Yeasts

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 eukaryotes, entry into mitosis from G2 phase is initiated by a complex of the cdc2 kinase and a B-type cyclin . It has now been shown that, in fission yeast, B-type cyclins also activate cdc2 in G1, thus governing cell-cycle commitment, as well as the onset of S phase . In this article, Karim Labib and Sergio Moreno review the evidence that ruml inhibits the kinase activity of cdc2 associated with B-type cyclins and is an important regulator o f G1 progression in fission yeast.

Trends Cell Biol, 1996 Jul, 6(7), 260 - 6
Phospholipids: synthesis, sorting, subcellular traffic - the yeast approach; Kohlwein SD et al.; Most of the enzymes and genes required for lipid biosynthesis and degradation in the budding yeast Saccharomyces cerevisiae have now been identified and the global mechanisms that regulate their activity are being established . Synthesis of phospholipids is restricted to specific subcellular compartments, and the lipids migrate from their site of formation to their final destination . In addition to synthesis, remodelling and degradation of phospholipids controls the content of the lipid portion of cellular membranes, while highly specific phospholipases catalyse the release of lipid-based second messengers . In this review, we describe the current understanding of the organization and regulation of phospholipid metabolism in yeast, and discuss the mechanisms that have been proposed for intracellular lipid transport.

Biochemistry, 2004 Jun 1, 43(21), 6745 - 50
LC-MS/MS identification and yeast polymerase eta bypass of a novel gamma-irradiation-induced intrastrand cross-link lesion G{8-5}C; Gu C et al.; Reactive oxygen species can give rise to intrastrand cross-link lesions, where two neighboring nucleobases are covalently bonded . Here, we employed LC-MS/MS and demonstrated for the first time that gamma irradiation of a synthetic duplex oligodeoxyribonucleotide can give rise to an intrastrand cross-link lesion G{8-5}C, where the C8 carbon atom of guanine and the C5 carbon atom of its 3'-neighboring cytosine are covalently bonded . We also carried out in vitro replication studies of a substrate containing a site-specifically incorporated G{8-5}C, and our results showed that yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion . Steady-state kinetic analyses for nucleotide incorporation by pol eta showed that the 3'-cytosine moiety of the cross-link did not significantly affect either the efficiency or the fidelity of nucleotide incorporation . The 5' guanine portion of the cross-link lesion, however, markedly reduced both the efficiency and the fidelity of nucleotide incorporation; the insertion of dGMP or dAMP was slightly favored over the insertion of the correct nucleotide, dCMP, which was in turn favored over the insertion of dTMP . The above results support that the oxidative cross-link lesion, if not repaired, can be mutagenic.

J Biol Chem, 2004 Jul 23, 279(30), 31190 - 6 Epub 2004 May 21.
A yeast strain lacking lipid particles bears a defect in ergosterol formation; Sorger D et al.; Lipid particles of the yeast Saccharomyces cerevisiae are storage compartments for triacylglycerols (TAG) and steryl esters (STE) . Four gene products, namely the TAG synthases Dga1p and Lro1p, and the STE synthases Are1p and Are2p contribute to storage lipid synthesis . A yeast strain lacking the four respective genes is devoid of lipid particles thus providing a valuable tool to study the physiological role of storage lipids and lipid particles . Using a dga1lro1are1are2 quadruple mutant transformed with plasmids bearing inducible DGA1, LRO1, or ARE2 we demonstrate that TAG synthesis contributes more efficiently to lipid particle proliferation than synthesis of STE . Moreover, we show that proteins typically located to lipid particles in wild type such as Erg1p, Erg6p, Erg7p, and Ayr1p are refined to microsomal fractions of the dga1lro1are1are2 quadruple mutant . This result confirms the close relationship between lipid particles and endoplasmic reticulum . Most interestingly, the amount of the squalene epoxidase Erg1p, which is dually located in lipid particles and endoplasmic reticulum of wild type, is decreased in the quadruple mutant, whereas amounts of other lipid particle proteins tested were not reduced . This decrease is not caused by down-regulation of ERG1 transcription but by the low stability of Erg1p in the quadruple mutant . Because a similar effect was also observed in are1are2 mutants this finding can be mainly attributed to the lack of STE . The quadruple mutant, however, was more sensitive to terbinafine, an inhibitor of Erg1p, than the are1are2 strain suggesting that the presence of TAG and/or intact lipid particles has an additional protective effect . In a strain lacking the two STE synthases, Are1p and Are2p, incorporation of ergosterol into the plasma membrane was reduced, although the total cellular amount of free ergosterol was higher in the mutant than in wild type . Thus, an esterification/deacylation mechanism appears to contribute to the supply of ergosterol to the plasma membrane.

Methods Mol Biol, 2004, 270, 403 - 22
Yeast two-hybrid assay for studying protein-protein interactions; Osman A; Protein-protein interactions occur in a wide variety of biological processes and essentially control the cell fate from division to death . Today, the identification of proteins that interact with a protein of interest is a focus of intensive research and is an essential element of the rapidly growing field of proteomics . Yeast two-hybrid assays represent a versatile tool to study protein interactions in vivo . GAL4-based assay, for example, uses yeast transcription factor GAL4 for detection of protein interactions by transcriptional activation . Some transcription factors (such as GAL4) possess a characteristic phenomenon that the transactivation function can be restored when the factor's DNA-binding domain (DBD) and its transcription-activation domain (AD) are brought together by two interacting, heterologous proteins . GAL4-yeast two-hybrid assay uses two expression vectors, one uses GAL4-DBD and the other uses GAL4-AD . DNA sequences encoding the two proteins of interest (or a protein and a complementary DNA library) can be cloned in the GAL4-DBD and GAL4-AD vectors to form the bait and the target of the interaction trap, respectively . A selection of host cells with different reporter genes and different growth selection markers provides a means to detect and confirm protein-protein interactions and highlight the flexibility of these assays to fit different applications . This chapter presents an outline for the GAL4-based yeast two-hybrid system with a detailed description of the vectors, host cells, and methods for detection and verifying protein interactions.

Biotechniques, 2004 May, 36(5), 872 - 6
Quantitative beta-galactosidase assay suitable for high-throughput applications in the yeast two-hybrid system; Mockli N et al.; Measurement of beta-galactosidase (beta-gal) activity is an important step in every yeast two-hybrid assay, yet many commonly used methods have distinct disadvantages, such as being only qualitative, time-consuming, and cumbersome when processing large numbers of samples . To overcome these drawbacks, we have implemented a novel technique, termed pellet X-gal assay, that allows simultaneous quantitative measurements from large numbers of samples with a minimum of hands-on time . The method was tested using five different, previously described protein-protein interactions and compared to two standard methods, the colony filter lift and the liquid ONPG assay . Our assay allows accurate quantitative measurements of protein-protein interactions and covers a greater dynamic range than the classic ONPG assay . The novel assay is robust and requires very little handling, making it suitable for applications in which several hundreds of individual protein interaction pairs need to be measured simultaneously.

Curr Genet, 2004 Aug, 46(2), 72 - 81 Epub 2004 May 19.
The yeast phosphotyrosyl phosphatase activator protein, yPtpa1/Rrd1, interacts with Sit4 phosphatase to mediate resistance to 4-nitroquinoline-1-oxide and UVA; Douville J et al.; We previously reported the isolation of mutants hypersensitive to the genotoxic agent 4-nitroquinoline-1-oxide, a potent inducer of oxidative stress . One of the mutants was defective in a gene designated yPTPA1, encoding a protein related to the human phosphotyrosyl phosphatase activator hPTPA, which is believed to play a role in activating the serine/threonine phosphatase PP2A . Yeast yptpa1Delta mutants are also sensitive to the UVA component of sunlight known to produce reactive oxygen species, suggesting a role for yPtpa1 in oxidative stress response . We now report the characterization of another 4-nitroquinoline-1-oxide-sensitive mutant, EBY20 . We show that this mutant is defective in the SIT4 gene encoding a catalytic subunit of the PP2A phosphatases and that sit4Delta mutants exhibit hypersensitivity to 4-nitroquinoline-1-oxide and UVA, but not to UVC at 254 nm . Like the yptpa1Delta mutants, sit4Delta mutants are also defective in the repair of 4-nitroquinoline-1-oxide-induced DNA lesions . Genetic analysis revealed that both yPtpa1 and Sit4 function in the same pathway to protect cells against the lethal effects of 4-nitroquinoline-1-oxide and UVA . Moreover, we demonstrate that yPtpa1-affinity columns specifically retain Sit4, confirming a previous report that these two proteins indeed belong to a complex . Cellular localization studies using GFP-tagged proteins reveals that yPtpa1 is localized to the cytoplasm and the nucleus, while the Sit4 protein shows an intense staining spot in the cytoplasm and diffused staining in this organelle . We suggest that the yPtpa1-Sit4 complex may participate in a novel mechanism that mediates repair of oxidative DNA damage caused by 4-nitroquinoline-1-oxide and UVA.

