Biochem Biophys Res Commun, 2004 Jul 23, 320(2), 416 - 23 Increased glycogen storage in yeast results in less branched glycogen; Wilson WA et al.; Glycogen is a branched polymer of glucose, synthesized as a reserve of both energy and carbon . The branched nature of glycogen is important for its function and polyglucosan bodies, particles that contain a glycogen-like polymer with reduced branching, are a feature of several disease states . The degree of glycogen branching is thought to be governed by the balance between glycogen synthesis and branching activities . However, there have been reports that the intrinsic properties of individual branching enzymes govern the degree of branching . To address the relationship between synthesis and branching more fully, we made use of the yeast Saccharomyces cerevisiae . The glycogen content of yeast cells was manipulated by using different growth conditions or by the introduction of specific mutations . Whenever glycogen storage was elevated, the polysaccharide formed was found to be less branched but normal branching could be restored by overexpression of branching enzyme. Biochem Biophys Res Commun, 2004 Jul 23, 320(2), 359 - 65 Functional differences between human and yeast protein disulfide isomerase family proteins; Kimura T et al.; Previously, it has been reported that a mammalian protein disulfide isomerase (PDI), when expressed on a single copy number plasmid, can rescue growth of a PDI1-disrupted yeast . However, here, for the first time we demonstrated by tetrad analysis that human PDI (hPDI) is unable to replace yeast PDI (yPDI) when hPDI cDNA is integrated into the yeast chromosome . This observation indicates that hPDI is not functionally equivalent to yPDI . Estimation of the actual copy number of the plasmid, as well as comparison of isomerase and chaperone activities between human and yeast PDI homologues, indicates that one copy of hPDI cDNA is not sufficient to rescue the PDI1-disrupted strain . Notably, the isomerase activities of yPDI family proteins, Mpd1p, Mpd2p, and Eug1p, were extremely low, although yPDI itself exhibited twice as much isomerase activity as hPDI in vitro . Moreover, with the exception of Mpd1p, all hPDI and yPDI family proteins had chaperone activity, this being particularly strong in the case of yPDI and Mpd2p . These observations indicate that the growth of Saccharomyces cerevisiae is completely dependent on the isomerase activity of yPDI. Biochem J, 2004 Aug 1, 381(Pt 3), 581 - 5 The yeast peroxisomal adenine nucleotide transporter: characterization of two transport modes and involvement in DeltapH formation across peroxisomal membranes; Lasorsa FM et al.; The yeast peroxisomal adenine nucleotide carrier, Ant1p, was shown to catalyse unidirectional transport in addition to exchange of substrates . In both transport modes, proton movement occurs . Nucleotide hetero-exchange is H+-compensated and electroneutral . Furthermore, microscopic fluorescence imaging of a pH-sensitive green fluorescent protein targeted to peroxisomes shows that Ant1p is involved in the formation of a DeltapH across the peroxisomal membrane, acidic inside. Acta Biochim Pol, 2004, 51(2), 323 - 47 Dynamics of neutral lipid storage in yeast; Mullner H et al.; Since energy storage is a basic metabolic process, the synthesis of neutral lipids occurs in all kingdoms of life . The yeast, Saccharomyces cerevisiae, widely accepted as a model eukaryotic cell, contains two classes of neutral lipids, namely steryl esters and triacylglycerols . Triacylglycerols are synthesized through two pathways governed by the acyl-CoA diacylglycerol acyltransferase Dga1p and the phospholipid diacylglycerol acyltransferase Lro1p, respectively . Steryl esters are formed by the two steryl ester synthases Are1p and Are2p, two enzymes with overlapping function which also catalyze triacylglycerol formation, although to a minor extent . Storage of neutral lipids is tightly linked to the biogenesis of so called lipid particles . The role of this compartment in lipid homeostasis and its interplay with other organelles involved in neutral lipid dynamics, especially the endoplasmic reticulum and the plasma membrane, are subject of current investigations . In contrast to neutral lipid formation, mobilization of triacylglycerols and steryl esters in yeast are less characterized at the molecular level . Only recently, the triacylglycerol lipase Tgl3p was identified as the first yeast enzyme of this kind by function . Genes and gene products governing steryl ester mobilization still await identification . Besides biochemical properties of enzymes involved in yeast neutral lipid synthesis and degradation, regulatory aspects of these pathways and cell biological consequences of neutral lipid depletion will be discussed in this minireview. J Membr Biol, 2004 Apr 1, 198(3), 177 - 92 Chloride channel function in the yeast TRK-potassium transporters; Kuroda T et al.; The TRK proteins-Trk1p and Trk2p- are the main agents responsible for "active" accumulation of potassium by the yeast Saccharomyces cerevisiae . In previous studies, inward currents measured through those proteins by whole-cell patch-clamping proved very unresponsive to changes of extracellular potassium concentration, although they did increase with extracellular proton concentration-qualitatively as expected for H(+) coupling to K(+) uptake . These puzzling observations have now been explored in greater detail, with the following major findings: a) the large inward TRK currents are not carried by influx of either K(+) or H(+), but rather by an efflux of chloride ions; b) with normal expression levels for Trk1p and Trk2p in potassium-replete cells, the inward TRK currents are contributed approximately half by Trk1p and half by Trk2p; but c) strain background strongly influences the absolute magnitude of these currents, which are nearly twice as large in W303-derived spheroplasts as in S288c-derived cells (same cell-size and identical recording conditions); d) incorporation of mutations that increase cell size (deletion of the Golgi calcium pump, Pmr1p) or that upregulate the TRK2 promoter, can further substantially increase the TRK currents; e) removal of intracellular chloride (e.g., replacement by sulfate or gluconate) reveals small inward currents that are K(+)-dependent and can be enhanced by K(+) starvation; and f) finally, the latter currents display two saturating kinetic components, with preliminary estimates of K(0.5) at 46 micro M {K(+)}(out) and 6.8 m M {K(+)}(out), and saturating fluxes of approximately 5 m M/min and approximately 10 m M/min (referred to intracellular water) . These numbers are compatible with the normal K(+)-transport properties of Trk1p and Trk2p, respectively. Mol Biol Cell, 2004 Sep, 15(9), 4203 - 14 Epub 2004 Jun 23. Antagonistic roles of ESCRT and Vps class C/HOPS complexes in the recycling of yeast membrane proteins; Bugnicourt A et al.; In Saccharomyces cerevisiae, deficiencies in the ESCRT machinery trigger the mistargeting of endocytic and biosynthetic ubiquitinated cargoes to the limiting membrane of the vacuole . Surprisingly, impairment of this machinery also leads to the accumulation of various receptors and transporters at the plasma membrane in both yeast and higher eukaryotes . Using the well-characterized yeast endocytic cargo uracil permease (Fur4p), we show here that the apparent stabilization of the permease at the plasma membrane in ESCRT mutants results from an efficient recycling of the protein . Whereas several proteins as well as internalized dyes are known to be recycled in yeast, little is known about the machinery and molecular mechanisms involved . The SNARE protein Snc1p is the only cargo for which the recycling pathway is well characterized . Unlike Snc1p, endocytosed Fur4p did not pass through the Golgi apparatus en route to the plasma membrane . Although ubiquitination of Fur4p is required for its internalization, deubiquitination is not required for its recycling . In an attempt to identify actors in this new recycling pathway, we found an unexpected phenotype associated with loss of function of the Vps class C complex: cells defective for this complex are impaired for recycling of Fur4p, Snc1p, and the lipophilic dye FM4-64 . Genetic analyses indicated that these phenotypes were due to the functioning of the Vps class C complex in trafficking both to and from the late endosomal compartment. Mol Biol Cell, 2004 Sep, 15(9), 4321 - 36 Epub 2004 Jun 23. Transcriptional response of yeast to aflatoxin B1: recombinational repair involving RAD51 and RAD1; Keller-Seitz MU et al.; The potent carcinogen aflatoxin B(1) is a weak mutagen but a strong recombinagen in Saccharomyces cerevisiae . Aflatoxin B(1) exposure greatly increases frequencies of both heteroallelic recombination and chromosomal translocations . We analyzed the gene expression pattern of diploid cells exposed to aflatoxin B(1) using high-density oligonucleotide arrays comprising specific probes for all 6218 open reading frames . Among 183 responsive genes, 46 are involved in either DNA repair or in control of cell growth and division . Inducible growth control genes include those in the TOR signaling pathway and SPO12, whereas PKC1 is downregulated . Eleven of the 15 inducible DNA repair genes, including RAD51, participate in recombination . Survival and translocation frequencies are reduced in the rad51 diploid after aflatoxin B(1) exposure . In mec1 checkpoint mutants, aflatoxin B(1) exposure does not induce RAD51 expression or increase translocation frequencies; however, when RAD51 is constitutively overexpressed in the mec1 mutant, aflatoxin B(1) exposure increased translocation frequencies . Thus the transcriptional profile after aflatoxin B(1) exposure may elucidate the genotoxic properties of aflatoxin B(1). Mol Biol Cell, 2004 Sep, 15(9), 3994 - 4002 Epub 2004 Jun 23. A type V myosin (Myo2p) and a Rab-like G-protein (Ypt11p) are required for retention of newly inherited mitochondria in yeast cells during cell division; Boldogh IR et al.; Two actin-dependent force generators contribute to mitochondrial inheritance: Arp2/3 complex and the myosin V Myo2p (together with its Rab-like binding partner Ypt11p) . We found that deletion of YPT11, reduction of the length of the Myo2p lever arm (myo2-Delta6IQ), or deletion of MYO4 (the other yeast myosin V), had no effect on mitochondrial morphology, colocalization of mitochondria with actin cables, or the velocity of bud-directed mitochondrial movement . In contrast, retention of mitochondria in the bud was compromised in YPT11 and MYO2 mutants . Retention of mitochondria in the bud tip of wild-type cells results in a 60% decrease in mitochondrial movement in buds compared with mother cells . In ypt11Delta mutants, however, the level of mitochondrial motility in buds was similar to that observed in mother cells . Moreover, the myo2-66 mutant, which carries a temperature-sensitive mutation in the Myo2p motor domain, exhibited a 55% decrease in accumulation of mitochondria in the bud tip, and an increase in accumulation of mitochondria at the retention site in the mother cell after shift to restrictive temperatures . Finally, destabilization of actin cables and the resulting delocalization of Myo2p from the bud tip had no significant effect on the accumulation of mitochondria in the bud tip. Mol Biol Cell, 2004 Sep, 15(9), 4125 - 35 Epub 2004 Jun 23. Cystic fibrosis transmembrane conductance regulator degradation depends on the lectins Htm1p/EDEM and the Cdc48 protein complex in yeast; Gnann A et al.; Cystic fibrosis is the most widespread hereditary disease among the white population caused by different mutations of the apical membrane ATP-binding cassette transporter cystic fibrosis transmembrane conductance regulator (CFTR) . Its most common mutation, DeltaF508, leads to nearly complete degradation via endoplasmic reticulum-associated degradation (ERAD) . Elucidation of the quality control and degradation mechanisms might give rise to new therapeutic approaches to cure this disease . In the yeast Saccharomyces cerevisiae, a variety of components of the protein quality control and degradation system have been identified . Nearly all of these components share homology with mammalian counterparts . We therefore used yeast mutants defective in the ERAD system to identify new components that are involved in human CFTR quality control and degradation . We show the role of the lectin Htm1p in the degradation process of CFTR . Complementation of the HTM1 deficiency in yeast cells by the mammalian orthologue EDEM underlines the necessity of this lectin for CFTR degradation and highlights the similarity of quality control and ERAD in yeast and mammals . Furthermore, degradation of CFTR requires the ubiquitin protein ligases Der3p/Hrd1p and Doa10p as well as the cytosolic trimeric Cdc48p-Ufd1p-Npl4p complex . These proteins also were found to be necessary for ERAD of a mutated yeast "relative" of CFTR, Pdr5(*)p. Tsitologiia, 2004, 46(3), 277 - 82 {Phylogenetic analysis of yeast genes with large stereospecific anomalies in their promoter region}; Lukina NI et al.; Using an original computer program, we analysed 3 yeast Saccharomyces cerevisiae