduction of cysteine residues at positions Glu(106) and Asp(199) led to cross-linking of subunits B and E, at positions Asp(341) and Ala(424) to cross-linking of subunits B and D, and at positions Ala(15) and Lys(45) to cross-linking of subunits B and G . Using a molecular model of subunit B constructed on the basis of sequence homology between the V- and F-ATPases, the X-ray coordinates of the F(1)-ATPase, and energy minimization, Glu(106), Asp(199), Ala(15), and Lys(45) are all predicted to be located on the outer surface of the complex, with Ala(15) and Lys(45) located near the top of the complex furthest from the membrane . By contrast, Asp(341) and Ala(424) are predicted to face the interior of the A(3)B(3) hexamer . These results suggest that subunits E and G form part of a peripheral stalk connecting the V(1) and V(0) domains whereas subunit D forms part of a central stalk . Subunit D is thus the most likely homologue to the gamma subunit of F(1), which undergoes rotation during ATP hydrolysis and serves an essential function in rotary catalysis.

Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 83 - 91
Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I; Kerscher S et al.; The obligate aerobic yeast Yarrowia lipolytica is introduced as a powerful new model for the structural and functional analysis of mitochondrial complex I . A brief introduction into the biology and the genetics of this nonconventional yeast is given and the relevant genetic tools that have been developed in recent years are summarized . The respiratory chain of Y . lipolytica contains complexes I-IV, one "alternative" NADH-dehydrogenase (NDH2) and a non-heme alternative oxidase (AOX) . Because the NADH binding site of NDH2 faces the mitochondrial intermembrane space rather than the matrix, complex I is an essential enzyme in Y . lipolytica . Nevertheless, complex I deletion strains could be generated by attaching the targeting sequence of a matrix protein, thereby redirecting NDH2 to the matrix side . Deletion strains for several complex I subunits have been constructed that can be complemented by shuttle plasmids carrying the deleted gene . Attachment of a hexa-histidine tag to the NUGM (30 kDa) subunit allows fast and efficient purification of complex I from Y . lipolytica by affinity-chromatography . The purified complex has lost most of its NADH:ubiquinone oxidoreductase activity, but is almost fully reactivated by adding 400-500 molecules of phosphatidylcholine per complex I . The established set of genetic tools has proven useful for the site-directed mutagenesis of individual subunits of Y . lipolytica complex I . Characterization of a number of mutations already allowed for the identification of several functionally important amino acids, demonstrating the usefulness of this approach.

Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 71 - 4
Structure, subunit function and regulation of the coated vesicle and yeast vacuolar (H(+))-ATPases; Arata Y et al.; The vacuolar (H(+))-ATPases (or V-ATPases) are ATP-dependent proton pumps that function to acidify intracellular compartments in eukaryotic cells . This acidification is essential for such processes as receptor-mediated endocytosis, intracellular targeting of lysosomal enzymes, protein processing and degradation and the coupled transport of small molecules . V-ATPases in the plasma membrane of specialized cells also function in such processes as renal acidification, bone resorption and pH homeostasis . Work from our laboratory has focused on the V-ATPases from clathrin-coated vesicles and yeast vacuoles.Structurally, the V-ATPases are composed of two domains: a peripheral complex (V(1)) composed of eight different subunits (A-H) that is responsible for ATP hydrolysis and an integral complex (V(0)) composed of five different subunits (a, d, c, c' and c") that is responsible for proton translocation . Electron microscopy has revealed the presence of multiple stalks connecting the V(1) and V(0) domains, and crosslinking has been used to address the arrangement of subunits in the complex . Site-directed mutagenesis has been employed to identify residues involved in ATP hydrolysis and proton translocation and to study the topology of the 100 kDa a subunit . This subunit has been shown to control intracellular targeting of the V-ATPase and to influence reversible dissociation and coupling of proton transport and ATP hydrolysis.

Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 21 - 8
Electron transfer between yeast cytochrome bc(1) complex and cytochrome c: a structural analysis; Hunte C et al.; The structure of the complex between cytochrome c (CYC) and the cytochrome bc(1) complex (QCR) from yeast crystallized with an antibody fragment has been recently determined at 2.97 A resolution {Proc . Natl . Acad . Sci . U . S . A . 99 (2002) 2800} . CYC binds to subunit cytochrome c(1) of the enzyme stabilized by hydrophobic interactions surrounding the heme crevices creating a small, compact contact site . A central cation-pi interaction is an important and conserved feature of CYC binding . Peripheral patches with highly conserved complementary charges further stabilize the enzyme-substrate complex by long-range electrostatic forces and may affect the orientation of the substrate . Size and characteristics of the contact site are optimal for a transient electron transfer complex . Kinetic data show a bell-shaped ionic strength dependence of the cytochrome c reduction with a maximum activity near physiological ionic strength . The dependence is less pronounced in yeast compared to horse heart CYC indicating less impact of electrostatic interactions in the yeast system . Interestingly, a local QCR activity minimum is found for both substrates at 120-140 mM ionic strength . The architecture of the complex results in close distance of both c-type heme groups allowing the rapid reduction of cytochrome c by QCR via direct heme-to-heme electron transfer . Remarkably, CYC binds only to one of the two possible binding sites of the homodimeric complex and binding appears to be coordinated with the presence of ubiquinone at the Q(i) site . Regulatory aspects of CYC reduction are discussed.

Biol Cell, 2002 Jun, 94(3), 147 - 56
Heterozygosity in MAT locus affects stability and function of microtubules in yeast; Steinberg-Neifach O et al.; The dynamic nature of microtubules is important for their cellular function and is tightly regulated by the cell cycle machinery and through other pathways that act on microtubule-associated proteins . Recently, it was reported that simultaneous expression of MATa and MATa genes in haploid cells of Saccharomyces cerevisiae increases microtubule stability {Mol . Gen . Genet . 264 (2000) 300} . In order to investigate the effect of zygosity in the MAT loci on microtubules independent of the effect of the actual ploidy, we compared microtubule stability and function in homozygous (MATa/MATa) and heterozygous (MATa/MATa) wild-type diploid cells . It was found that homozygosity in the MAT locus decreases stability of both cytoplasmic and nuclear microtubules . This was expressed in a more symmetrical distribution in the placement of the spindle relative to the neck, a delay in cytokinesis and reduced fidelity of chromosome segregation in these cells compared to the heterozygotes . Our results suggest that expression of both MAT loci initiates a pathway that results in an increase in microtubule stability and the fidelity of chromosome segregation in diploids . This pathway is independent of the pathway that determines the budding pattern in haploids and diploids, which is also initiated by simultaneous expression of MATa and MATa genes.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1997, 29(2), 122 - 128
Analysis of Activation Activity of Yeast PHO2, PHO4 Protein and Their Interaction; Yang J et al.; Both PHO2 and PHO4 are positive regulatory factors of yeast PHO5 gene . Here we show that the PHO2 fused to yeast transcriptional factor GAL4 DNA-binding domain activates the expression of the reporter gene (lacZ), and the lacZ activities were regulated by Pi concentration, therefore it could be suggested that there are acidic activation domains on the PHO2 protein . Acidic amino acid rich region of 287-326 aa in PHO2 is not a transcriptional activation domain . PHO2 maintained its activation activity only if Ser230 is phosphorylated, thus the phosphorylated site may play a key role in the transcriptional activation function of PHO2 . PHO4 fused to the GAL4 DNA-binding domain also activates the expression of lacZ . A segment of 1-97 aa at its N-terminal is responsible for the transcriptional activation activity . A two-hybrid assay reveals that there exists interaction between PHO2 and PHO4 protein, and the interaction affects their transcriptional activation function.