Mol Cell, 2004 May 21, 14(4), 515 - 22
Reciprocal association of the budding yeast ATM-related proteins Tel1 and Mec1 with telomeres in vivo; Takata H et al.; The phosphoinositide (PI)-3-kinase-related kinase (PIKK) family proteins Tel1p and Mec1p have been implicated in the telomere integrity of Saccharomyces cerevisiae . However, the mechanism of PIKK-mediated telomere length control remains unclear . Here, we show that Tel1p and Mec1p are recruited to the telomeres at specific times in the cell cycle in a mutually exclusive manner . In particular, Mec1p interacts with the telomeres during late S phase and is associated preferentially with shortened telomeres . We propose a model in which telomere integrity is maintained by the reciprocal association of PIKKs, and Mec1p acts as a sensor for structural abnormalities in the telomeres . Our study suggests a mechanistic similarity between telomere length regulation and DNA double-strand break repair, both of which are achieved by the direct association of PIKKs.

Proc Natl Acad Sci U S A, 2004 May 25, 101(21), 8114 - 9 Epub 2004 May 17.
The structure of a yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1; Xie K et al.; Activation-induced deaminase (AID) uses base deamination for class-switch recombination and somatic hypermutation and is related to the mammalian RNA-editing enzyme apolipoprotein B editing catalytic subunit 1 (APOBEC-1) . CDD1 is a yeast ortholog of APOBEC-1 that exhibits cytidine deaminase and RNA-editing activity . Here, we present the crystal structure of CDD1 at 2.0-A resolution and its use in comparative modeling of APOBEC-1 and AID . The models explain dimerization and the need for trans-acting loops that contribute to active site formation . Substrate selectivity appears to be regulated by a central active site "flap" whose size and flexibility accommodate large substrates in contrast to deaminases of pyrimidine metabolism that bind only small nucleosides or free bases . Most importantly, the results suggested both AID and APOBEC-1 are equally likely to bind single-stranded DNA or RNA, which has implications for the identification of natural AID targets.

Proc Natl Acad Sci U S A, 2004 May 25, 101(21), 7862 - 7 Epub 2004 May 17.
Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells; Mauzeroll J et al.; The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy . The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV-visible) behavior . By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution, the export of the conjugate from the cells with time could be measured . Similar experiments were performed on immobilized yeast cell aggregates stressed by a menadione solution . From the export of the menadione-glutathione conjugate detected at a 1-microm-diameter electrode situated 10 microm from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted . Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster.

FEBS Lett, 2004 May 21, 566(1-3), 100 - 4
Expression of Bax in yeast affects not only the mitochondria but also vacuolar integrity and intracellular protein traffic; Dimitrova I et al.; Bax-induced lethality in yeast is accompanied by morphological changes in mitochondria, giving rise to a reduced number of swollen tubules . Although these changes are completely abolished upon coexpression of the Bax inhibitor, Bcl-2, coexpression of Bax with Bax inhibiting-glutathione S-transferase (BI-GST) leads to aggregation, but not fusion of the mitochondria . In addition, Bax affects the integrity of yeast vacuoles, resulting in the disintegration and eventual loss of the organelles, and the disruption of intracellular protein traffic . While Bcl-2 coexpression only partially corrects this phenotype, coexpression of BI-GST fully restores the organelles, indicating a different mode of protection exerted by Bcl-2 and BI-GST.

FEBS Lett, 2004 May 21, 566(1-3), 77 - 82
Fission yeast Skp1 is required for spindle morphology and nuclear membrane segregation at anaphase; Lehmann A et al.; Skp1 is a core component of the Skp1-Cullin-1-F-box ubiquitin ligase . Here, we show a novel role for fission yeast Skp1 in mitotic progression . Temperature-sensitive skp1-A7 mutants enter mitosis, but fail to execute anaphase . Time-lapse imaging shows that spindles in this mutant form intranuclear arch-like structures, which eventually collapse abruptly . The two spindle poles are also seen to move backward to the cell centre rather than towards the cell ends . These abnormal phenotypes appear to stem from defects in nuclear membrane segregation . Our results show that Skp1 is required for coordinated structural alterations of mitotic spindles and nuclear membranes.

Genes Cells, 2004 May, 9(5), 399 - 418
The C-terminus of Bfa1p in budding yeast is essential to induce mitotic arrest in response to diverse checkpoint-activating signals; Kim J et al.; During mitosis, genomic integrity is maintained by the proper coordination of anaphase entry and mitotic exit through mitotic checkpoints . In budding yeast, exit from mitosis is triggered by the activation of the small GTPase Tem1p . Bfa1p in association with Bub2p negatively regulates Tem1p in response to spindle damage, spindle misorientation, and DNA damage, resulting in cell cycle arrest . To delineate the Bfa1p domains that respond to distinct checkpoint signals, we constructed 13 Bfa1 deletion mutants . The C-terminal 184 amino acids of Bfa1p (Bfa1-D8(391-574)) contained the entire capacity of Bfa1p to generate mitotic arrest in response to spindle damage, spindle misorientation, and DNA damage . This domain was also enough to interact with the mitotic exit network proteins Tem1p, Bub2p, and Cdc5p, and to localize to the spindle pole body (SPB) . Over-expression of Bfa1-D8(391-574) induced late anaphase arrest as efficient as the full-length Bfa1p in a Bub2p-dependent manner . In contrast, the N-terminal portion of Bfa1p (Bfa1-D16(1-376)) could not localize to SPB and did not block mitotic exit in response to diverse checkpoint signals . Bfa1-D16(1-376) interacted with Tem1p but not with Bub2p and its over-expression partially arrested cells in mitosis in the absence of Bub2p . By random mutagenesis of Bfa1-D8(391-574) with hydroxylamine, we isolated a point mutant of D8, D8(E438K), which interacts with both Tem1p and Bub2p but cannot respond to checkpoint signals . This mutant also showed reduced efficiency in the localization to SPB . Taken together, our study demonstrated that various checkpoint signals are transmitted to the C-terminal domain of Bfa1 (Bfa1-D8(391-574)) and that Bfa1p localization to SPB is necessary but not sufficient to regulate mitotic exit in response to various checkpoint signals.

Genes Cells, 2004 May, 9(5), 367 - 82
Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpoint; Lehmann A et al.; Skp1 is a central component of the E3 ubiquitin ligase SCF (Skp1-Cullin-1-F-box) . It forms an adapter bridge between Cullin-1 and the substrate-determining component, the F-box protein . In order to establish the role of Skp1, a temperature sensitive (ts) screen was carried out using mutagenic PCR (polymerase chain reaction) and 9 independent ts mutants were isolated . Mapping the mutated residues on the 3-D structure of human Skp1 suggested that the mutants would be compromised in binding to F-box proteins but not Cullin-1 (Pcu1) . In order to assess the binding properties of ts Skp1, 12 F-box proteins and Pcu1 were epitope-tagged, and co-immunoprecipitation performed . This systematic analysis showed that ts Skp1 retains binding to Pcu1 . However, binding to three specific F-box proteins, essential Pof1, Pof3 involved in maintaining genome integrity, and nonessential Pof10, was reduced . skp1ts cells exhibit a G2 cell cycle delay, which is attributable to activation of the DNA damage checkpoint . Intriguingly, contrary to pof3 mutants, in which this checkpoint is required for survival, checkpoint abrogation in skp1(ts) suppresses a G2 delay and furthermore almost rescues the ts phenotype . The activation mechanism of the DNA damage checkpoint therefore differs between pof3Delta and skp1(ts), implicating a novel role for Skp1 in the checkpoint-signalling cascade.