genes that contain large stereospecific anomalies (SA) in their promoter regions . Homologous genes for higher eukaryotic organisms contain large SA either in the same promoter regions or in one of their introns, the involved dinucleotide and DNA helical repeat being often conserved . We suppose that both promoter and enhancer-like sequences for these genes are evolutionary related and/or are regulated by related proteins. J Biol Chem, 2004 Sep 17, 279(38), 39677 - 85 Epub 2004 Jun 21. Distinct intracellular localization of Gpd1p and Gpd2p, the two yeast isoforms of NAD+-dependent glycerol-3-phosphate dehydrogenase, explains their different contributions to redox-driven glycerol production; Valadi A et al.; During anaerobiosis Saccharomyces cerevisiae strongly increases glycerol production to provide for non-respiratory oxidation of NADH to NAD(+) . We here report that respiratory-deficient cells become strictly dependent on the Gpd2p isoform of the NAD(+)-linked glycerol-3-phosphate dehydrogenase (Gpd) . The growth inhibition of respiratory incompetent cox18Delta cells lacking GPD2 is reversed by the addition of acetoin, an alternative sink for NADH oxidation . Growth is also restored by addition of lysine or glutamic acid/glutamine, the synthesis of which involves production of mitochondrial NADH . Lysine produced a stronger growth stimulating effect than glutamic acid consistent with an upregulated expression of the IDP3 gene for peroxisomal synthesis of the glutamate precursor alpha-ketoglutarate . Gpd2p is known to be a cytosolic protein but possesses a classical mitochondrial presequence, which we show is sufficient for mitochondrial targeting . A partial mitochondrial localization of Gpd2p will provide for establishment of intramitochondrial redox balance under non-respiratory conditions . Gpd1p, the other Gpd isoform, is partly cytosolic and partly peroxisomal and becomes more strictly peroxisomal in respiratory-deficient mutants . The different cellular distribution of Gpd1p and Gpd2p thus appears to be the main reason Gpd1p cannot substitute for Gpd2p in cox18Deltagpd2Delta cells, despite similar kinetic characteristics of the two iso-enzymes. Cell, 2004 Jun 25, 117(7), 899 - 913 CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast; Costanzo M et al.; Cyclin-dependent kinase (CDK) activity initiates the eukaryotic cell division cycle by turning on a suite of gene expression in late G1 phase . In metazoans, CDK-dependent phosphorylation of the retinoblastoma tumor suppressor protein (Rb) alleviates repression of E2F and thereby activates G1/S transcription . However, in yeast, an analogous G1 phase target of CDK activity has remained elusive . Here we show that the cell size regulator Whi5 inhibits G1/S transcription and that this inhibition is relieved by CDK-mediated phosphorylation . Deletion of WHI5 bypasses the requirement for upstream activators of the G1/S transcription factors SBF/MBF and thereby accelerates the G1/S transition . Whi5 is recruited to G1/S promoter elements via its interaction with SBF/MBF in vivo and in vitro . In late G1 phase, CDK-dependent phosphorylation dissociates Whi5 from SBF and drives Whi5 out of the nucleus . Elimination of CDK activity at the end of mitosis allows Whi5 to reenter the nucleus to again repress G1/S transcription . These findings harmonize G1/S control in eukaryotes. PLoS Biol . 2004 Jun;2(6):e160 . Epub 2004 Jun 15. Integrative analysis of the mitochondrial proteome in yeast; Prokisch H et al.; In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle . Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins . By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins . By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach . We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets . We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome . These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species . Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans. J Biol Chem, 2004 Aug 20, 279(34), 35384 - 91 Epub 2004 Jun 18. Nuclear export of the yeast mRNA-binding protein Nab2 is linked to a direct interaction with Gfd1 and to Gle1 function; Suntharalingam M et al.; Nuclear export of mRNA is mediated by interactions between soluble factors and nuclear pore complex (NPC) proteins . In Saccharomyces cerevisiae, Nab2 is an essential RNA-binding protein that shuttles between the nucleus and cytoplasm . The mechanism for trafficking of Nab2-bound mRNA through the NPC has not been defined . Gle1 is also required for mRNA export, and Gle1 interactions with NPC proteins, the RNA helicase Dbp5, and Gfd1 have been reported . Here we report that Nab2, Gfd1, and Gle1 associate in a complex . By using immobilized recombinant Gfd1, Nab2 was isolated from total yeast lysate . A similar biochemical assay with immobilized recombinant Nab2 resulted in coisolation of Gfd1 and Gle1 . A Nab2-Gfd1 complex was also identified by coimmunoprecipitation from yeast lysates . In vitro binding assays with recombinant proteins revealed a direct association between Nab2 and Gfd1, and two-hybrid assays delineated Gfd1 binding to the N-terminal Nab2 domain . This N-terminal Nab2 domain is distinct from its RNA binding domains suggesting Nab2 could bind Gfd1 and RNA simultaneously . As Nab2 export was blocked in a gle1 mutant at the restrictive temperature, we propose a model wherein Gfd1 serves as a bridging factor between Gle1 and Nab2-bound mRNA during export. J Biol Chem, 2004 Aug 27, 279(35), 36390 - 6 Epub 2004 Jun 18. HOR7, a multicopy suppressor of the Ca2+-induced growth defect in sphingolipid mannosyltransferase-deficient yeast; Lisman Q et al.; Yeast mutants defective in sphingolipid mannosylation accumulate inositol phosphorylceramide C (IPC-C), which renders cells Ca(2+)-sensitive . A screen for loss of function suppressors of the Ca(2+)-sensitive phenotype previously led to the identification of numerous genes involved in IPC-C synthesis . To better understand the molecular basis of the Ca(2+)-induced growth defect in IPC-C-overaccumulating cells, we searched for genes whose overexpression restored Ca(2+) tolerance in a mutant lacking the IPC mannosyltransferases Csg1p and Csh1p . Here we report the isolation of HOR7 as a multicopy suppressor of the Ca(2+)-sensitive phenotype of Deltacsg1Deltacsh1 cells . HOR7 belongs to a group of hyperosmolarity-responsive genes and encodes a small (59-residue) type I membrane protein that localizes at the plasma membrane . Hor7p is not required for high Ca(2+) or Na(+) tolerance . Instead, we find that Hor7p-overproducing cells display an increased resistance to high salt, sensitivity to low pH, and a reduced uptake of methylammonium, an indicator of the plasma membrane potential . These phenotypes are induced through a mechanism independent of the plasma membrane H(+)-ATPase, Pma1p . Our findings suggest that induction of Hor7p causes a depolarization of the plasma membrane that may counteract a Ca(2+)-induced influx of toxic cations in IPC-C-overaccumulating cells. J Biol Chem, 2004 Aug 20, 279(34), 36083 - 92 Epub 2004 Jun 18. Human Smp3p adds a fourth mannose to yeast and human glycosylphosphatidylinositol precursors in vivo; Taron BW et al.; Yeast and human glycosylphosphatidylinositol (GPI) precursors differ in the extent to which a fourth mannose is present as a side branch of the third core mannose . A fourth mannose addition to GPIs has scarcely been detected in studies of mammalian GPI synthesis but is an essential step in the Saccharomyces cerevisiae pathway . We report that human SMP3 encodes a functional homolog of the yeast Smp3 GPI fourth mannosyl-transferase . Expression of hSMP3 in yeast complements growth and biochemical defects of smp3 mutants and permits in vivo mannosylation of trimannosyl (Man(3))-GPIs . Immunolocalization shows that hSmp3p resides in the endoplasmic reticulum in human cells . Northern analysis of mRNA from human tissues and cell lines indicates that hSMP3 is expressed in most tissues, with the highest levels in brain and colon, but its mRNA is nearly absent from cultured human cell lines . Correspondingly, increasing expression of hSMP3 in cultured HeLa cells causes abundant formation of three putative tetramannosyl (Man(4))-GPIs . Our data indicate that hSmp3p functions as a mannosyltransferase that adds a fourth mannose to certain Man(3)-GPIs during biosynthesis of the human GPI precursor, and suggest it may do so in a tissue-specific manner. Virology, 2004 Jul 1, 324(2), 430 - 8 Analysis of murine leukemia virus replication complemented by yeast tRNA(Phe) reveals inherent preferences for the tRNA primer selected for reverse transcription; Palmer MT et al.; The replication of murine leukemia virus (MuLV) requires the capture of a cellular tRNA(Pro) as a primer for reverse transcription . To further study the specificity of primer selection, we have utilized a defective MuLV in which the primer-binding site (PBS) has been altered to be complementary to a nonmammalian tRNA, yeast tRNA(Phe) . Infectivity of the defective MuLV is dependent upon co-expression of yeast tRNA(Phe) in the cell . Defective MuLV genomes have been constructed in which the PBS was altered to be complementary to tRNA(Phe) that also encoded the cDNA for tRNA(Phe) . Transfection of these defective proviral genomes into cells resulted in the production of infectious MuLV as determined by a single-round assay . The amount of infectious virus produced using this complementation system, though, was approximately 6-fold lower than that produced following transfection of defective proviral genomes with a wild-type PBS complementary to tRNA(Pro) . The lower infectivity was not due to reduced expression of tRNA(Phe) in the transfected cells as compared to endogenous tRNA(Pro) or tRNA(Lys,3) . Serial passage of the MuLV genome with a PBS complementary to tRNA(Phe) that encoded tRNA(Phe) resulted in amplification of the virus . Using this rescue system, we have passaged the virus for four serial passages, after which time a revertant genome in which the PBS was altered to be complementary to tRNA(Gln) was detected that grew to high titers following subsequent serial passage . The results of these studies suggest that MuLV has preferences for the tRNA primer used in reverse transcription and are discussed with respect to the mechanism of primer selection. J Theor Biol, 2004 Jul 21, 229(2), 189 - 96 A mathematical model of ageing in yeast; Gillespie CS et al.; Budding yeast, Saccharomyces cerevisiae, is commonly used as a system to study cellular ageing . Yeast mother cells are capable of only a limited number of divisions before they undergo senescence, whereas newly formed daughters usually have their replicative age "reset" to zero . Accumulation of extrachromosomal ribosomal DNA circles (ERCs) appears to be an important contributor to ageing in yeast, and we describe a mathematical model that we developed to examine this process . We show that an age-related accumulation of ERCs readily explains the observed features of yeast ageing but that in order to match the experimental survival curves quantitatively, it is necessary that the probability of ERC formation increases with the age of the cell . This implies that some other mechanism(s), in addition to ERC accumulation, must underlie yeast ageing . We also demonstrate that the model can be used to gain insight into how an extra copy of the Sir2 gene might extend lifespan and we show how the model makes novel, testable predictions about patterns of age-specific mortality in yeast populations. Bioresour Technol, 2004 Oct, 95(1), 15 - 8 Application of oil refinery waste in the biosynthesis of glycolipids by yeast; Bednarski W et al.; Candida antarctica or Candida apicola synthesized surfactants (glycolipids) in the cultivation medium supplemented with oil refinery waste, either with soapstock (from 5.0% to 12.0% v/v) or post-refinery fatty acids (from 2.0% to 5.0% v/v) . The efficiency of glycolipids synthesis was determined by the amount of waste supplemented to the medium and was from 7.3 to 13.4 g/l and from 6.6 to 10.5 g/l in the medium supplemented with soapstock and post-refinery fatty acids, respectively . The studied yeast also synthesized glycolipids in the non-supplemented medium however, by the enrichment of medium with the oil refinery waste, a 7.5-8.5-fold greater concentration of glycolipids was obtained in the post-culture liquid then in the medium without addition of oil refinery waste . The yeast synthesized from 6.6 to 10.3 g dry biomass/l and the intra-cellular fat content was from 16.8% to 30.2% . The efficiency of glycolipids synthesis was determined by yeast species, medium acidity and culture period . The surface tension of the post-culture liquid separated from yeast biomass was reduced to 35.6 mN/m, which corresponded to the surface tension obtained at the critical micelle concentration of glycolipids. Bioorg Med Chem Lett, 2004 Jul 16, 14(14), 3841 - 5 Yeast and mammalian alpha-glucosidase inhibitory constituents from Himalayan rhubarb Rheum emodi Wall.ex Meisson; Suresh Babu K et al.; The methanolic extract of rhizome of Himalayan rhubarb Rheum emodi displayed mild yeast as well as mammalian intestinal