Methods, 2002 Aug, 27(4), 301 - 10
Heterodimerization of gamma-aminobutyric acid B receptor subunits as revealed by the yeast two-hybrid system; White JH et al.; Several lines of evidence suggested that the first gamma-aminobutyric acid B receptor to be cloned required an additional factor for functional expression . GABA(B1) was retained within the endoplasmic reticulum and failed to couple to signal transduction pathways on stimulation with agonists . In radioligand binding experiments it was found that although the affinity of antagonists showed a close agreement between rat brain membranes and membranes expressing the cloned receptor, agonist ligands were significantly weaker at recombinant receptors . Using the C-terminal tail as bait, a yeast two-hybrid screen was run against a human brain cDNA library and identified a second receptor, GABA(B2), as a major interacting protein . This interaction was confirmed by coimmunoprecipitation as well as extensive colocalization studies . Coexpression of the two seven-transmembrane proteins generated a fully functional receptor, which was expressed at the cell surface confirming the importance of receptor heterodimerization for GABA(B) receptor activity.

J Chromatogr A, 2002 Aug 9, 966(1-2), 135 - 43
Improved performance of gravitational field-flow fractionation for screening wine-making yeast varieties; Sanz R et al.; Performance of gravitational field-flow fractionation (GFFF) is improved here with respect to the ability to fractionate and distinguish different varieties of wine-making yeast from Saccharomyces cerevisiae . A new GFFF channel with non-polar walls has been employed to enhance fractionation selectivity and reproducibility . Since GFFF retention depends from first principles on particle size, Coulter counter measurements were performed in order to compare size distribution profiles with GFFF profiles . From such a comparison, GFFF was shown to be able to reveal differences in yeast cells other than size . This could make use of GFFF for screening different varieties of wine-making yeast towards future quality assessment procedures based on a possible correlation between yeast cell morphology indexes and quality indexes.

Annu Rev Microbiol, 2002, 56, 769 - 92 Epub 2002 Jan 30.
Growing old: metabolic control and yeast aging; Jazwinski SM; The metabolic characteristics of a yeast cell determine its life span . Depending on conditions, stress resistance can have either a salutary or a deleterious effect on longevity . Gene dysregulation increases with age, and countering it increases life span . These three determinants of yeast longevity may be interrelated, and they are joined by a potential fourth, genetic stability . These factors can also operate in phylogenetically diverse species . Adult longevity seems to borrow features from the genetic programs of dormancy to provide the metabolic and stress resistance resources necessary for extended survival . Both compensatory and preventive mechanisms determine life span, while epigenetic factors and the element of chance contribute to the role that genes and environment play in aging.

Microbiology, 2002 Sep, 148(Pt 9), 2697 - 704
Physiological role of the glutathione-dependent formaldehyde dehydrogenase in the methylotrophic yeast Candida boidinii; Lee B et al.; The methylotrophic yeast Candida boidinii exhibits formaldehyde dehydrogenase activity (FLD, EC 1.2.1.1) during growth on methanol as a sole carbon source . The structural gene, FLD1, was cloned from a genomic library of C . boidinii . The 1263 bp FLD1 gene contained a 123 bp intron and its exon encoded a gene product of 380 amino acids, whose predicted amino acid sequence showed high similarity to the sequences of FLDs from other organisms . The FLD1 gene was disrupted in the C . boidinii genome by one-step gene disruption . The fld1Delta strain could not grow on methanol as a carbon source under methanol-limited chemostat culture conditions, even with low dilution rates (D<0.05 x h(-1)), whereas a strain with a disruption in the gene for formate dehydrogenase (FDH; another NADH-generating dehydrogenase involved in the formaldehyde oxidation pathway) could survive . These results indicated that FLD, but not FDH, is essential for growth of C . boidinii on methanol.