Mol Biol Cell, 2004 Jul, 15(7), 3345 - 56 Epub 2004 May 14.
Disruption of astral microtubule contact with the cell cortex activates a Bub1, Bub3, and Mad3-dependent checkpoint in fission yeast; Tournier S et al.; In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division . This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring . In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton . In this article, we show that addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B . Moreover, we find that whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed . By analyzing the timing of kinetochore separation, we find that this anaphase delay requires the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins . In agreement with this, we find that Bub1 remains associated with kinetochores when spindles are mispositioned . These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins . We propose that this checkpoint ensures spindles are properly oriented before anaphase takes place.

J Biol Chem, 2004 Jul 30, 279(31), 32093 - 9 Epub 2004 May 15.
Effect of pH on the stability and structure of yeast hexokinase A . Acidic amino acid residues in the cleft region are critical for the opening and the closing of the structure; Kumar DP et al.; pH and salts have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions . In this work, calorimetry, CD, and fluorescence studies have been carried out to understand the pH-dependent conformational changes of the two-domain protein yeast hexokinase A . In conjunction with the crystal structural data available, the present results have enabled the complete characterization and analysis of the pH-dependent conformational changes of the enzyme that have strong implications in understanding its structure-function relationship . The calorimetric profiles show a single thermal transition in the acidic pH range, whereas two independent transitions were observed in the alkaline pH range, suggesting the structural merger of the domains at the acidic pH . Comparison of the thermal transitions at pH 8.5 studied by different techniques suggests that the first transition corresponds to the smaller domain, and the second transition corresponds to the larger domain . The acid-denatured state of hexokinase A has high secondary structure content with little or no tertiary interactions and binds to the hydrophobic dye 8-anilinonaphthalene-1-sulfonic acid, suggesting that it is a molten globule-like state, whereas the alkali-denatured state is less structured than the acid-denatured state but more structured than the urea-denatured state, suggestive of a premolten globule-like state . Structural analysis using the published hexokinase B structure as well as the hexokinase A structure with the revised amino acid sequence in conjunction with the results obtained by us suggests that the ionization state of the acidic residues at the active site could regulate domain movements that are responsible for the opening and the closure of the cleft between the two domains and in turn affect the structure and function of the enzyme.

Mol Cell Biol, 2004 Jun, 24(11), 4929 - 42
Molecular and genetic characterization of a Taf1p domain essential for yeast TFIID assembly; Singh MV et al.; Yeast Taf1p is an integral component of the multiprotein transcription factor TFIID . By using coimmunoprecipitation assays, coupled with a comprehensive set of deletion mutants encompassing the entire open reading frame of TAF1, we have discovered an essential role of a small portion of yeast Taf1p . This domain of Taf1p, termed region 4, consisting of amino acids 200 to 303, contributes critically to the assembly and stability of the 15-subunit TFIID holocomplex . Region 4 of Taf1p is mutationally sensitive, can assemble several Tafps into a partial TFIID complex, and interacts directly with Taf4p and Taf6p . Mutations in Taf1p-region 4 induce temperature-conditional growth of yeast cells . At the nonpermissive temperature these mutations have drastic effects on both TFIID integrity and mRNA synthesis . These data are consistent with the hypothesis that Taf1p subserves a critical scaffold function within the TFIID complex . The significance of these data with regard to TFIID structure and function is discussed.

Mol Cell Biol, 2004 Jun, 24(11), 4848 - 57
The yeast scaffold proteins Isu1p and Isu2p are required inside mitochondria for maturation of cytosolic Fe/S proteins; Gerber J et al.; Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol, and nucleus . Mitochondrial Fe/S proteins are matured by the iron-sulfur cluster (ISC) assembly machinery . Little is known about the formation of Fe/S proteins in the cytosol and nucleus . A function of mitochondria in cytosolic Fe/S protein maturation has been noted, but small amounts of some ISC components have been detected outside mitochondria . Here, we studied the highly conserved yeast proteins Isu1p and Isu2p, which provide a scaffold for Fe/S cluster synthesis . We asked whether the Isu proteins are needed for biosynthesis of cytosolic Fe/S clusters and in which subcellular compartment the Isu proteins are required . The Isu proteins were found to be essential for de novo biosynthesis of both mitochondrial and cytosolic Fe/S proteins . Several lines of evidence indicate that Isu1p and Isu2p have to be located inside mitochondria in order to perform their function in cytosolic Fe/S protein maturation . We were unable to mislocalize Isu1p to the cytosol due to the presence of multiple, independent mitochondrial targeting signals in this protein . Further, the bacterial homologue IscU and the human Isu proteins (partially) complemented the defects of yeast Isu protein-depleted cells in growth rate, Fe/S protein biogenesis, and iron homeostasis, yet only after targeting to mitochondria . Together, our data suggest that the Isu proteins need to be localized in mitochondria to fulfill their functional requirement in Fe/S protein maturation in the cytosol.

Mol Cell Biol, 2004 Jun, 24(11), 4801 - 9
Identification of a distinctive mutation spectrum associated with high levels of transcription in yeast; Lippert MJ et al.; High levels of transcription are associated with increased mutation rates in Saccharomyces cerevisiae, a phenomenon termed transcription-associated mutation (TAM) . To obtain insight into the mechanism of TAM, we obtained LYS2 forward mutation spectra under low- versus high-transcription conditions in which LYS2 was expressed from either the low-level pLYS2 promoter or the strong pGAL1-10 promoter, respectively . Because of the large size of the LYS2 locus, forward mutations first were mapped to specific LYS2 subregions, and then those mutations that occurred within a defined 736-bp target region were sequenced . In the low-transcription strain base substitutions comprised the majority (64%) of mutations, whereas short insertion-deletion mutations predominated (56%) in the high-transcription strain . Most notably, deletions of 2 nucleotides (nt) comprised 21% of the mutations in the high-transcription strain, and these events occurred predominantly at 5'-(G/C)AAA-3' sites . No -2 events were present in the low-transcription spectrum, thus identifying 2-nt deletions as a unique mutational signature for TAM.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 975 - 86
Systematics of the anamorphic basidiomycetous yeast genus Trichosporon Behrend with the description of five novel species: Trichosporon vadense, T . smithiae, T . dehoogii, T . scarabaeorum and T . gamsii; Middelhoven WJ et al.; Phylogenetic trees of the anamorphic basidiomycetous yeast genus Trichosporon Behrend, based on molecular sequence analysis of the internal transcribed spacer region and the D1/D2 region of the large subunit of ribosomal (26S) DNA, are presented . This study includes three novel species from soils, Trichosporon vadense sp . nov . (type strain, CBS 8901(T)), Trichosporon smithiae sp . nov . (type strain, CBS 8370(T)) and Trichosporon gamsii sp . nov . (type strain, CBS 8245(T)), one novel species from an insect, Trichosporon scarabaeorum sp . nov . (type strain, CBS 5601(T)) and one species of unknown origin, Trichosporon dehoogii sp . nov . (type strain, CBS 8686(T)) . The phylogenetic positions and physiological characteristics that distinguish the new taxa from related species, based partly on growth tests that are not traditionally used in yeast taxonomy (uric acid, ethylamine, L-4-hydroxyproline, tyramine and L-phenylalanine as sources of carbon and nitrogen, and polygalacturonate, quinate, 4-ethylphenol, phloroglucinol, 2,3-dihydroxybenzoate and orcinol as sole carbon sources), are discussed . Assimilation of L-rhamnose and erythritol and maximum growth temperature were also used to delineate species.