alpha-glucosidase inhibitory activity . However, further fractionation of active extract led to the isolation of several potent molecules in excellent yields, displaying varying degrees of inhibition on two test models of alpha-glucosidase . Rhapontigenin, desoxyrhapontigenin, chrysophanol-8-O-beta-d-glucopyranoside, torachrysone-8-O-beta-d-glucopyranoside displayed potent yeast alpha-glucosidase inhibition . However chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside displayed potent to moderate mammalian alpha-glucosidase inhibitory activity . Other compounds displayed mild activity on both the tests . Except desoxyrhapontigenin and rhapontigenin that increased Vmax, other compounds including crude extract decreased the Vmax significantly (p<0.02) in yeast alpha-glucosidase test . Further kinetic analysis on mammalian alpha-glucosidase inhibition showed that chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside may be classified as mixed-noncompetitive inhibitors . However, desoxyrhapontigenin and rhapontigenin may be classified as modulators of enzyme activity . Presence and position of glycoside moiety in compounds appear important for better inhibition of mammalian alpha-glucosidase . This is the first report assigning particularly, mammalian intestinal alpha-glucosidase inhibitory activity to these compounds . Chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin, desoxyrhapontigenin and rhapontigenin have been isolated in substantial yields from R . emodi for the first time . Therefore, these compounds may have value in the treatment and prevention of hyperglycemia associated diabetes mellitus. EMBO J, 2004 Jul 7, 23(13), 2520 - 30 Epub 2004 Jun 17. Mmr1p is a mitochondrial factor for Myo2p-dependent inheritance of mitochondria in the budding yeast; Itoh T et al.; Class V myosins play a pivotal role in organelle distribution . In the budding yeast, Myo2p, a class V myosin, is essential for mitochondrial distribution . We identified MMR1 as a high-dose suppressor of the myo2 mitochondrial defect and that Mmr1p resides restrictively on the bud-localizing mitochondria and forms a complex with Myo2p tail . Mmr1p loss delayed mitochondrial transfer to buds and completely abolished mitochondrial distribution in the absence of Ypt11p, which promotes mitochondrial distribution by complex formation with Myo2p tail . The myo2-573 mutation, which causes a mitochondrial distribution defect and inactivates the Mmr1p function, reduced association between Myo2p and Mmr1p and depolarized Mmr1p localization on mitochondria . These strongly suggest that Mmr1p is a key mitochondrial component of the link between Myo2p and mitochondria for Myo2p-dependent mitochondrial distribution . Genetical analysis revealed that the Mmr1p-Myo2p pathway is independent of the Ypt11p-Myo2p pathway, suggesting that an essential system for mitochondrial distribution is composed of two independent Myo2p pathways. Methods Mol Biol, 2004, 277, 29 - 45 Genetic assays for triplet repeat instability in yeast; Dixon MJ et al.; The unusual genetic features of trinucleotide repeat (TNR) diseases have stimulated a substantial body of research into the underlying molecular mechanisms of repeat instability . As one useful tool to study TNR instability, selectable genetic assays for expansions and contractions were developed in the yeast Saccharomyces cerevisiae . These assays are sensitive, quantitative, easy to manipulate, and reproducible . Once colonies are identified through genetic selection, follow-up experiments with PCR help detail the precise molecular changes that occurred at the TNR tract . This chapter describes these yeast assays and provides useful technical insights into creating and testing triplet repeat instability in a classic model system. Mol Cell, 2004 Jun 18, 14(6), 825 - 32 Sho1 and Pbs2 Act as Coscaffolds Linking Components in the Yeast High Osmolarity MAP Kinase Pathway; Zarrinpar A et al.; Scaffold proteins mediate efficient and specific signaling in several mitogen-activated protein (MAP) kinase cascades . In the yeast high osmolarity response pathway, the MAP kinase kinase Pbs2 is thought to function as a scaffold, since it binds the osmosensor Sho1, the upstream MAP kinase kinase kinase Ste11, and the downstream MAP kinase Hog1 . Nonetheless, previous work has shown that Ste11 can be activated even when Pbs2 is deleted, resulting in inappropriate crosstalk to the mating pathway . We have found a region in the C terminus of Sho1 that binds Ste11 independently of Pbs2 and is required for crosstalk . These data support a model in which Sho1 has at least two separable interaction regions: one that binds Ste11 and mediates its activation, and one that binds Pbs2, directing Ste11 to act on Pbs2 . Thus, a network of interactions provided by both Sho1 and Pbs2 appears to direct pathway information flow. Mol Cell, 2004 Jun 18, 14(6), 813 - 23 Protein-protein interaction affinity plays a crucial role in controlling the sho1p-mediated signal transduction pathway in yeast; Marles JA et al.; Protein-protein interactions are required for most cellular functions, yet little is known about the relationship between protein-protein interaction affinity and biological activity . To investigate this issue, we engineered a series of mutants that incrementally reduced the affinity of the yeast Sho1p SH3 domain for its in vivo target, the MAP kinase kinase Pbs2p . We demonstrate a strong linear correlation between the binding energy of these mutants and quantitative in vivo outputs from the HOG high-osmolarity response pathway controlled by Sho1p . In addition, we find that reduction in binding affinity for the correct target within this pathway causes a proportional increase in misactivation of the related mating pheromone response pathway and that strong binding affinity alone does not guarantee efficient biological activity . Our experiments also indicate that a second binding surface on the Sho1p SH3 domain is required for its proper in vivo function. J Biol Chem, 2004 Aug 20, 279(34), 35334 - 40 Epub 2004 Jun 15. Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome C oxidase; Horng YC et al.; The assembly of the copper sites in cytochrome c oxidase involves a series of accessory proteins, including Cox11, Cox17, and Sco1 . The two mitochondrial inner membrane proteins Cox11 and Sco1 are thought to be copper donors to the Cu(B) and Cu(A) sites of cytochrome oxidase, respectively, whereas Cox17 is believed to be the copper donor to Sco1 within the intermembrane space . In this report we show Cox17 is a specific copper donor to both Sco1 and Cox11 . Using in vitro studies with purified proteins, we demonstrate direct copper transfer from CuCox17 to Sco1 or Cox11 . The transfer is specific because no transfer occurs to heterologous proteins, including bovine serum albumin and carbonic anhydrase . In addition, a C57Y mutant of Cox17 fails to transfer copper to Sco1 but is competent for copper transfer to Cox11 . The in vitro transfer studies were corroborated by a yeast cytoplasm expression system . Soluble domains of Sco1 and Cox11, lacking the mitochondrial targeting sequence and transmembrane domains, were expressed in the yeast cytoplasm . Metallation of these domains was strictly dependent on the co-expression of Cox17 . Thus, Cox17 represents a novel copper chaperone that delivers copper to two proteins. Genes Dev, 2004 Jun 15, 18(12), 1452 - 65 A new yeast PUF family protein, Puf6p, represses ASH1 mRNA translation and is required for its localization; Gu W et al.; In yeast Saccharomyces cerevisiae, Ash1p, a protein determinant for mating-type switching, is segregated within the daughter cell nucleus to establish asymmetry of HO expression . The accumulation of Ash1p results from ASH1 mRNA that is sorted as a ribonucleoprotein particle (mRNP or locasome) to the distal tip of the bud where translation occurs . To study the mechanism regulating ASH1 mRNA translation, we isolated the ASH1 locasome and characterized the associated proteins by MALDI-TOF . One of these proteins was Puf6p, a new member of the PUF family of highly conserved RNA-binding proteins such as Pumilio in Drosophila, responsible for translational repression, usually to effect asymmetric expression . Puf6p-bound PUF consensus sequences in the 3'UTR of ASH1 mRNA and repressed the translation of ASH1 mRNA both in vivo and in vitro . In the puf6 Delta strain, asymmetric localization of both Ash1p and ASH1 mRNA were significantly reduced . We propose that Puf6p is a protein that functions in the translational control of ASH1 mRNA, and this translational inhibition is necessary before localization can proceed. Genes Dev, 2004 Jun 15, 18(12), 1391 - 6 The generation of proper constitutive G-tails on yeast telomeres is dependent on the MRX complex; Larrivee M et al.; The precise DNA arrangement at chromosomal ends and the proteins involved in its maintenance are of crucial importance for genome stability . For the yeast Saccharomyces cerevisiae, this constitutive DNA configuration has remained unknown . We demonstrate here that G-tails of 12-14 bases are present outside of S phase on normal yeast telomeres . Furthermore, the Mre11p protein is essential for the proper establishment of this constitutive end-structure . However, the timing of extended G-tails occurring during S phase is not affected in strains lacking Mre11p . Thus, G-tails are present on yeast chromosomes throughout the cell cycle and the MRX complex is required for their normal establishment. Cell Cycle, 2004 Jun, 3(6), 817 - 22 Epub 2004 Jun 28. Temporal coupling of spindle disassembly and cytokinesis is disrupted by deletion of LTE1 in budding yeast; Jensen S et al.; The mitotic exit network (MEN) is a signal transduction cascade that controls exit from mitosis in budding yeast by triggering the nucleolar release and hence activation of the Cdc14 phosphatase . Activation of the MEN is tightly coordinated with spindle position in such a way that Cdc14 is only fully released upon spindle pole body (SPB) migration into the daughter cell . This temporal regulation of the MEN has been proposed to rely in part on the spatial separation of the G-protein Tem1 at the SPB and its nucleotide exchange factor Lte1 confined to the daughter cell cortex . However, the dispensability of LTE1 for survival has raised questions regarding this model . Here using real-time microscopy we show that lte1Delta mutants not only delay exit from mitosis but also uncouple the normal coordination between spindle disassembly and contraction of the actomyosin ring at cell division . These mitotic defects can be suppressed by a bub2Delta mutation or by Cdc14 over-expression suggesting that they are caused by compromised MEN activity . Thus Lte1 function is important to fine-tune the timing of mitotic exit and to couple this event with cytokinesis in budding yeast. J Cell Biol, 2004 Jun 21, 165(6), 759 - 65 Epub 2004 Jun 14. The fission yeast heterochromatin protein Rik1 is required for telomere clustering during meiosis; Tuzon CT et al.; Telomeres share the ability to silence nearby transcription with heterochromatin, but the requirement of heterochromatin proteins for most telomere functions is unknown . The fission yeast Rik1 protein is required for heterochromatin formation at centromeres and the mating-type locus, as it recruits the Clr4 histone methyltransferase, whose modification of histone H3 triggers binding by Swi6, a conserved protein involved in spreading of heterochromatin . Here, we demonstrate that Rik1 and Clr4, but not Swi6, are required along with the telomere protein Taz1 for crucial chromosome movements during meiosis . However, Rik1 is dispensable for the protective roles of telomeres in preventing chromosome end-fusion . Thus, a Swi6-independent heterochromatin function distinct from that at centromeres and the mating-type locus operates at telomeres during sexual differentiation. Zhonghua Kou Qiang Yi Xue Za Zhi, 2004 May, 39(3), 214 - 7; discussion 217 {Construction and confirmation of the fused plasmid with Rb bait gene in yeast two-hybrid system}; Xu Q et al.; OBJECTIVE: To screen and clone the novel genes related to cellular proliferation of oral squamous cell carcinoma . METHODS: We selected the Rb gene as the bait protein gene to construct the fusion bait plasmid of yeast two-hybrid . The whole code sequence of Rb gene was acquired by digestion with restricted enzyme EcoRI and BamH1 and reclaimed from its original vector pGBT9-pRb . After being confirmed by electrophoresis, the Rb gene was cloned into the MCS of the plasmid pGBKT7 to construct a recombined plasmid pGBKT7-pRb and the sequence of the recombined plasmid was detected in company . According to the protocol of yeast two hybrid system III, the competent Y187 yeast was prepared, and transformed with recombined plasmid pGBKT7-pRb . Following that, the toxicity and transcriptional activation of this recombined plasmid pGBKT7-pRb in Y187 yeast were tested . RESULTS: The sequence of the recombined plasmid was correct compared with the sequence provided in Genbank . The protein could be correctly synthesized in vitro, and no self-activating transcriptional activation and toxicity was observed in Y187 