Exp Gerontol, 2002 Aug-Sep, 37(8-9), 1023 - 30
Distinct roles of processes modulated by histone deacetylases Rpd3p, Hda1p, and Sir2p in life extension by caloric restriction in yeast; Jiang JC et al.; Caloric restriction has been demonstrated to extend life span and postpone aging in a variety of species . The recent extension of the caloric restriction paradigm to yeast places the emphasis of the search for the longevity effectors at the cellular level . To narrow the range of potential effectors of the caloric restriction response, we have examined the effects of the histone deacetylases Rpd3p, Hda1p, and Sir2p, which have distinguishable but partially overlapping influences on global patterns of gene expression, on the life extension afforded by caloric restriction . Deletion of the RPD3 gene extended life span, and there was no additive effect of caloric restriction . Deletion of HDA1 had no effect of its own on longevity but acted synergistically with caloric restriction to increase life span . SIR2 deletion shortened life span but did not prevent extension of life span by caloric restriction . The results suggest that Rpd3p affects both processes that play an obligate and those that play a synergistic role in life extension by caloric restriction, while Hda1p and Sir2p affect processes that are not the obligate longevity effectors of caloric restriction but instead synergize with them, although in opposite directions . From the known patterns of gene expression elicited by rpd3delta, hda1delta, and sir2delta, we propose that the major longevity effectors of caloric restriction in yeast involve carbohydrate/energy metabolism and mitochondrial function.

Exp Cell Res, 2002 Sep 10, 279(1), 71 - 9
The spindle pole body duplicates in early G1 phase in the pathogenic yeast Exophiala dermatitidis: an ultrastructural study; Yamaguchi M et al.; The spindle pole body of the pathogenic yeast Exophiala dermatitidis was observed during the cell cycle using freeze-substitution and serial ultrathin sectioning electron microscopy . The spindle pole body was located on the outer membrane of the nuclear envelope and consisted of two disk elements connected by an intervening midpiece in G1 through G2 phases . Each disk element was composed of filamentous materials and measured 150 nm in diameter and 100 nm in thickness . The midpiece had higher electron density and measured 60 nm in length and 40 nm in thickness . At the beginning of prophase, each disk element of the spindle pole body enlarged to more than double in size . They were separated on the nuclear envelope, and associated with numerous cytoplasmic microtubules . At mitosis, the spindle pole body entered the nuclear envelope, associated with numerous nuclear microtubules, and was located at the spindle poles . At the end of telophase, it was extruded back into the cytoplasm from the nuclear envelope . Three-dimensional analysis of cells in different cell cycles suggested that duplication of the spindle pole body took place in early G1 phase . Thus, the location, structure, and duplication cycle of the E . dermatitidis spindle pole body were different from those of Saccharomyces cerevisiae.

RNA, 2002 Aug, 8(8), 997 - 1010
The conserved central domain of yeast U6 snRNA: importance of U2-U6 helix Ia in spliceosome assembly; Ryan DE et al.; In the pre-mRNA processing machinery of eukaryotic cells, U6 snRNA is located at or near the active site for pre-mRNA splicing catalysis, and U6 is involved in catalyzing the first chemical step of splicing . We have further defined the roles of key features of yeast U6 snRNA in the splicing process . By assaying spliceosome assembly and splicing in yeast extracts, we found that mutations of yeast U6 nt 56 and 57 are similar to previously reported deletions of U2 nt 27 or 28, all within yeast U2-U6 helix Ia . These mutations lead to the accumulation of yeast A1 spliceosomes, which form just prior to the Prp2 ATPase step and the first chemical step of splicing . These results strongly suggest that, at a late stage of spliceosome assembly, the presence of U2-U6 helix Ia is important for promoting the first chemical step of splicing, presumably by bringing together the 5' splice site region of pre-mRNA, which is base paired to U6 snRNA, and the branchsite region of the intron, which is base paired to U2 snRNA, for activation of the first chemical step of splicing, as previously proposed by Madhani and Guthrie {Cell, 1992, 71: 803-817} . In the 3' intramolecular stem-loop of U6, mutation G81C causes an allele-specific accumulation of U6 snRNP . Base pairing of the U6 3' stem-loop in yeast spliceosomes does not extend as far as to include the U6 sequence of U2-U6 helix Ib, in contrast to the human U6 3' stem-loop structure.

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