Res Microbiol, 2004 May, 155(4), 217 - 23
Capturing the adaptive mutation in yeast; Zeyl C; An accurate view of adaptive mutations is essential to evolutionary genetics, but their rarity makes them difficult to study . This can be partially overcome using the many tools of yeast genetics and the ability to study very large populations over many generations . Adaptation to laboratory environments has occurred primarily by chromosomal rearrangements, often involving retrotransposons and apparently selected for their effects on gene regulation . Estimated rates of adaptive mutation are on the order of 1 in 10(11) cell divisions . There remains great potential for the genomic study of variation within yeast species to contribute to our understanding of adaptive mutation.

Ai Zheng, 2004 May, 23(5), 508 - 11
{Screening hepatitis B virus X-interactive proteins by yeast two-hybrid system}; Li D et al.; BACKGROUND & OBJECTIVE: Hepatitis B virus-encoded X protein is a promiscuous transactivator and contributes to the development of hepatocellular carcinoma . Protein-protein interaction seems to be crucial for HBx transactivation . The aim of this study was to screen and identify the proteins which interact with hepatitis B virus (HBV) X protein by yeast two-hybrid system . METHODS: HBV X gene was amplified by polymerase chain reaction (PCR) . HBV X bait plasmid, named pAS2-1-X, was constructed by yeast-two hybridization system 3 and verified by sequencing . pAS2-1-X was transformed into the yeast AH109, and X-BD fusion protein expressed in the yeast cells was confirmed by Western blot analysis . Yeast cells cotransformed with pAS2-1-X and normal human liver cDNA library were cultured in selective SC/-trp-leu-his-ade medium . The second screening was performed with beta-gal activity detection . The false positive clones were eliminated by segregation analysis and mating experiment . The real positive clones were amplified, sequenced, and analyzed with bioinformatics . RESULTS: Bait plasmid pAS2-1-X was successfully constructed . The result of Western blot analysis confirmed that pAS2-1-X correctly expressed X-BD fusion protein in the transformed yeast AH109 . Ninety-seven clones grew in the selective SC/-trp-leu-his-ade medium; however, only one clone past through the beta-gal activity detection, segregation analysis, and mating experiment . The inserted cDNA fragment of positive clone showed high homology with Fis gene . CONCLUSION: Fis protein is a novel protein which can interact with X protein in vivo by yeast two-hybrid system.

Cell Mol Life Sci, 2004 May, 61(10), 1208 - 20
Characterization of the yeast tricalbins: membrane-bound multi-C2-domain proteins that form complexes involved in membrane trafficking; Creutz CE et al.; In a survey of yeast genomic sequences encoding calcium- and phospholipid-binding C2 domains, three homologous genes were identified that encode proteins that each have three C2 domains and an apparent transmembrane domain near the N terminus . The name tricalbins is suggested for these proteins, corresponding to the open reading frames YOR086c (TCB1), YNL087w (TCB2), and YML072c (TCB3) . An antiserum was raised against the C-terminal portion of tricalbin 2 and used on Western blots to demonstrate that the corresponding protein is expressed in yeast and appears as a high-molecular-weight band at 130 kDa with smaller fragments at 39 kDa and 46 kDa . A fusion protein consisting of full length tricalbin 2 fused to the green fluorescent protein was expressed in cells and found to traffic from the cell surface to intracellular vesicles near the vacuole . A two-hybrid interaction screen with the C-terminal portion of tricalbin 2 indicated that tricalbin 2 binds the C-terminal portions of tricalbins 1 and 3 suggesting that the tricalbins may form heterodimers in vivo . Tricalbin 2 also interacted with the activation domain of the pleiotropic drug resistance transcription factor Pdr1p . Combinatorial disruptions of the tricalbin genes revealed that tcb2 single mutants or tcb1, tcb3 double mutants have an altered vacuole morphology and are hypersensitive to cycloheximide . A screen for single-copy suppressors of the cycloheximide sensitivity of tricalbin mutants yielded RSP5, which encodes a C2-domain-containing, ubiquitin-conjugating ligase essential for receptor-mediated and fluid phase endocytosis . The results suggest that the tricalbins function as multimers in membrane-trafficking events and may provide insights into the roles of multi-C2-domain proteins, such as the synaptotagmins, in other organisms.

Proc Natl Acad Sci U S A, 2004 May 25, 101(21), 7999 - 8004 Epub 2004 May 12.
Methionine sulfoxide reductase regulation of yeast lifespan reveals reactive oxygen species-dependent and -independent components of aging; Koc A et al.; Aging is thought to be caused by the accumulation of damage, primarily from oxidative modifications of cellular components by reactive oxygen species (ROS) . Here we used yeast methionine sulfoxide reductases MsrA and MsrB to address this hypothesis . In the presence of oxygen, these antioxidants could increase yeast lifespan and did so independent of the lifespan extension offered by caloric restriction . However, under ROS-deficient, strictly anaerobic conditions, yeast lifespan was shorter, not affected by MsrA or MsrB, and further reduced by caloric restriction . In addition, we identified changes in the global gene expression associated with aging in yeast, and they did not include oxidative stress genes . Our findings suggest how the interplay between ROS, antioxidants, and efficiency of energy production regulates the lifespan . The data also suggest a model wherein factors implicated in aging (for example, ROS) may influence the lifespan yet not be the cause of aging.

Nucleic Acids Res, 2004 May 11, 32(8), 2578 - 85 Print 2004.
Functions of yeast helicase Ssl2p that are essential for viability are also involved in protection from the toxicity of adriamycin; Furuchi T et al.; We have found that, in the yeast Saccharomyces cerevisiae, overexpression of the DNA helicase Ssl2p confers resistance to adriamycin . Ssl2p is involved, as a subunit of the basic transcription factor TFIIH, in the initiation of transcription and in nucleotide-excision repair (NER), and this helicase is essential for the survival of yeast cells . An examination of the relationship between the known functions of Ssl2p and adriamycin resistance indicated that overexpression of Ssl2p caused little or no increase in the rate of RNA synthesis and in NER . The absence of any involvement of NER in adriamycin resistance was supported by the finding that yeast cells that overexpressed the mutant form of Ssl2p that lacked the carboxy-terminal region, which is necessary for NER, remained resistant to adriamycin . When we examined the effects of overexpression in yeast of other mutant forms of Ssl2p with various deletions, we found that, of the 843 amino acids of Ssl2p, the entire amino acid sequence from position 81 to position 750 was necessary for adriamycin resistance . This region is identical to the region of Ssl2p that is necessary for the survival of yeast cells . Although this region contains helicase motifs, the overexpression of other yeast helicases, such as Rad3 and Sgs1, had little or no effect on adriamycin resistance, indicating that a mere increase in the intracellular level of helicases does not result in adriamycin resistance . Our results suggest that the functions of Ssl2p that are essential for yeast survival are also required for protection against adriamycin toxicity.

Eur J Pharmacol, 2004 May 3, 491(2-3), 101 - 10
Characterization of phorbol esters activity on individual mammalian protein kinase C isoforms, using the yeast phenotypic assay; Saraiva L et al.; An alternative in vivo assay, based on growth inhibition of yeast expressing an individual mammalian protein kinase C (PKC) isoform (proportional to the degree of PKC activation), was used to characterize the activities of phorbol-12-myristate-13-acetate (PMA) and its analogues on classical (alpha and betaI), novel (delta and eta) and atypical (zeta) PKC isoforms . Effects of PMA, 4alpha-PMA, phorbol-12-myristate-13-acetate-4-O-methyl-ether (MPMA), phorbol-12-monomyristate (PMM), phorbol-12,13-diacetate (PDA), phorbol-13-monoacetate (PA), phorbol-12,13-dibutyrate (PDB), phorbol-12,13-didecanoate (PDD) and 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA), on growth of yeast expressing individual PKC isoforms was determined . PMA-induced growth inhibition on all isoforms tested (except on PKC-zeta) . PDD and PDB presented an efficacy similar to PMA; the other PMA-analogues presented lower efficacies . MPMA and 4alpha-PMA stimulated growth of yeast expressing classical PKCs and reduced the PMA-induced growth inhibition, effects similar to those exhibited by the PKC inhibitors chelerythrine and R-2,6-diamino-N-{{1-(1-oxotridecyl)-2-piperidinyl}methyl}-hexanamide dihydrochloride (NPC 15437) . This study reveals that phorbol esters differ on their potency to activate a given PKC isoform, and presents their isoform-selectivity . Furthermore, MPMA and 4alpha-PMA caused effects similar to those expected from PKC inhibition .