yeast . CONCLUSIONS: The construction of the recombined plasmid was capable to be used as the fusion bait plasmid in yeast two-hybrid system III, and the recombined Rb-protein could be used as the bait protein successfully. Biochemistry, 2004 Jun 22, 43(24), 7736 - 42 Structures of the yeast ribonucleotide reductase Rnr2 and Rnr4 homodimers; Sommerhalter M et al.; Class I ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides . Eukaryotic RNRs comprise two subunits, the R1 subunit, which contains substrate and allosteric effector binding sites, and the R2 subunit, which houses a catalytically essential diiron-tyrosyl radical cofactor . In Saccharomyces cerevisiae, there are two variants of the R2 subunit, called Rnr2 and Rnr4 . Rnr4 is unique in that it lacks three iron-binding residues conserved in all other R2s . Nevertheless, Rnr4 is required to activate Rnr2, and the functional species in vivo is believed to be a heterodimeric complex between the two proteins . The crystal structures of the Rnr2 and Rnr4 homodimers have been determined and are compared to that of the heterodimer . The homodimers are very similar to the heterodimer and to mouse R2 in overall fold, but there are several key differences . In the Rnr2 homodimer, one of the iron-binding helices, helix alphaB, is not well-ordered . In the heterodimer, interactions with a loop region connecting Rnr4 helices alphaA and alpha3 stabilize this Rnr2 helix, which donates iron ligand Asp 145 . Sequence differences between Rnr2 and Rnr4 prevent the same interactions from occurring in the Rnr2 homodimer . These findings provide a structural rationale for why the heterodimer is the preferred complex in vivo . The active-site region in the Rnr4 homodimer reveals interactions not apparent in the heterodimer, supporting previous conclusions that this subunit does not bind iron . When taken together, these results support a model in which Rnr4 stabilizes Rnr2 for cofactor assembly and activity. Biol Chem, 2004 May, 385(5), 389 - 95 Characterization of pancreatic ERj3p, a homolog of yeast DnaJ-like protein Scj1p; Bies C et al.; We have previously identified in the human EST sequence data base four overlapping clones that could be aligned with both a predicted protein sequence, deduced from the C . elegans genomic sequence, and partial amino acid sequences, obtained for a protein from canine pancreatic microsomes . We suggested that these proteins are homologs of yeast microsomal and DnaJ-like protein Scj1p and termed them ERj3p . Here we verified the predicted protein sequence of human ERj3p by sequence analysis of the corresponding cDNA . Multiple alignment of related sequences identified these proteins as true homologs of yeast Scj1p . Biochemical analysis of the canine protein characterized ERj3p as a soluble glycoprotein of the pancreatic endoplasmic reticulum . This pancreatic DnaJ-like protein was shown to interact with lumenal DnaK-like proteins, such as BiP . Furthermore, we found that ERj3p interacts with SDF2L1 protein that may be involved in protein O-glycosylation . We propose that ERj3p represents a cochaperone of DnaK-like chaperones of the mammalian endoplasmic reticulum and is involved in folding and maturation of newly synthesized proteins. Biotechnol Lett, 2004 May, 26(9), 757 - 62 Changes in volatile compounds and aromatic series in sherry wine with high gluconic acid levels subjected to aging by submerged flor yeast cultures; Peinado RA et al.; Volatile compounds of sherry wine containing gluconic acid under aging by submerged flor yeast cultures were analyzed . The aroma profile was obtained by grouping the compounds in nine aromatic series . The balsamic, fatty, herbaceous and empyreumatic series increased significantly as consequence of the increase of pantolactone, acids (butanoic, 2-methylbutanoic and 3-methylbutanoic), methionol and gamma-butyrolactone compounds, respectively . The decrease of higher alcohols promoted solvent series diminished . These changes are consistent with those observed in the production of commercial sherry wine using traditional biological aging. Mol Biol Cell, 2004 Aug, 15(8), 3740 - 50 Epub 2004 Jun 11. A novel intermediate in initiation complex assembly for fission yeast DNA replication; Yamada Y et al.; Assembly of initiation factors on individual replication origins at onset of S phase is crucial for regulation of replication timing and repression of initiation by S-phase checkpoint control . We dissected the process of preinitiation complex formation using a point mutation in fission yeast nda4-108/mcm5 that shows tight genetic interactions with sna41(+)/cdc45(+) . The mutation does not affect loading of MCM complex onto origins, but impairs Cdc45-loading, presumably because of a defect in interaction of MCM with Cdc45 . In the mcm5 mutant, however, Sld3, which is required for Cdc45-loading, proficiently associates with origins . Origin-association of Sld3 without Cdc45 is also observed in the sna41/cdc45 mutant . These results suggest that Sld3-loading is independent of Cdc45-loading, which is different from those observed in budding yeast . Interestingly, returning the arrested mcm5 cells to the permissive temperature results in immediate loading of Cdc45 to the origin and resumption of DNA replication . These results suggest that the complex containing MCM and Sld3 is an intermediate for initiation of DNA replication in fission yeast. Curr Opin Struct Biol, 2004 Jun, 14(3), 292 - 9 Protein interaction networks from yeast to human; Bork P et al.; Protein interaction networks summarize large amounts of protein-protein interaction data, both from individual, small-scale experiments and from automated high-throughput screens . The past year has seen a flood of new experimental data, especially on metazoans, as well as an increasing number of analyses designed to reveal aspects of network topology, modularity and evolution . As only minimal progress has been made in mapping the human proteome using high-throughput screens, the transfer of interaction information within and across species has become increasingly important . With more and more heterogeneous raw data becoming available, proper data integration and quality control have become essential for reliable protein network reconstruction, and will be especially important for reconstructing the human protein interaction network. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 May, 20(3), 269 - 71 {Inhibitory effect of hDAF displayed on yeast cells on deposition of complement on the yeast cells}; Guo B et al.; AIM: To investigate whether the hDAF displayed on the surface of yeast cells can inhibit the deposition of C5b-9 . METHODS: After treatment with normal human serum, deposition of human complement C5b-9 and DAF expression on yeast cells EBY100 were analyzed by flow cytometry . RESULTS: Yeast cells EBY100 could activate the complement molecules in human serum and lead to the deposition of C5b-9, while the deposition of C5b-9 on the surface of yeast cells with hDAF could significantly reduce the deposition of C5b-9 . CONCLUSION: hDAF displayed on the surface of yeast cells can fold correctly and exhibit activity of complement's inhibition. EMBO J, 2004 Jul 7, 23(13), 2620 - 31 Epub 2004 Jun 10. Biochemical analysis of TREX complex recruitment to intronless and intron-containing yeast genes; Abruzzi KC et al.; The TREX complex is involved in both transcription elongation and mRNA export and is recruited to nascent transcription complexes . We have examined Yra1p, Sub2p and Hpr1p recruitment to nine genes of varying lengths and transcription frequencies . All three proteins increase from the 5' to the 3' ends of the four intronless genes examined . A modified chromatin immunoprecin Golgi export (PIK1 and YPT31/32) . Importantly, prominent defects in the actin cytoskeleton were observed in all of these strains, thus implicating a known causal relationship between the deregulation of actin and the inhibition of mRNA transport . Our novel observations suggest that vesicular transport regulates the actin cytoskeleton in yeast (and not just vice versa) leading to subsequent defects in mRNA transport and localization. J Biol Chem, 2004 Aug 13, 279(33), 35113 - 20 Epub 2004 Jun 10. In vitro assembly of the characteristic chromatin organization at the yeast PHO5 promoter by a replication-independent extract system; Korber P et al.; An extensive set of analyses of the yeast PHO5 gene, mostly performed in vivo, has made this gene a model for the role of chromatin structure in gene regulation . In the repressed state, the PHO5 promoter shows a characteristic chromatin organization with four positioned nucleosomes and a short hypersensitive site . So far the basis for this nucleosome positioning has remained unresolved . We have therefore decided to complement the in vivo studies by an in vitro approach . As a first step, we have asked whether the characteristic PHO5 promoter chromatin structure depends on the cellular context including replication or higher order nuclear chromatin organization or whether it can be reconstituted in vitro in a cell-free system . To this end we have established an in vitro chromatin assembly system based on yeast extracts . It is capable of generating extensive regular nucleosomal arrays with physiological spacing . Assembly requires supplementation with exogenous histones and is dependent on energy leading to chromatin with dynamic properties due to ATP-dependent activities of the extrain Golgi export (PIK1 and YPT31/32) . Importantly, prominent defects in the actin cytoskeleton were observed in all of these strains, thus implicating a known causal relationship between the deregulation of actin and the inhibition of mRNA transport . Our novel observations suggest that vesicular transport regulates the actin cytoskeleton in yeast (and not just vice versa) leading to subsequent defects in mRNA transport and localization. J Biol Chem, 2004 Aug 13, 279(33), 35113 - 20 Epub 2004 Jun 10. In vitro assembly of the characteristic chromatin organization at the yeast PHO5 promoter by a replication-independent extract system; Korber P et al.; An extensive set of analyses of the yeast PHO5 gene, mostly performed in vivo, has made this gene a model for the role of chromatin structure in gene regulation . In the repressed state, the PHO5 promoter shows a characteristic chromatin organization with four positioned nucleosomes and a short hypersensitive site . So far the basis for this nucleosome positioning has remained unresolved . We have therefore decided to complement the in vivo studies by an in vitro approach . As a first step, we have asked whether the characteristic PHO5 promoter chromatin structure depends on the cellular context including replication or higher order nuclear chromatin organization or whether it can be reconstituted in vitro in a cell-free system . To this end we have established an in vitro chromatin assembly system based on yeast extracts . It is capable of generating extensive regular nucleosomal arrays with physiological spacing . Assembly requires supplementation with exogenous histones and is dependent on energy leading to chromatin with dynamic properties due to ATP-dependent activities of the extract . Using the PHO5 promoter sequence as template in this replication independent system, we obtain a nucleosomal pattern over the PHO5 promoter region that is very similar to the in vivo pattern of the repressed state . This shows that the chromatin structure at the PHO5 promoter represents a self-organizing system in cell-free yeast extracts and provides a promising substrate for in vitro studies with a direct in vivo correlate. Nature, 2004 Jun 10, 429(6992), 661 - 4 Metabolic network analysis of the causes and evolution of enzyme dispensability in yeast; Papp B et al.; Under laboratory conditions 80% of yeast genes seem not to be essential for viability . This raises the question of what the mechanistic basis for dispensability is, and whether it is the result of selection for buffering or an incidental side product . Here we analyse these issues using an in silico flux model of the yeast metabolic network . The model correctly predicts the knockout fitness effects in 88% of the genes studied and in vivo fluxes . Dispensable genes might be important, but under conditions not yet examined in the laboratory . Our model indicates that this is the dominant explanation for apparent dispensability, accounting for 37-68% of dispensable genes, whereas 15-28% of them are compensated by a duplicate, and only 4-17% are buffered by metabolic network flux reorganization . For over one-half of those not important under nutrient-rich conditions, we can predict conditions when they will be important . As expected, such condition-specific genes have a more restricted phylogenetic distribution . Gene duplicates catalysing the same reaction are not more common for indispensable reactions, suggesting that the reason for their retention is not to provide compensation . Instead their presence is better explained by selection for high enzymatic flux. Nature, 2004 Jul 1, 430(6995), 88 - 93 Epub 2004 Jun 09. Evidence for dynamically organized modularity in the yeast protein-protein interaction