Oncol Rep, 2004 Jun, 11(6), 1281 - 6
Spectrum of p53 mutations in biopsies from breast cancer patients selected for preoperative chemotherapy analysed by the functional yeast assay to predict therapeutic response; Deissler H et al.; The most frequent alteration detected in breast cancer cells is an inactivation of the tumor protein p53 . Numerous studies revealed that p53 mutations are an independent prognostic indicator of a shorter period of overall and disease-free survival . Meta-analysis of these investigations clearly showed that prognostic significance cannot be achieved by indirect assessment of the p53 status by immunohistochemistry . Therefore, the tumor RNA-based functional p53 assay in yeast (functional assay of separated alleles in yeast, FASAY) appears to be an attractive option to generate clinically relevant information without the need of microdissection . We describe FASAY analyses of 50 biopsies taken before pre-operative anthracycline/taxane chemotherapy to evaluate the predictive value of p53 mutations for this common combination of substances . Wild-type p53, present in 22 samples, resulted in numerous white colonies with a low background of red colonies that was consistently below 8% . Biopsy samples containing mutated alleles gave rise to many red colonies accompanied with variable numbers of white colonies . With one single exception, all biopsies containing mutated p53 resulted in more than 8% red colonies . In 25 samples (53%), 23 single and 2 double mutations of the p53 gene were confirmed by sequencing of DNA from the yeast colonies . Six of the observed sequence insertions or deletions and 2 of the point mutations have not been reported previously . In accordance with an abolished or altered function as a transcriptional activator, most mutations affected the p53 DNA-binding domain, and one the tetramerization domain . Our results confirmed that the FASAY is sufficiently reliable to detect p53 mutations in breast tumor biopsies . In this pilot study with a limited number of patients to evaluate the predictive value of p53 mutations for an anthracycline/taxane combination therapy in the neoadjuvant setting, stable disease was observed more often in patients with wild-type p53 but statistical significance was not quite reached for this clear trend.

PLoS Biol . 2004 May;2(5):E128 . Epub 2004 May 11.
Ras and Gpa2 mediate one branch of a redundant glucose signaling pathway in yeast; Wang Y et al.; Addition of glucose to starved yeast cells elicits a dramatic restructuring of the transcriptional and metabolic state of the cell . While many components of the signaling network responsible for this response have been identified, a comprehensive view of this network is lacking . We have used global analysis of gene expression to assess the roles of the small GTP-binding proteins, Ras2 and Gpa2, in mediating the transcriptional response to glucose . We find that 90% of the transcriptional changes in the cell attendant on glucose addition are recapitulated by activation of Ras2 or Gpa2 . In addition, we find that protein kinase A (PKA) mediates all of the Ras2 and Gpa2 transcriptional effects . However, we also find that most of the transcriptional effects of glucose addition to wild-type cells are retained in strains containing a PKA unresponsive to changes in cAMP levels . Thus, most glucose-responsive genes are regulated redundantly by a Ras/PKA-dependent pathway and by one or more PKA-independent pathways . Computational analysis extracted RRPE/PAC as the major response element for Ras and glucose regulation and revealed additional response elements mediating glucose and Ras regulation . These studies provide a paradigm for extracting the topology of signal transduction pathways from expression data.

Cell, 2004 May 14, 117(4), 471 - 82
Cdc14 phosphatase induces rDNA condensation and resolves cohesin-independent cohesion during budding yeast anaphase; Sullivan M et al.; At anaphase onset, the protease separase triggers chromosome segregation by cleaving the chromosomal cohesin complex . Here, we show that cohesin destruction in metaphase is sufficient for segregation of much of the budding yeast genome, but not of the long arm of chromosome XII that contains the rDNA repeats . rDNA in metaphase, unlike most other sequences, remains in an undercondensed and topologically entangled state . Separase, concomitantly with cleaving cohesin, activates the phosphatase Cdc14 . We find that Cdc14 exerts two effects on rDNA, both mediated by the condensin complex . Lengthwise condensation of rDNA shortens the chromosome XII arm sufficiently for segregation . This condensation depends on the aurora B kinase complex . Independently of condensation, Cdc14 induces condensin-dependent resolution of cohesin-independent rDNA linkage . Cdc14-dependent sister chromatid resolution at the rDNA could introduce a temporal order to chromosome segregation.

Cell, 2004 May 14, 117(4), 441 - 53
SIR2 regulates recombination between different rDNA repeats, but not recombination within individual rRNA genes in yeast; Kobayashi T et al.; It is known that mutations in gene SIR2 increase and those in FOB1 decrease recombination within rDNA repeats as assayed by marker loss or extrachromosomal rDNA circle formation . SIR2-dependent chromatin structures have been thought to inhibit access and/or function of recombination machinery in rDNA . We measured the frequency of FOB1-dependent arrest of replication forks, consequent DNA double-strand breaks, and formation of DNA molecules with Holliday junction structures, and found no significant difference between sir2Delta and SIR2 strains . Formal genetic experiments measuring mitotic recombination rates within individual rRNA genes also showed no significant difference between these two strains . Instead, we found a significant decrease in the association of cohesin subunit Mcd1p (Scc1p) to rDNA in sir2Delta relative to SIR2 strains . From these and other experiments, we conclude that SIR2 prevents unequal sister-chromatid recombination, probably by forming special cohesin structures, without significant effects on recombinational events within individual rRNA genes.

Photochem Photobiol, 2004 Apr, 79(4), 349 - 55
Survival and recovery of yeast cells after simultaneous treatment of UV light radiation and heat; Kim JK et al.; Cell survival, synergistic interaction, liquid-holding recovery (LHR) kinetics and inactivation forms after the simultaneous treatment with UV light (254 nm) and various high temperatures were studied in diploid yeast cells Saccharomyces cerevisiae . The synergistic interaction was observed within a certain temperature range in which there was a temperature that maximizes the synergistic effect . The LHR study revealed that both the extent and the rate of recovery greatly decreased with the increase in exposure temperature . A quantitative approach describing the LHR process as a decrease in the effective radiation dose was used to estimate the probability of recovery per unit time and the irreversible component of damage . Using the experimental data obtained and the mathematical model described, it was shown that the irreversible component, i.e . the fraction of cells incapable of recovery, increased with the exposure temperature, whereas the recovery constant, i.e . the probability of recovery per unit time, was independent of the exposure temperature . The increase in the irreversible component was accompanied by an increase in cell death without postirradiation division . It is concluded based on this that the synergistic interaction of UV light radiation and hyperthermia in yeast cells is not related to the impairment of the recovery process itself and that it may be attributed to an increased yield of the irreversible damage.

J Leukoc Biol, 2004 Aug, 76(2), 451 - 61 Epub 2004 May 10.
Uptake of Abeta 1-40- and Abeta 1-42-coated yeast by microglial cells: a role for LRP; Laporte V et al.; Artificial diffuse and amyloid core of neuritic plaques {beta-amyloid peptide (Abeta) deposits} could be prepared using heat-killed yeast particles opsonized with Abeta 1-40 or Abeta 1-42 peptides . Interaction and fate of these artificial deposits with microglial cells could be followed using a method of staining that allows discrimination of adherent and internalized, heat-killed yeast particles . Using this system, it was possible to show that nonfibrillar or fibrillar (f)Abeta peptides, formed in solution upon heating (aggregates), could not impair the internalization of heat-killed yeast particles opsonized with fAbeta 1-40 or fAbeta 1-42 . This indicated that depending on their physical state, Abeta peptide(s) do not recognize the same receptors and probably do not follow the same internalization pathway . Using competitive ligands of class A scavenger receptors (SR-A) or low-density lipoprotein-related receptor protein (LRP), it has been shown that SR-A were not involved in the recognition of amyloid peptide deposits, whereas LRP specifically recognized deposits of fAbeta 1-42 (but not fAbeta 1-40) and mediated their phagocytosis.

J Biol Chem, 2004 Jul 2, 279(27), 27980 - 5 Epub 2004 May 10.
Specific residues of the GDP/GTP exchange factor Bud5p are involved in establishment of the cell type-specific budding pattern in yeast; Kang PJ et al.; Cells of the budding yeast undergo oriented cell division by choosing a specific site for growth depending on their cell type . Haploid a and alpha cells bud in an axial pattern whereas diploid a/alpha cells bud in a bipolar pattern . The Ras-like GTPase Rsr1p/Bud1p, its GDP-GTP exchange factor Bud5p, and its GTPase-activating protein Bud2p are essential for selecting the proper site for polarized growth in all cell types . Here we showed that specific residues at the N terminus and the C terminus of Bud5p were important for bipolar budding, while some residues were involved in both axial and bipolar budding . These bipolar-specific mutations of BUD5 disrupted proper localization of Bud5p in diploid a/alpha cells without affecting Bud5p localization in haploid alpha cells . In contrast, Bud5p expressed in the bud5 mutants defective in both budding patterns failed to localize in all cell types . Thus, these results identify specific residues of Bud5p that are likely to be involved in direct interaction with spatial landmarks, which recruit Bud5p to the proper bud site . Finally, we found a new start codon of BUD5, which extends the open reading frame to 210 bp upstream of the previously estimated start site, thus encoding a polypeptide of 608 amino acid residues . Bud5p with these additional N-terminal residues interacted with Bud8p, a potential bipolar landmark, suggesting that the N-terminal region is necessary for recognition of the spatial cues.

DNA Repair (Amst), 2004 Jun 3, 3(6), 593 - 601
The role of TDP1 from budding yeast in the repair of DNA damage; Liu C et al.; The TDP1 gene encodes a protein that can hydrolyze certain types of 3'-terminal phosphodiesters, but the relevance of these catalytic activities to gene function has not been previously tested . In this work we engineered a point mutation in TDP1 and present evidence that, as per design, it severely diminishes tyrosyl-DNA phosphodiesterase enzyme activity without affecting protein folding . The phenotypes of yeast strains that express this mutant show that the contribution of TDP1 to the repair of two kinds of damaged termini-induced, respectively, by camptothecin (CPT) and by bleomycin-strongly depends on enzyme activity . In routine assays of cell survival and growth the contribution of this activity is often overshadowed by other repair pathways . However, the value of TDP1 in the economy of the cell is highlighted by our discovery of several phenotypes that are evident even without deliberate inactivation of parallel pathways . These non-redundant mutant phenotypes include increased spontaneous mutation rate, transient accumulation of cells in a mid-anaphase checkpoint after exposure to camptothecin and, in cells that overexpress topoisomerase I (Top1), decreased survival of camptothecin-induced damage . The relationship between the role of TDP1 in Saccharomyces and its role in metazoans is discussed.

FEBS Lett, 2004 May 7, 565(1-3), 148 - 54
Regulation of the yeast phospholipid hydroperoxide glutathione peroxidase GPX2 by oxidative stress is mediated by Yap1 and Skn7; Tsuzi D et al.; The GPX2 gene encodes a homologue of phospholipid hydroperoxide glutathione peroxidase in Saccharomyces cerevisiae . The GPX2 promoter contains three elements the sequence of which is completely consistent with the optimal sequence for the Yap1 response element (YRE) . Here, we identify the intrinsic YRE that functions in the oxidative stress response of GPX2 . In addition, we discovered a cis-acting element (5'-GGCCGGC-3') within the GPX2 promoter proximal to the functional YRE that is necessary for H(2)O(2)-induced expression of GPX2 . We present evidence showing that Skn7 is necessary for the oxidative stress response of GPX2 and is able to bind to this sequence . We determine the optimal sequence for Skn7 to regulate GPX2 under conditions of oxidative stress to be 5'-GGC(C/T)GGC-3', and we designate this sequence the oxidative stress-responsive Skn7 response element.

J Biochem Biophys Methods, 2004 Apr 30, 59(1), 61 - 4
Immobilization of catalase by entrapment of permeabilized yeast cells in hen egg white using glutaraldehyde; Kubal BS et al.; An immobilized preparation of whole cell-based catalase was obtained by cross-linking the yeast cells permeabilized with toluene in hen egg white using glutaraldehyde . Optimal preparations were obtained when cross-linking was carried out for 2 h at 4 degrees C . Immobilized cells could be reused for the removal of H2O2 from milk.

Exp Gerontol, 2004 May, 39(5), 707 - 15
Expression of the human ferritin light chain in a frataxin mutant yeast affects ageing and cell death; Desmyter L et al.; Ferritin is one of the major eukaryotic proteins involved in regulating iron metabolism and maintaining iron homeostasis . However, Saccaromyces cerevisiae is an exception, possessing no ferritin and using other means to store excess iron . The only potential iron storage protein identified in yeast so far is the homologue of human frataxin (YFH1p) . In this study, we found that dysfunction of yeast frataxin shortens mean lifespan by 49% compared to the WT control . Interestingly, the human ferritin L gene can, at least partially, complement the function of yeast frataxin, extending lifespan and protecting cells from death induced by oxidative stress or excess iron . Our findings indicate that ferritin L can perform functions in yeast that are similar to its functions in mammals, and suggest that common mechanisms may exist for preventing iron and oxidative damage in single- and multi-cellular eukaryotic organisms . Clearly, elucidation of the function of human ferritin in yeast would help in gaining a better understanding the molecular basis of iron storage diseases.

J Cell Sci, 2004 May 15, 117(Pt 12), 2461 - 8 Epub 2004 May 05.
A role for the Cdc14-family phosphatase Flp1p at the end of the cell cycle in controlling the rapid degradation of the mitotic inducer Cdc25p in fission yeast; Esteban V et al.; The Schizosaccaromyces pombe protein Flp1p belongs to a conserved family of serine-threonine-phosphatases . The founding member of this family, Saccharomyces cerevisiae Cdc14p, is required for inactivation of mitotic CDKs and reversal of CDK mediated phosphorylation at the end of mitosis, thereby bringing about the M-G1 transition . Initial studies of Flp1p suggest that it may play a different role to Cdc14p . Here we show that Flp1p is required for rapid degradation of the mitotic inducer Cdc25p at the end of mitosis, and that Cdc25p is a substrate of Flp1p in vitro . Down-regulation of Cdc25p activity by Flp1p may ensure a prompt inactivation of mitotic CDK complexes to trigger cell division . Our results suggest a regulatory mechanism, and a universal role, for Cdc14p like proteins in coordination of cytokinesis with other cell cycle events.

Genome Biol . 2004;5(5):R33 . Epub 2004 Apr 30.
Transcriptional regulation of protein complexes in yeast; Simonis N et al.; BACKGROUND: Multiprotein complexes play an essential role in many cellular processes . But our knowledge of the mechanism of their formation, regulation and lifetimes is very limited . We investigated transcriptional regulation of protein complexes in yeast using two approaches . First, known regulons, manually curated or identified by genome-wide screens, were mapped onto the components of multiprotein complexes . The complexes comprised manually curated ones and those characterized by high-throughput analyses . Second, putative regulatory sequence motifs were identified in the upstream regions of the genes involved in individual complexes and regulons were predicted on the basis of these motifs . RESULTS: Only a very small fraction of the analyzed complexes (5-6%) have subsets of their components mapping onto known regulons . Likewise, regulatory motifs are detected in only about 8-15% of the complexes, and in those, about half of the components are on average part of predicted regulons . In the manually curated complexes, the so-called 'permanent' assemblies have a larger fraction of their components belonging to putative regulons than 'transient' complexes . For the noisier set of complexes identified by high-throughput screens, valuable insights are obtained into the function and regulation of individual genes . CONCLUSIONS: A small fraction of the known multiprotein complexes in yeast seems to have at least a subset of their components co-regulated on the transcriptional level . Preliminary analysis of the regulatory motifs for these components suggests that the corresponding genes are likely to be co-regulated either together or in smaller subgroups, indicating that transcriptionally regulated modules might exist within complexes.