network; Han JD et al.; In apparently scale-free protein-protein interaction networks, or 'interactome' networks, most proteins interact with few partners, whereas a small but significant proportion of proteins, the 'hubs', interact with many partners . Both biological and non-biological scale-free networks are particularly resistant to random node removal but are extremely sensitive to the targeted removal of hubs . A link between the potential scale-free topology of interactome networks and genetic robustness seems to exist, because knockouts of yeast genes encoding hubs are approximately threefold more likely to confer lethality than those of non-hubs . Here we investigate how hubs might contribute to robustness and other cellular properties for protein-protein interactions dynamically regulated both in time and in space . We uncovered two types of hub: 'party' hubs, which interact with most of their partners simultaneously, and 'date' hubs, which bind their different partners at different times or locations . Both in silico studies of network connectivity and genetic interactions described in vivo support a model of organized modularity in which date hubs organize the proteome, connecting biological processes--or modules--to each other, whereas party hubs function inside modules. J Biomol Tech, 2004 Jun, 15(2), 134 - 43 Improved 2D nano-LC/MS for proteomics applications: a comparative analysis using yeast proteome; Nagele E et al.; The most commonly used method for protein identification with two-dimensional (2D) online liquid chromatography-mass spectrometry (LC/MS) involves the elution of digest peptides from a strong cation exchange column by an injected salt step gradient of increasing salt concentration followed by reversed phase separation . However, in this approach ion exchange chromatography does not perform to its fullest extent, primarily because the injected volume of salt solution is not optimized to the SCX column . To improve the performance of strong cation exchange chromatography, we developed a new method for 2D online nano-LC/MS that replaces the injected salt step gradient with an optimized semicontinuous pumped salt gradient . The viability of this method is demonstrated in the results of a comparative analysis of a complex tryptic digest of the yeast proteome using the injected salt solution method and the semicontinuous pump salt method . The semicontinuous pump salt method compares favorably with the commonly used injection method and also with an offline 2D-LC method. Eukaryot Cell, 2004 Jun, 3(3), 785 - 94 Sequence elements necessary for transcriptional activation of BAD1 in the yeast phase of Blastomyces dermatitidis; Rooney PJ et al.; Blastomyces dermatitidis is a dimorphic fungal pathogen that converts from mycelia or conidia to a host-adapted yeast morphotype upon infection . Conversion to the yeast form is accompanied by the production of the virulence factor BAD1 . Yeast-phase-specific expression of BAD1 is transcriptionally regulated, and its promoter shares homology with that of the yeast-phase-specific gene YPS3 of Histoplasma capsulatum . Serial truncations of the BAD1 upstream region were fused to the lacZ reporter to define functional areas in the promoter . Examination of PBAD1-lacZ fusions in B . dermatitidis indicated that BAD1 transcription is upregulated in the yeast phase . The 63-nucleotide box A region conserved in the YPS3 upstream region was shown to be an essential component of the minimal BAD1 promoter . A matched PYPS3-lacZ construct indicated that this same region was needed for minimal YPS3 promoter activity in B . dermatitidis transformants . Reporter activity in H . capsulatum transformants similarly showed a requirement for box A in the minimal BAD1 promoter . Several putative transcription factor binding sites were identified within box A of BAD1 . Replacement of two of these predicted sites within box A--a cAMP responsive element and a Myb binding site--sharply reduced transcriptional activity, indicating that these regions are critical in dictating the yeast-phase-specific expression of this crucial virulence determinant of B . dermatitidis . Antonie Van Leeuwenhoek, 2000 Apr, 77(3), 293 - 302 Emendation of the basidiomycetous yeast genus Kondoa and the description of Kondoa aeria sp . nov; Fonseca A et al.; The genus Kondoa Y . Yamada, Nakagawa & Banno was erected to accommodate a single taxon, K . malvinella (Fell & Hunter) Y . Yamada, Nakagawa & Banno, which was transferred from the teliospore-forming genus Rhodosporidium Banno based on pronounced differences in the 5S and 26S ribosomal RNA (rRNA) nucleotide sequences to R . toruloides Banno . In contrast with the original description, reinvestigation of K . malvinella revealed the formation of transversely septate (auricularioid) basidia that did not arise on teliospores, but formed directly on the dikaryotic mycelium . The four-celled basidia developed sterigmata on which forcibly discharged asymmetric basidiospores (ballistospores) were produced . Additionally, a new taxon emerged from the study of recent isolates, for which the name K . aeria sp . nov . is proposed . This new species produced two-celled auricularioid basidia on hyphae with incomplete clamp connections . Ballistospores arose on the basidia at the tip of sterigmata and, after ejection, germinated by budding . These observations led us to present an emended diagnosis for the genus Kondoa . Analysis of the sequence data from the D1/D2 region of the 26S rRNA gene showed a very close resemblance between K . aeria and K . malvinella in a cluster that also contained several Bensingtonia species . Taxa in this cluster share specific physiological traits and produce characteristic pinkish-cream to mauve colonies; in contrast, formation of ballistoconidia is only observed in the Bensingtonia species . Sequence data supported placement of K . malvinella and K . aeria in the 'Agaricostilbum clade' of the Urediniomycetes. Methods Mol Biol, 2004, 280, 83 - 98 Analyzing the spindle checkpoint in yeast and frogs; Stukenberg PT et al.; The spindle checkpoint is an evolutionarily conserved regulatory mechanism that ensures correct segregation of chromosomes at mitosis and meiosis . The kinetochore plays an integral role in spindle checkpoint signaling by integrating chromosome attachment to the spindle with cell cycle progression . A single kinetochore can inhibit cell cycle progression in the absence of proper spindle attachment or tension from bipolar orientation . Recent advances have shed light on how the kinetochore measures these situations, transduces a signal, and inhibits the entry into anaphase. J Agric Food Chem, 2004 Jun 16, 52(12), 3761 - 71 Identification and synthesis of a novel selenium-sulfur amino acid found in selenized yeast: Rapid indirect detection NMR methods for characterizing low-level organoselenium compounds in complex matrices; Block E et al.; After proteolytic digestion, aqueous extraction, and derivatization with diethyl pyrocarbonate or ethyl chloroformate, HPLC-inductively coupled plasma (ICP)-MS, GC-atomic emission detection (AED), and GC-MS analysis of high-selenium yeast stored at room temperature for more than 10 years showed selenomethionine as the major Se product along with substantial amounts of selenomethionine selenoxide hydrate and the previously unreported selenoamino acid having a Se-S bond, S-(methylseleno)cysteine . The identity of the latter compound was confirmed by synthesis . The natural product was shown to be different from a synthetic sample of the isomeric compound Se-(methylthio)selenocysteine . Selenium-specific NMR spectroscopic methods were developed to directly analyze the aqueous extracts of the hydrolyzed selenized yeast without derivatization or separation . Selenomethionine and S-(methylseleno)cysteine were identified by 77Se-1H HMQC-TOCSY experiments. J Cell Biol, 2004 Jun 7, 165(5), 685 - 95 Myosin-II reorganization during mitosis is controlled temporally by its dephosphorylation and spatially by Mid1 in fission yeast; Motegi F et al.; Cytokinesis in many eukaryotes requires an actomyosin contractile ring . Here, we show that in fission yeast the myosin-II heavy chain Myo2 initially accumulates at the division site via its COOH-terminal 134 amino acids independently of F-actin . The COOH-terminal region can access to the division site at early G2, whereas intact Myo2 does so at early mitosis . Ser1444 in the Myo2 COOH-terminal region is a phosphorylation site that is dephosphorylated during early mitosis . Myo2 S1444A prematurely accumulates at the future division site and promotes formation of an F-actin ring even during interphase . The accumulation of Myo2 requires the anillin homologue Mid1 that functions in proper ring placement . Myo2 interacts with Mid1 in cell lysates, and this interaction is inhibited by an S1444D mutation in Myo2 . Our results suggest that dephosphorylation of Myo2 liberates the COOH-terminal region from an intramolecular inhibition . Subsequently, dephosphorylated Myo2 is anchored by Mid1 at the medial cortex and promotes the ring assembly in cooperation with F-actin . Copyright the Rockefeller University Press Biochem Biophys Res Commun, 2004 Jul 2, 319(3), 840 - 6 The yeast genes, ARL1 and CCZ1, interact to control membrane traffic and ion homeostasis; Love SL et al.; The yeast ARL1 gene, encoding a guanine-nucleotide binding protein of the Arf-like family, exhibits a synthetic genetic interaction with CCZ1 . An arl1 Delta ccz1 Delta double mutant was viable but grew slowly, was more sensitive to caffeine, Ca(2+), Zn(2+), and hygromycin B than either single mutant, and had a more severe vacuolar protein sorting phenotype . Overexpression of ARL1 did not suppress ccz1 Delta mutant phenotypes, nor did overexpression of CCZ1 suppress arl1 Delta mutant phenotypes . We conclude that ARL1 and CCZ1 independently contribute to both ion homeostasis and protein sorting. J Mol Biol, 2004 Jun 25, 340(1), 15 - 27 Complementation of yeast Arc1p by the p43 component of the human multisynthetase complex does not require its association with yeast MetRS and GluRS; Golinelli-Cohen MP et al.; Yeast Arc1p, human p43 and plant methionyl-tRNA synthetase (MetRS) possess an EMAPII-like domain capable of non-specific interactions with tRNA . Arc1p interacts with MetRS (MES1) and GluRS and operates as a tRNA-interacting factor (tIF) in trans of these two synthetases . In plant MetRS, the EMAPII-like domain is fused to the catalytic core of the synthetase and acts as a cis-acting tIF for aminoacylation . We observed that the catalytic core of plant MetRS expressed from a centromeric plasmid cannot complement a yeast arc1(-) mes1(-) strain . Overexpression of the mutant enzyme from a high-copy number plasmid restored cell growth, suggesting that deletion of its C-terminal tIF domain was responsible for the poor aminoacylation efficiency of that enzyme in vivo . Accordingly, expression of full-size plant MetRS from a centromeric plasmid, but also of fusion proteins between its catalytic core and the EMAPII-like domains of yeast Arc1p or of human p43 restored cell viability . These data showed that homologous tIF domains from different origins are interchangeable and may act indifferently in trans or in cis of the catalytic domain of a synthetase . Unexpectedly, co-expression of Arc1p with the catalytic core of plant MetRS restored cell viability as well, even though Arc1p did not associate with plant MetRS . Because Arc1p also interacts with yeast GluRS, restoration of cell growth could be due at least in part to its role of cofactor for that enzyme . However, co-expression of human p43, a tIF that did not associate with plant MetRS or with yeast GluRS and MetRS, also restored cell viability of a yeast strain that expressed the catalytic core of plant MetRS . These results show that p43 and Arc1p are able to facilitate tRNA aminoacylation in vivo even if they do not interact physically with the synthetases . We propose that p43/Arc1p may be involved in sequestering tRNAs in the cytoplasm of eukaryotic cells, thereby increasing their availability for protein synthesis. Bioresour Technol, 2004 Sep, 94(3), 245 - 9 Removal of chromate anions from aqueous stream by a cationic surfactant-modified yeast; Bingol A et al.; The removal of chromate anions (CrO(4)(2-)) from aqueous solution by a cationic surfactant-modified yeast was studied in a batch system . Cetyl trimethyl ammonium bromide (CTAB) was used for biomass modification; it substantially improved the biosorption efficiency . The influences of solution pH, CrO(4)(2-) anion concentrations and biomass concentration on the biosorption efficiency were investigated . The biosorption of chromate anions by modified yeast was strongly affected by pH . The optimum pH for biosorption of CrO(4)(2-) by modified yeast was 4.5-5.5 . Zeta potential values of modified and unmodified yeast were determined at various pH values . Concentrations ranging from 5.2 to 208 mg/l Cr(VI) were tested and the biosorptive removal efficiency of the metal ions from aqueous solution was more than 99.5% . Freundlich and Langmuir isotherms were used to evaluate the data and the regression constants were determined. FEBS Lett, 2004 Jun 4, 567(2-3), 270 - 4 Kinetic study of the H103A mutant yeast transketolase; Selivanov VA et al.; Data from site-directed mutagenesis and X-ray crystallography show that His103 of holotransketolase (holoTK) does not come into contact with thiamin diphosphate (ThDP) but stabilizes the transketolase (TK) reaction intermediate, alpha,beta-dihydroxyethyl-thiamin diphosphate, by forming a hydrogen bond with the oxygen of its beta-hydroxyethyl group {Eur . J . Biochem . 