Eur J Biochem, 2004 May, 271(10), 1963 - 70
Functional transitions of F0F1-ATPase mediated by the inhibitory peptide IF1 in yeast coupled submitochondrial particles; Galkin M et al.; The mechanism of inhibition of yeast F(0)F(1)-ATPase by its naturally occurring protein inhibitor (IF1) was investigated in submitochondrial particles by studying the IF1-mediated ATPase inhibition in the presence and absence of a protonmotive force . In the presence of protonmotive force, IF1 added during net NTP hydrolysis almost completely inhibited NTPase activity . At moderate IF1 concentration, subsequent uncoupler addition unexpectedly caused a burst of NTP hydrolysis . We propose that the protonmotive force induces the conversion of IF1-inhibited F(0)F(1)-ATPase into a new form having a lower affinity for IF1 . This form remains inactive for ATP hydrolysis after IF1 release . Uncoupling simultaneously releases ATP hydrolysis and converts the latent form of IF1-free F(0)F(1)-ATPase back to the active form . The relationship between the different steps of the catalytic cycle, the mechanism of inhibition by IF1 and the interconversion process is discussed.

J Mol Neurosci, 2004, 23(1-2), 13 - 22
Amyloid formation of a yeast prion determinant; Scheibel T; The {PSI+} factor of the yeast Saccharomyces cerevisiae is a cytoplasmic, epigenetic regulator of translation termination and can be transmitted from mother to daughter cells in a non-Mendelian manner . The transmission is caused by self-perpetuating noncovalent changes in the physical state of the protein determinant Sup35p, rather than by changes in its encoding gene . This phenomenon is reminiscent of the protein-only mechanism proposed for the infectious agent in a group of unusual, fatal neurodegenerative diseases in mammals . These diseases, known as prion diseases, are thought to involve a self-perpetuating change in the conformation of the prion protein (PrP) from a soluble form to one reflecting amyloid structure . In contrast to mammalian PrPs, Sup35p{PSI+} is not associated with disease in yeast and is not infectious for humans . Because of the mechanistic similarities of transmission of a specific, nonsoluble protein conformation, the epigenetic inheritance of {PSI+} in yeast was called a yeast prion phenomenon, and the yeast prion hypothesis was born . The elucidation of the mechanism by which alternative protein conformations transmit their structural information is key to understanding how proteins function as elements of epigenetic inheritance and how amyloidogenic conformations can be propagated . Yeast provides an ideal system to analyze both the epigenetic traits in vivo and the phenomenon of amyloid formation in vitro . The combination of these tools will help to determine the general mechanism of prion and amyloid appearance and propagation.

J Cell Sci, 2004 May 1, 117(Pt 11), 2309 - 20
Yeast ARL1 encodes a regulator of K+ influx; Munson AM et al.; A molecular genetic approach was undertaken in Saccharomyces cerevisiae to examine the functions of ARL1, encoding a G protein of the Ras superfamily . We show here that ARL1 is an important component of the control of intracellular K(+) . The arl1 mutant was sensitive to toxic cations, including hygromycin B and other aminoglycoside antibiotics, tetramethylammonium ions, methylammonium ions and protons . The hygromycin-B-sensitive phenotype was suppressed by the inclusion of K(+) and complemented by wild-type ARL1 and an allele of ARL1 predicted to be unbound to nucleotide in vivo . The arl1 mutant strain internalized approximately 25% more {(14)C}-methylammonium ion than did the wild type, consistent with hyperpolarization of the plasma membrane . The arl1 strain took up 30-40% less (86)Rb(+) than did the wild type, showing an inability to regulate K(+) import properly, contributing to membrane hyperpolarity . By contrast, K(+) and H(+) efflux were undisturbed . The loss of ARL1 had no effect on the steady-state level or the localization of a tagged version of Trk1p . High copy suppressors of the hygromycin-B phenotype included SAP155, encoding a protein that interacts with the cell cycle regulator Sit4p, and HAL4 and HAL5, encoding Ser/Thr kinases that regulate the K(+)-influx mediators Trk1p and Trk2p . These results are consistent with a model in which ARL1, via regulation of HAL4/HAL5, governs K(+) homeostasis in cells.

Genetics, 2004 Apr, 166(4), 1995 - 9
Genomic background predicts the fate of duplicated genes: evidence from the yeast genome; Zhang Z et al.; Gene duplication with subsequent divergence plays a central role in the acquisition of genes with novel function and complexity during the course of evolution . With reduced functional constraints or through positive selection, these duplicated genes may experience accelerated evolution . Under the model of subfunctionalization, loss of subfunctions leads to complementary acceleration at sites with two copies, and the difference in average rate between the sequences may not be obvious . On the other hand, the classical model of neofunctionalization predicts that the evolutionary rate in one of the two duplicates is accelerated . However, the classical model does not tell which of the duplicates experiences the acceleration in evolutionary rate . Here, we present evidence from the Saccharomyces cerevisiae genome that a duplicate located in a genomic region with a low-recombination rate is likely to evolve faster than a duplicate in an area of high recombination . This observation is consistent with population genetics theory that predicts that purifying selection is less effective in genomic regions of low recombination (Hill-Robertson effect) . Together with previous studies, our results suggest the genomic background (e.g., local recombination rate) as a potential force to drive the divergence between nontandemly duplicated genes . This implies the importance of structure and complexity of genomes in the diversification of organisms via gene duplications.

Genetics, 2004 Apr, 166(4), 1673 - 86
Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from Homologous recombination, but not from postreplicative repair; Schurer KA et al.; The MPH1 gene from Saccharomyces cerevisiae, encoding a member of the DEAH family of proteins, had been identified by virtue of the spontaneous mutator phenotype of respective deletion mutants . Genetic analysis suggested that MPH1 functions in a previously uncharacterized DNA repair pathway that protects the cells from damage-induced mutations . We have now analyzed genetic interactions of mph1 with a variety of mutants from different repair systems with respect to spontaneous mutation rates and sensitivities to different DNA-damaging agents . The dependence of the mph1 mutator phenotype on REV3 and REV1 and the synergy with mutations in base and nucleotide excision repair suggest an involvement of MPH1 in error-free bypass of lesions . However, although we observed an unexpected partial suppression of the mph1 mutator phenotype by rad5, genetic interactions with other mutations in postreplicative repair imply that MPH1 does not belong to this pathway . Instead, mutations from the homologous recombination pathway were found to be epistatic to mph1 with respect to both spontaneous mutation rates and damage sensitivities . Determination of spontaneous mitotic recombination rates demonstrated that mph1 mutants are not deficient in homologous recombination . On the contrary, in an sgs1 background we found a pronounced hyperrecombination phenotype . Thus, we propose that MPH1 is involved in a branch of homologous recombination that is specifically dedicated to error-free bypass.

Genetics, 2004 Apr, 166(4), 1641 - 9
EXO1 plays a role in generating type I and type II survivors in budding yeast; Maringele L et al.; Telomerase-defective budding yeast cells escape senescence by using homologous recombination to amplify telomeric or subtelomeric structures . Similarly, human cells that enter senescence can use homologous recombination for telomere maintenance, when telomerase cannot be activated . Although recombination proteins required to generate telomerase-independent survivors have been intensively studied, little is known about the nucleases that generate the substrates for recombination . Here we demonstrate that the Exo1 exonuclease is an initiator of the recombination process that allows cells to escape senescence and become immortal in the absence of telomerase . We show that EXO1 is important for generating type I survivors in yku70delta mre11delta cells and type II survivors in tlc1delta cells . Moreover, in tlc1delta cells, EXO1 seems to contribute to the senescence process itself.

Mol Cell, 2004 May 7, 14(3), 331 - 42
The carboxy-terminal extension of yeast ribosomal protein S14 is necessary for maturation of 43S preribosomes; Jakovljevic J et al.; Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known . Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing . Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants . These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins . These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway-the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3' end of 18S rRNA, consistent with a role for rpS14 in 3' end processing of 20S pre-rRNA.