233 (1995) 750; Proc . Natl . Acad . Sci . USA 99 (2002) 591} . We studied the influence of His103 mutation on ThDP-binding and enzymatic activity . It was found that mutation does not affect the affinity of the coenzyme to apotransketolase (apoTK) in the presence of Ca(2+) (a cation found in the native holoenzyme) but changes all the kinetic parameters of the ThDP-apoTK interaction in the presence of Mg(2+) (a cation commonly used in ThDP-dependent enzymes studies) . It was concluded that the structures of TK active centers formed in the presence of Mg(2+) and Ca(2+) are not identical . Mutation of His103 led to a significant acceleration of the one-substrate reaction but a slow down of the two-substrate reaction so that the rates of both types of catalysis became equal . Our results provide evidence for the intermediate-stabilizing function of His103. Gene, 2004 May 26, 333, 151 - 5 Short-range compositional correlation in the yeast genome depends on transcriptional orientation; Marin A et al.; This article reports an analysis of composition of about 5000 intergenic regions and neighboring ORFs in the nuclear genome of Saccharomyces cerevisiae, and their correlation . Intergenic regions flanked by divergently transcribed ORFs are GC richer (36%) than those separating convergent ORFs (29%) . This difference in GC content cannot be fully attributed to its location upstream or downstream the ORFs, since no such strong compositional bias is found within 3' and 5' segments of intergenic regions between ORFs transcribed in the same direction . We have also found that the GC content of intergenic regions is positively correlated to that of its flanking ORFs in tandem and divergent orientations, but not in convergent orientations, and that the correlation coefficient between the GC content of nearby ORFs is higher for divergent pairs . Our observations are discussed in the light of recent work stressing the relationships between base composition, chromatin structure and meiotic recombination. DNA Repair (Amst), 2004 Mar 4, 3(3), 277 - 87 The yeast Rad7/Rad16/Abf1 complex generates superhelical torsion in DNA that is required for nucleotide excision repair; Yu S et al.; Nucleotide excision repair (NER) in eukaryotes removes DNA base damage as an oligonucleotide in a complex series of reactions . The nature of the dual incision reactions on either side of the damaged base has been extensively investigated . However, the precise mechanism of cleavage of the phosphodiester backbone of the DNA by the NER endonucleases and how this relates to removal of the damage-containing oligonucleotide during the excision process has not been determined . We previously isolated a stable heterotrimeric complex of Rad7/Rad16/Abf1 from yeast which functions in the conserved global genome repair (GGR) pathway . GGR removes lesions from DNA that is not actively transcribing . We have shown previously that the Rad7/Rad16/Abf1 heterotrimer is required to observe DNA repair synthesis and oligonucleotide excision during in vitro NER, but not needed to detect NER-dependent incision in such reactions . Here we report that this protein complex generates superhelicity in DNA through the catalytic activity of the Rad16 component . The torsion generated in the DNA by this complex is necessary to remove the damage-containing oligonucleotide during NER--a process referred to as excision . We conclude that in yeast the molecular mechanism of NER includes the generation of superhelical torsion in DNA . Biotechnol Prog, 2004 May-Jun, 20(3), 688 - 91 Improvement of cellulose-degrading ability of a yeast strain displaying Trichoderma reesei endoglucanase II by recombination of cellulose-binding domains; Ito J et al.; To improve the cellulolytic activity of a yeast strain displaying endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414, the genes encoding the cellulose-binding domain (CBD) of EGII, cellobiohydrolase I (CBHI) and cellobiohydrolase II (CBHII) from T . reesei QM9414, were fused with the catalytic domain of EGII and expressed in Saccharomyces cerevisiae . Display of each of the recombinant EGIIs was confirmed using immunofluorescence microscopy . In the case of EGII-displaying yeast strains in which the CBD of EGII was replaced with the CBD of CBHI or CBHII, the binding affinity to Avicel and hydrolytic activity toward phosphoric acid swollen Avicel were similar to that of a yeast strain displaying wild-type EGII . On the other hand, the three yeast strains displaying EGII with two or three tandemly aligned CBDs showed binding affinity and hydrolytic activity higher than that of the yeast strain displaying wild-type EGII . This result indicates that the hydrolytic activity of yeast strains displaying recombinant EGII increases with increased binding ability to cellulose. Mol Cell, 2004 Jun 4, 14(5), 657 - 66 Chromatin disassembly mediated by the histone chaperone Asf1 is essential for transcriptional activation of the yeast PHO5 and PHO8 genes; Adkins MW et al.; Nucleosome loss from a promoter region has recently been described as a potential mechanism for transcriptional regulation . We investigated whether H3/H4 histone chaperones mediate the loss of nucleosomes from the promoter of the yeast PHO5 gene during transcriptional activation . We found that antisilencing function 1 (Asf1p) mediates nucleosome disassembly from the PHO5 promoter in vivo . We show that nucleosome disassembly also occurs at a second promoter, that of the PHO8 gene, during activation, and we demonstrate that this is also mediated by Asf1p . Furthermore, we show that nucleosome disassembly is essential for PHO5 and PHO8 activation . Contrary to the current dogma, we demonstrate that nucleosome disassembly is not required to enable binding of the Pho4p activator to its PHO5 UASp2 site in vivo . Finally, we show that nucleosomes are reassembled over the PHO5 promoter during repression . As such, nucleosome disassembly and reassembly are important mechanisms for transcriptional activation and repression, respectively. J Environ Monit, 2004 Jun, 6(6), 546 - 51 Epub 2004 Mar 05. Improved application of recombinant yeast assays on environmental samples by size exclusion chromatography; Tashiro Y et al.; The utility of HPLC with a size exclusion chromatography (SEC) column for the fractionation of environmental samples was examined to expand the applicability of recombinant yeast assays (RYAs) for the measurement of estrogenic activity in environmental samples . The elution of steroidal hormone standards through SEC was tested, and river water and sediment samples were subjected to SEC fractionation . The retention times of all estrogen standards were longer than those of the major components with absorbance at 230 nm of the environmental sample matrix . The estrogenic activity of SEC fractions of environmental samples from highly polluted sites was measured using RYA and revealed the existence of estrogenic compounds that could not be detected or quantified in extracted samples before fractionation . The fractions from environmental samples that corresponded to the retention time of estrone, 17{small beta}-estradiol, and estriol were analyzed with LC/MS/MS . These three estrogens were separated into three different fractions, and the concentration of estrone coincided with the estrogenic activity of the fraction in which it was detected . The profile of estrogenic activity of SEC fractions indicated the molecular size of the estrogenic compounds and the yeast growth inhibitors in samples. J Cell Sci, 2004 Jun 15, 117(Pt 14), 2983 - 96 Epub 2004 Jun 01. A role for yeast oxysterol-binding protein homologs in endocytosis and in the maintenance of intracellular sterol-lipid distribution; Beh CT et al.; The seven yeast OSH genes (OSH1-OSH7) encode a family of orthologs of the mammalian oxysterol-binding protein (OSBP) . The OSH genes share at least one essential overlapping function, potentially linked to the regulation of secretory trafficking and membrane lipid composition . To investigate the essential roles of the OSH genes, we constructed conditional OSH mutants and analyzed their cellular defects . Elimination of all OSH function altered intracellular sterol-lipid distribution, caused vacuolar fragmentation, and resulted in an accumulation of lipid droplets in the cytoplasm and within vacuolar fragments . Gradual depletion of Osh proteins also caused cell budding defects and abnormal cell wall deposition . In OSH mutant cells endocytosis was severely impaired, but protein transport to the vacuole and the plasma membrane was largely unaffected . Other mutants affecting sterol-lipid function and distribution, namely erg2Delta and arv1Delta, shared similar defects . These findings suggested that OSH genes, through effects on intracellular sterol distribution, establish a plasma membrane lipid composition that promotes endocytosis. Novartis Found Symp, 2004, 259, 48 - 56; discussion 56-62, 163-9 A model for step-wise assembly of heterochromatin in yeast; Moazed D et al.; Gene silencing involves the assembly of DNA into specialized chromatin domains that are inaccessible to trans-acting factors and are epigenetically inherited . In the budding yeast Saccharomyces cerevisiae, silent heterochromatic DNA domains occur at telomeres, the silent mating type loci, and the rDNA repeats . At telomeres and the mating type loci, silencing requires the Sir2, Sir3 and Sir4 proteins, the conserved N-termini of histones H3 and H4, and a number of chromatin assembly factors . The Sir proteins form a multimeric complex that binds preferentially to deacetylated nucleosomes through the Sir3 and Sir4 subunits . The Sir2 subunit possesses an unusual NAD-dependent deacetylase activity that is required for silencing at each of the above loci . Recent studies have shown that silent chromatin domains are assembled in a step-wise manner involving sequential cycles of deacetylation and SIR complex binding . Sir2-dependent deacetylation is specifically required for the spreading of the complex to regions beyond nucleation sites but not for its initial binding to DNA at the mating type loci and telomeres . A distinct Sir2 complex called RENT is required for silencing at rDNA . In contrast to telomeres and the mating type loci, Sir2 activity is not required for association of RENT with rDNA. Cytometry A, 2004 Jun, 59(2), 246 - 53 Single-cell analysis of yeast, mammalian cells, and fungal spores with a microfluidic pressure-driven chip-based system; Palkova Z et al.; BACKGROUND: Cytomics aims at understanding the function of cellular systems by analysis of single cells . Recently, there has been a growing interest in single cell measurements being performed in microfluidic systems . These systems promise to integrate staining, measurement, and analysis in a single system . One important aspect is the limitation of allowable cell sizes due to microfluidic channel dimensions . Here we want to demonstrate the broad applicability of microfluidic chip technology for the analysis of many different cell types . METHODS: We have developed a microfluidic chip and measurement system that allows flow cytometric analysis of fluorescently stained cells from different organisms . In this setup, the cells are moved by pressure-driven flow inside a network of microfluidic channels and are analyzed individually by fluorescence detection . RESULTS: We have successfully applied the system to develop a methodology to detect viable and dead cells in yeast cell populations . Also, we have measured short interfering RNA (siRNA) mediated silencing of protein expression in mammalian cells . In addition, we have characterized the infection state of Magnaportae grisea fungal spores . CONCLUSIONS: Results obtained with the microfluidic system demonstrate a broad applicability of microfluidic flow cytometry to measurement of various cell types . Mol Cell Biol, 2004 Jun, 24(12), 5197 - 208 Glucose and nitrogen regulate the switch from histone deacetylation to acetylation for expression of early meiosis-specific genes in budding yeast; Pnueli L et al.; In eukaryotes, the switch between alternative developmental pathways is mainly attributed to a switch in transcriptional programs . A major mode in this switch is the transition between histone deacetylation and acetylation . In budding yeast, early meiosis-specific genes (EMGs) are repressed in the mitotic cell cycle by active deacetylation of their histones . Transcriptional activation of these genes in response to the meiotic signals (i.e., glucose and nitrogen depletion) requires histone acetylation . Here we follow how this regulated switch is accomplished, demonstrating the existence of two parallel mechanisms . (i) We demonstrate that depletion of glucose and nitrogen leads to a transient replacement of the histone deacetylase (HDAC) complex on the promoters of EMG by the transcriptional activator Ime1 . The occupancy by either component occurs independently of the presence or absence of the other . Removal of the HDAC complex depends on the protein kinase Rim15, whose activity in the presence of nutrients is inhibited by protein kinase A phosphorylation . (ii) In the absence of glucose, HDAC loses its ability to repress transcription, even if this repression complex is directly bound to a promoter . We show that this relief of repression depends on Ime1, as well as on the kinase activity of Rim11, a glycogen synthase kinase 3beta homolog that phosphorylates Ime1 . We further show that the glucose signal is transmitted through Rim11 . In cells expressing the constitutive active rim11-3SA allele, HDAC repression in glucose medium is impaired. Mol Biol Cell, 2004 Aug, 15(8), 3567 - 79 Epub 2004 May 28. Essential role for the myotubularin-related phosphatase Ymr1p and the synaptojanin-like phosphatases Sjl2p and Sjl3p in regulation of phosphatidylinositol 3-phosphate in yeast; Parrish WR et al.; The requirement of Vps34p, the sole phosphatidylinositol (PI) 3-kinase in Saccharomyces cerevisiae, for protein sorting to the vacuole in yeast has exemplified the essential role for phosphoinositides, phosphorylated derivatives of PI, in membrane trafficking . To better understand mechanisms that regulate PI 3-phosphate {PI(3)P}-mediated signaling, the role of the yeast myotubularin-related PI(3)P phosphatase Ymr1p was investigated . We found that Ymr1p and the synaptojanin-like phosphatase Sjl3p function as key regulators of the localization and levels of PI(3)P . Our data indicated that the ymr1Delta sjl3Delta double mutant aberrantly accumulated PI(3)P and demonstrated a steady-state redistribution of this lipid that leads to enrichment on the vacuolar membrane . This resulted in vacuole protein sorting defects, vacuolar fragmentation, and the misregulation of PI(3)P-specific effectors . Triple deletion of YMR1, SJL2, and SJL3 was lethal, suggesting an essential requirement for phosphatase-mediated PI(3)P regulation . Consistent with this, growth was restored to a ymr1Delta sjl2Delta sjl3Delta triple mutant by a PI(3)P-targeted Sac1p domain chimera (GFP-Sac1DeltaC-FYVE(EEA1)) that returned PI(3)P to levels comparable with wild-type cells . Together, this study demonstrated that Ymr1p, a myotubularin phosphatase family member, functions in the control of PI(3)P-dependent signaling and the maintenance of endosomal system integrity . In addition, this work defined an essential overlapping role for lipid phosphatases in the regulation of 3' phosphoinositides in yeast. Mol Biol Cell, 2004 Aug, 15(8), 3841 - 62 Epub 2004 May 28. Integrative analysis of cell cycle control in budding yeast; Chen KC et al.; The adaptive responses of a living cell to internal and external signals are controlled by networks of proteins whose interactions are so complex that the functional integration of the network cannot be comprehended by intuitive reasoning alone . Mathematical modeling, based on biochemical rate equations, provides a rigorous and reliable tool for unraveling the complexities of molecular regulatory networks . The budding yeast cell cycle is a challenging test case for this approach, because the control system is known in exquisite detail and its function is constrained by the phenotypic properties of >100 genetically engineered strains . We show that a mathematical model built on a consensus picture of this control system is largely successful in explaining the phenotypes of mutants described so far . A few inconsistencies between the model and experiments indicate aspects of the mechanism that require revision . In addition, the model allows one to frame and critique hypotheses about how the division cycle is regulated in wild-type and mutant cells, to predict the phenotypes of new mutant combinations, and to estimate the effective values of biochemical rate constants that are difficult to measure directly in vivo. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Sep, 19(5), 437 - 9 {Construction,identification and amplification of a yeast two-hybrid random cycle peptide library}; Xu X et al.; AIM: To construct a cycle peptide library composed of 16 random amino acids with yeast two-hybrid system . METHODS: Random oligonucleotides encoding 16-mer peptides were designed and synthesized artificially, and then were amplified by PCR . The amplified products were digested with BamH I and EcoR I and cloned into yeast expression plasmid pGADT(7) GH to construct the cycle library plasmids pGADT(7) GH-RP Then the number of different recombinants and the randomness of the library were tested, and the cycle peptide library plasmids were amplified, extracted and purified . RESULTS: A random cycle peptide library with 1.28 x10(7) different recombinant clones was obtained . No significant difference was found between amino acid distribution in the cycle peptide library and the expected frequency . CONCLUSION: The random cycle peptide library has been successfully constructed . And a lot of cycle peptide library plasmids with high purity were obtained. Inflamm Bowel Dis, 2004 Mar, 10(2), 97 - 105 Immune sensitization to yeast antigens in ASCA-positive patients with Crohn's disease; Konrad A et al.; BACKGROUND: Alimentary antigens may play a role in the perpetuation of inflammation in Crohn's disease (CD) . Yeast antigens are widespread components of food . A proportion of CD patients develop antibodies against the yeast Saccharomyces cerevisiae (ASCA), but little is known about the cellular immune reactivity against food antigens in antibody-positive and -negative patients . METHODS: Lymphocytes from patients with CD, ulcerative colitis, and healthy controls were tested for their proliferative response after stimulation with the yeast antigen mannan and ovalbumin . The cellular phenotypes and activation markers were analyzed via FACS . Cytokine concentrations and antibody titers were determined by ELISA . RESULTS: Only lymphocytes of ASCA-positive patients with CD proliferated after stimulation with mannan . These lymphocytes expressed increased activation markers (CD25, CD69) . Activation of T cells was mediated by antigen-presenting cells and was associated with increased tumor necrosis factor-alpha (TNF-alpha) levels . The immune reactivity to ovalbumin was predominantly found in CD patients . It was weaker compared with mannan, independent of ASCA status, and also present in healthy controls . CONCLUSIONS: A disturbed humoral and cellular response to the yeast antigen mannan is specifically seen in a subgroup of CD patients . This phenomenon may be due to a loss of tolerance toward yeast and is possibly genetically determined. EMBO J, 2004 Jun 16, 23(12), 2392 - 401 Epub 2004 May 27. Coupling between snoRNP assembly and 3' processing controls box C/D snoRNA biosynthesis in yeast; Morlando M et al.; RNA polymerase II transcribes genes encoding proteins and a large number of small stable RNAs . While pre-mRNA 3'-end formation requires a machinery ensuring tight coupling between cleavage and polyadenylation, small RNAs utilize polyadenylation-independent pathways . In yeast, specific factors required for snRNA and snoRNA 3'-end formation were characterized as components of the APT complex that is associated with the core complex of the cleavage/polyadenylation machinery (core-CPF) . Other essential factors were identified as independent components: Nrd1p, Nab3p and Sen1p . Here we report that mutations in the conserved box D of snoRNAs and in the snoRNP-specific factor Nop1p interfere with transcription and 3'-end formation of box C/D snoRNAs . We demonstrate that Nop1p is associated with box C/D snoRNA genes and that it interacts with APT components . These data suggest a mechanism of quality control in which efficient transcription and 3'-end formation occur only when nascent snoRNAs are successfully assembled into functional particles. EMBO J, 2004 Jun 16, 23(12), 2336 - 47 Epub 2004 May 27. The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast; Leger-Silvestre I et al.; We have conducted a genetic screen in order to identify ribosomal proteins of Saccharomyces cerevisiae involved in nuclear export of the small subunit precursors . This has led us to distinguish Rps15p as a protein dispensable for maturation of the pre-40S particles, but whose assembly into the pre-ribosomes is a prerequisite to their nuclear exit . Upon depletion of Rps15p, 20S pre-rRNA is released from the nucleolus and retained in the nucleus, without alteration of the pre-rRNA early cleavages . In contrast, Rps18p, which contacts Rps15p in the small subunit, is required upstream for pre-rRNA processing at site A2 . Most pre-40S specific factors are correctly associated with the intermediate particles accumulating in the nucleus upon Rps15p depletion, except the late-binding proteins Tsr1p and Rio2p . Here we show that these two proteins are dispensable for nuclear exit; instead, they participate in 20S pre-rRNA processing in the cytoplasm . We conclude that, during the final maturation steps in the nucleus, incorporation of the ribosomal protein Rps15p is specifically required to render the pre-40S particles competent for translocation to the cytoplasm. Genetics, 2004 May, 167(1), 51 - 63 In vivo analysis of synaptonemal complex formation during yeast meiosis; White EJ et al.; During meiotic prophase a synaptonemal complex (SC) forms between each pair of homologous chromosomes and is believed to be involved in regulating recombination . Studies on SCs usually destroy nuclear architecture, making it impossible to examine the relationship of these structures to the rest of the nucleus . In Saccharomyces cerevisiae the meiosis-specific Zip1 protein is found throughout the entire length of each SC . To analyze the formation and structure of SCs in living cells, a functional ZIP1::GFP fusion was constructed and introduced into yeast . The ZIP1::GFP fusion produced fluorescent SCs and rescued the spore lethality phenotype of zip1 mutants . Optical sectioning and fluorescence deconvolution light microscopy revealed that, at zygotene, SC assembly was initiated at foci that appeared uniformly distributed throughout the nuclear volume . At early pachytene, the full-length SCs were more likely to be localized to the nuclear periphery while at later stages the SCs appeared to redistribute throughout the nuclear volume . These results suggest that SCs undergo dramatic rearrangements during meiotic prophase and that pachytene can be divided into two morphologically distinct substages: pachytene A, when SCs are perinuclear, and pachytene B, when SCs are uniformly distributed throughout the nucleus . ZIP1::GFP also facilitated the enrichment of fluorescent SC and the identification of meiosis-specific proteins by MALDI-TOF mass spectroscopy. FEBS Lett, 2004 Jun 1, 567(1), 80 - 5 Yeast activator proteins and stress response: an overview; Rodrigues-Pousada CA et al.; Yeast, and especially Saccharomyces cerevisiae, are continuously exposed to rapid and drastic changes in their external milieu . Therefore, cells must maintain their homeostasis, which is achieved through a highly coordinated gene expression involving a plethora of transcription factors, each of them performing specific functions . Here, we discuss recent advances in our understanding of the function of the yeast activator protein family of eight basic-leucine zipper trans-activators that have been implicated in various forms of stress response. J Biomech, 2004 Jul, 37(7), 977 - 87 Numerical simulation of the mechanics of a yeast cell under high hydrostatic pressure; Hartmann C et al.; The mechanical effects of the compression of a yeast cell (Saccharomyces cerevisiae) under high hydrostatic pressure used for the processing of food and food ingredients are modelled and simulated with the finite-element method . The cell model consists of a cell wall, cytoplasm a lipid filled vacuole and the nucleus . Material parameters have been taken from literature or have been derived from thermodynamic relationships of water and lipids under high hydrostatic pressure . The model has been validated for a pressure load up to 250 MPa . Comparison of the volume reduction to in situ experimental observations reveals very good agreement . Dimensional analysis of the governing equations shows that transient pressure application in a high-pressure food process does not enhance structural inactivation (mechanical damage), unless pressure oscillation frequencies of 700 MHz are applied . The deformation of the cell under pressure deviates strongly from isotropic volume reduction . Especially, organelle membranes exhibit large effective strain values . Hydrostatic stress conditions are preserved in the interior part of the cell . A pressure load of 400 MPa, which is critical upon disruption of cell organelle membranes, generates an effective strain up to 80% . In the cell wall, the stress state is heterogeneous . Von-Mises stress reaches the critical value upon failure of the cell wall of 70+/-4 MPa at a pressure load between 415 and 460 MPa. J Mol Biol, 2004 Jun 11, 339(4), 681 - 93 Structural and functional characterization on the interaction of yeast TFIID subunit TAF1 with TATA-binding protein; Mal TK et al.; General transcription factor TFIID, consisting of TATA-binding protein (TBP) and TBP-associated factors (TAFs), plays a central role in both positive and negative regulation of transcription . The TAF N-terminal domain (TAND) of TAF1 has been shown to interact with TBP and to modulate the interaction of TBP with the TATA box, which is required for transcriptional initiation and activation of TATA-promoter operated genes . We have previously demonstrated that the Drosophila TAND region of TAF1 (residues 11-77) undergoes an induced folding from a largely unstructured state to a globular structure that occupies the DNA-binding surface of TBP thereby inhibiting the DNA-binding activity of TBP . In Saccharomyces cerevisiae, the TAND region of TAF1 displays marked differences in the primary structure relative to Drosophila TAF1 (11% identity) yet possesses transcriptional activity both in vivo and in vitro . Here we present structural and functional studies of yeast TAND1 and TAND2 regions (residues 10-37, and 46-71, respectively) . Our NMR data show that, in yeast, TAND1 contains two alpha-helices (residues 16-23, 30-36) and TAND2 forms a mini beta-sheet structure (residues 53-56, 61-64) . These TAND1 and TAND2 structured regions interact with the concave and convex sides of the saddle-like structure of TBP, respectively . Present NMR, mutagenesis and genetic data together elucidate that the minimal region (TAND1 core) required for GAL4-dependent transcriptional activation corresponds to the first helix region of TAND1, while the functional core region of TAND2, involved in direct interaction with TBP convex alpha-helix 2, overlaps with the mini beta-sheet region. Yeast, 2004 May, 21(7), 569 - 82 The trehalose pathway and intracellular glucose phosphates as modulators of potassium transport and general cation homeostasis in yeast; Mulet JM et al.; Trk, encoded by the partially redundant genes TRK1 and TRK2, is the major potassium transporter of Saccharomyces cerevisiae . This system is specific for potassium and rubidium but, by reducing the electrical membrane potential of the plasma membrane, Trk decreases the uptake of toxic cations such as lithium, calcium, aminoglycosides and polyamines, which are transported by other systems . Gain- and loss-of-function studies indicate that TPS1, a gene encoding trehalose-6-phosphate synthase and known to modulate glucose metabolism, activates Trk and reduces the sensitivity of yeast cells to many toxic cations . This effect is independent of known regulators of Trk, such as the Hal4 and Hal5 protein kinases and the protein phosphatase calcineurin . Mutants defective in isoform 2 of phosphoglucomutase (pgm2) and mutants defective in isoform 2 of hexokinase (hxk2) exhibit similar phenotypes of reduced Trk activity and increased sensitivity to toxic cations compared with tps1 mutants . In all cases Trk activity was positively correlated with levels of glucose phosphates (glc-1-P and glc-6-P) . These results indicate that Tps1, like Pgm2 and Hxk2, increases the levels of glucose phosphates and suggest that these metabolites, directly or indirectly, activate Trk . Cell, 2004 May 28, 117(5), 637 - 48 Yeast Ras regulates the complex that catalyzes the first step in GPI-anchor biosynthesis at the ER; Sobering AK et al.; The yeast ERI1 gene encodes a small ER-localized protein that associates in vivo with GTP bound Ras2 in an effector loop-dependent manner . We showed previously that loss of Eri1 function results in hyperactive Ras phenotypes . Here, we demonstrate that Eri1 is a component of the GPI-GlcNAc transferase (GPI-GnT) complex in the ER, which catalyzes transfer of GlcNAc from UDP-GlcNAc to an acceptor phosphatidylinositol, the first step in the production of GPI-anchors for cell surface proteins . We also show that GTP bound Ras2 associates with the GPI-GnT complex in vivo and inhibits its activity, indicating that yeast Ras uses the ER as a signaling platform from which to negatively regulate the GPI-GnT . We propose that diminished GPI-anchor protein production contributes to hyperactive Ras phenotypes. Cell, 2004 May 28, 117(5), 625 - 35 Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function; Jang HH et al.; Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function . We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones . The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes . Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes . This triggers a peroxidase-to-chaperone functional switch . These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells . The chaperone function of these proteins enhances yeast resistance to heat shock. Brief Funct Genomic Proteomic, 2004 Feb, 2(4), 298 - 307 Modelling the fission yeast cell cycle; Sveiczer A et al.; The molecular networks regulating basic physiological processes in a cell can be converted into mathematical equations (eg differential equations) and solved by a computer . The division cycle of eukaryotic cells is an important example of such a control system, and fission yeast is an excellent test organism for the computational modelling approach . The mathematical model is tested by simulating wild-type cells and many known cell cycle mutants . This paper describes an example where this approach is useful in understanding multiple rounds of DNA synthesis (endoreplication) in fission yeast cells that lack the main (B-type) mitotic cyclin, Cdc13 . It is proposed that the key physiological variable driving progression through the cell cycle during balanced growth and division is the mass/DNA ratio, rather than the mass/nucleus ratio. BMC Biol . 2004 May 26;2(1):11. Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations; Pavlov YI et al.; BACKGROUND: DNA polymerase epsilon (Pol epsilon) is essential for S-phase replication, DNA damage repair and checkpoint control in yeast . A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol epsilon active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain . Here we investigate the mechanism responsible for the mutator effect . RESULTS: Purified four-subunit Y831A Pol epsilon turns over more deoxynucleoside triphosphates to deoxynucleoside monophosphates than does wild-type Pol epsilon, suggesting altered coordination between the polymerase and exonuclease active sites . The pol2-Y831A mutation suppresses the mutator effect of the pol2-4 mutation in the exonuclease active site that abolishes proofreading by Pol epsilon, as measured in haploid strain with the pol2-Y831A,4 double mutation . Analysis of mutation rates in diploid strains reveals that the pol2-Y831A allele is recessive to pol2-4 . In addition, the mutation rates of strains with the pol2-4 mutation in combination with active site mutator mutations in Pol delta and Pol alpha suggest that Pol epsilon may proofread certain errors made by Pol alpha and Pol delta during replication in vivo . CONCLUSIONS: Our data suggest that Y831A replacement in Pol epsilon reduces replication fidelity and its participation in chromosomal replication, but without eliminating an additional function that is essential for viability . This suggests that other polymerases can substitute for certain functions of polymerase epsilon. J Biol Chem, 2004 Aug 6, 279(32), 33653 - 61 Epub 2004 May 25. A mitochondrial-vacuolar signaling pathway in yeast that affects iron and copper metabolism; Li L et al.; Mitochondria utilize iron, but the transporters that mediate mitochondrial iron uptake and efflux are largely unknown . Cells with a deletion in the vacuolar iron/manganese transporter Ccc1p are sensitive to high iron . Overexpression of MRS3 or MRS4 suppresses the high iron sensitivity of Deltaccc1 cells . MRS3 and MRS4 have recently been suggested to encode mitochondrial iron transporters . We demonstrate that deletion of MRS3 and MRS4 severely affects cellular and mitochondrial metal homeostasis, including a reduction in cytosolic and mitochondrial iron acquisition . We show that vacuolar iron transport is increased in Deltamrs3Deltamrs4 cells, resulting in decreased cytosolic iron and activation of the iron-sensing transcription factor Aft1p . Activation of Aft1p leads to increased expression of the high affinity iron transport system and increased iron uptake . Deletion of CCC1 in Deltamrs3Deltamrs4 cells restores cellular and mitochondrial iron homeostasis to near normal levels . Deltamrs3Deltamrs4 cells also show increased resistance to cobalt but decreased resistance to copper and cadmium . These phenotypes are also corrected by deletion of CCC1 in Deltamrs3Deltamrs4 cells . Decreased copper resistance in Deltamrs3Deltamrs4 cells results from activation of Aft1p by Ccc1p-mediated iron depletion, as deletion of CCC1 or AFT1 in Deltamrs3Deltamrs4 cells restores copper resistance . These results suggest that deletion of mitochondrial proteins can alter vacuolar metal homeostasis . The data also indicate that increased expression of the AFT1-regulated gene(s) can disrupt copper homeostasis. J Am Chem Soc, 2004 Jun 2, 126(21), 6751 - 8 Proton-mediated dynamics of the alkaline conformational transition of yeast iso-1-cytochrome c; Martinez RE et al.; The kinetics of the alkaline conformational transition of a Lys 73-->His variant of iso-1-cytochrome c have been investigated using pH jump stopped-flow methods to probe the nature of the ionizable "trigger" group for this conformational change . This mutation moves the pK(a) of the ligand replacing Met 80 from about 10.5 to approximately 6.6 and has unmasked two other ionizable groups, besides the ligand replacing Met 80, that modulate the kinetics of this process . The results are discussed in terms of the impact of ionization equilibria on protein folding mechanisms. Biochim Biophys Acta, 2004 May 24, 1689(1), 1 - 5 The prion-like protein Doppel fails to interact with itself, the prion protein and the 37 kDa/67 kDa laminin receptor in the yeast two-hybrid system; Hundt C et al.; The prion-like protein termed Doppel (Dpl) shows approx . 25% sequence identity with all known prion proteins (PrP) . We recently showed that the cellular PrP is dimeric under native conditions, a finding which was confirmed by the investigation of its crystal structure . Human PrP further interacts with its cellular receptor, the 37 kDa/67 kDa laminin receptor (LRP/LR) . Here we report that human Doppel fails to interact with (i) . itself, (ii) . the human 37 kDa/67 kDa LRP/LR, and (iii) . the human cellular prion protein (huPrP) in the yeast two-hybrid system . Our findings suggest that Dpl and PrP are not related or are only marginally related with respect to their ligand binding behaviour. Methods, 2004 Jul, 33(3), 252 - 9 Analysis of chromatin in fission yeast; Pidoux A et al.; The use of fission yeast as a model system for studies of chromosome biology has contributed to several key advances in the last few years . The structure of its large complex centromeres and composition of its transcriptionally silent heterochromatin resemble those of metazoa . The application of chromatin immunoprecipitation to fission yeast has been instrumental in these advances and we describe an improved version of this technique in detail . In addition, we describe several other techniques, which are useful in the analysis of chromatin in fission yeast. Trends Cell Biol, 1996 Jan, 6(1), 29 - 33 Telomere functions: lessons from yeast; Zakian VA; Telomeres are specialized DNA protein structures that form the ends of eukaryotic chromosomes . In yeast, loss of even a single telomere causes a prolonged, but transitory, cell-cycle arrest . During this arrest, many broken chromosomes acquire a new telomere by one of three pathways, although at the cost of a partial loss of heterozygosity . In addition, a substantial fraction of the chromosomes lacking a telomere is lost, which generates an aneuploid cell . In these cases, the broken chromosome is usually replicated and segregated for ten or more cell divisions in unstable form . Extrapolation from yeast suggests that the gradual loss of telomeric DNA that accompanies ageing in humans may initiate the kinds of chromosomal rearrangements and genetic changes that are associated with tumorigenesis. Trends Cell Biol, 1996 Nov, 6(11), 434 - 41 Selection of polarized growth sites in yeast; Roemer T et al.; The budding yeast Saccharomyces cerevisiae responds to intracellular and extracellular cues to direct cell growth . Genetic analysis has revealed many components that participate in this process and has provided insight into the mechanisms by which these proteins function . Several of these components, such as the septins, pheromone receptors and GTPase proteins, have homologues in multicellular eukaryotes, suggesting that many aspects of polarized cell growth may be conserved throughout evolution . This review discusses our current understanding of the molecular mechanisms of growth-site selection during the different stages of the yeast life cycle. Trends Cell Biol, 1996 Feb, 6(2), 62 - 6 rum1: a CDK inhibitor regulating G1 progression in fission yeast; Labib K et al.; In all