J Biol Chem, 2004 Jul 9, 279(28), 29513 - 8 Epub 2004 Apr 28.
Transcription of the yeast iron regulon does not respond directly to iron but rather to iron-sulfur cluster biosynthesis; Chen OS et al.; Saccharomyces cerevisiae responds to iron deprivation by increased transcription of the iron regulon, including the high affinity cell-surface transport system encoded by FET3 and FTR1 . Here we demonstrate that transcription of these genes does not respond directly to cytosolic iron but rather to the mitochondrial utilization of iron for the synthesis of iron-sulfur (Fe-S) clusters . We took advantage of a mutant form of an iron-dependent enzyme in the sterol pathway (Erg25-2p) to assess cytosolic iron levels . We showed that disruption of mitochondrial Fe-S biosynthesis, which results in excessive mitochondrial iron accumulation, leads to transcription of the iron transport system independent of the cytosolic iron level . There is an inverse correlation between the activity of the mitochondrial Fe-S-containing enzyme aconitase and the induction of FET3 . Regulation of transcription by Fe-S biosynthesis represents a mechanism by which cellular iron acquisition is integrated with mitochondrial iron metabolism.

Genome Res, 2004 May, 14(5), 829 - 34
A global view of the selection forces in the evolution of yeast cis-regulation; Tanay A et al.; The interaction between transcription factors and their DNA binding sites is key to understanding gene regulation . By performing a genome-wide study of the evolutionary dynamics in yeast promoters, we provide a first global view of the network of selection forces in the evolution of transcription factor binding sites . This analysis gives rise to new models for binding site activity, identifies families of related binding sites, and characterizes the functional similarities among them . We discovered rich and highly optimized selective pressures operating inside and around these families . In several cases, this organization reveals that a single transcription factor has multiple functional modes . We demonstrate how such functional heterogeneity is related to the binding site's affinity and how it is exploited in transcription programs.

Chem Biol, 2004 Mar, 11(3), 295 - 9
Rpd3p relocation mediates a transcriptional response to rapamycin in yeast; Humphrey EL et al.; Treating yeast cells with rapamycin, a small molecule that inhibits the TOR proteins, leads to the repression of many genes . Consistent with prior studies, we find that RPD3, which encodes a histone deacetylase (HDAC), is required for repression upon rapamycin treatment . To elucidate the mechanism underlying RPD3-mediated repression, we screened all promoters in yeast for occupancy by Rpd3p before and after treatment with rapamycin . We find that Rpd3p binds to the promoters of rapamycin-repressible genes only following treatment . These data conflict with a previously proposed model suggesting that Rpd3p is constitutively bound to rapamycin-repressible genes and becomes active only after a stimulus such as treatment with rapamycin . Rather, the comprehensive analysis presented here strongly supports a model in which recruitment of Rpd3p to gene promoters is a regulated step in the control of gene repression.

Nucleic Acids Res, 2004 Apr 30, 32(8), 2441 - 52 Print 2004.
Multiple protein/protein and protein/RNA interactions suggest roles for yeast DNA/RNA helicase Sen1p in transcription, transcription-coupled DNA repair and RNA processing; Ursic D et al.; Sen1p in Saccharomyces cerevisiae is a Type I DNA/RNA helicase . Mutations in the helicase domain perturb accumulation of diverse RNA classes, and Sen1p has been implicated in 3' end formation of non-coding RNAs . Using a combination of global and candidate-specific two hybrid screens, eight proteins were identified that interact with Sen1p . Interactions with three of the proteins were analyzed further: Rpo21p(Rpb1p), a subunit of RNA polymerase II, Rad2p, a deoxyribonuclease required in DNA repair, and Rnt1p (RNase III), an endoribonuclease required for RNA maturation . For all three interactions, the two-hybrid results were confirmed by co-immunoprecipitation experiments . Genetic tests designed to assess the biological significance of the interactions indicate that Sen1p plays functionally significant roles in transcription and transcription-coupled DNA repair . To investigate the potential role of Sen1p in RNA processing and to assess the functional significance of the Sen1p/Rnt1p interaction, we examined U5 snRNA biogenesis . We provide evidence that Sen1p functions in concert with Rnt1p and the exosome at a late step in 3' end formation of one of the two mature forms of U5 snRNA but not the other . The protein-protein and protein-RNA interactions reported here suggest that the DNA/RNA helicase activity of Sen1p is utilized for several different purposes in multiple gene expression pathways.

Mol Cell Biol, 2004 May, 24(10), 4151 - 65
The functions of budding yeast Sae2 in the DNA damage response require Mec1- and Tel1-dependent phosphorylation; Baroni E et al.; DNA damage checkpoint pathways sense DNA lesions and transduce the signals into appropriate biological responses, including cell cycle arrest, induction of transcriptional programs, and modification or activation of repair factors . Here we show that the Saccharomyces cerevisiae Sae2 protein, known to be involved in processing meiotic and mitotic double-strand breaks, is required for proper recovery from checkpoint-mediated cell cycle arrest after DNA damage and is phosphorylated periodically during the unperturbed cell cycle and in response to DNA damage . Both cell cycle- and DNA damage-dependent Sae2 phosphorylation requires the main checkpoint kinase, Mec1, and the upstream components of its pathway, Ddc1, Rad17, Rad24, and Mec3 . Another pathway, involving Tel1 and the MRX complex, is also required for full DNA damage-induced Sae2 phosphorylation, that is instead independent of the downstream checkpoint transducers Rad53 and Chk1, as well as of their mediators Rad9 and Mrc1 . Mutations altering all the favored ATM/ATR phosphorylation sites of Sae2 not only abolish its in vivo phosphorylation after DNA damage but also cause hypersensitivity to methyl methanesulfonate treatment, synthetic lethality with RAD27 deletion, and decreased rates of mitotic recombination between inverted Alu repeats, suggesting that checkpoint-mediated phosphorylation of Sae2 is important to support its repair and recombination functions.

Curr Biol, 2004 May 4, 14(9), 763 - 75
Microtubule nucleation at non-spindle pole body microtubule-organizing centers requires fission yeast centrosomin-related protein mod20p; Sawin KE et al.; BACKGROUND: Many types of differentiated eukaryotic cells display microtubule distributions consistent with nucleation from noncentrosomal intracellular microtubule organizing centers (MTOCs), although such structures remain poorly characterized . In fission yeast, two types of MTOCs exist in addition to the spindle pole body, the yeast centrosome equivalent . These are the equatorial MTOC, which nucleates microtubules from the cell division site at the end of mitosis, and interphase MTOCs, which nucleate microtubules from multiple sites near the cell nucleus during interphase . RESULTS: From an insertional mutagenesis screen we identified a novel gene, mod20+, which is required for microtubule nucleation from non-spindle pole body MTOCs in fission yeast . Mod20p is not required for intranuclear mitotic spindle assembly, although it is required for cytoplasmic astral microtubule growth during mitosis . Mod20p localizes to MTOCs throughout the cell cycle and is also dynamically distributed along microtubules themselves . We find that mod20p is required for the localization of components of the gamma-tubulin complex to non-spindle pole body MTOCs and physically interacts with the gamma-tubulin complex in vivo . Database searches reveal a family of eukaryotic proteins distantly related to mod20p; these are found in organisms ranging from fungi to mammals and include Drosophila centrosomin . CONCLUSIONS: Mod20p appears to act by recruiting components of the gamma-tubulin complex to non-spindle pole body MTOCs . The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells.

Physiol Res, 2004, 53 Suppl 1, S91 - 8
Yeast as a model organism to study transport and homeostasis of alkali metal cations; Sychrova H; To maintain an optimum cytoplasmic K(+)/Na+ ratio, cells employ three distinct strategies: 1) strict discrimination among alkali metal cations at the level of influx, 2) efficient efflux of toxic cations from cells, and 3) selective sequestration of cations in organelles . Cation efflux and influx are mediated in cells by systems with different substrate specificities and diverse mechanisms, e.g . ATPases, symporters, antiporters, and channels . Simple eukaryotic yeast Saccharomyces cerevisiae cells proved to be an excellent model for studying the transport properties and physiological function of alkali-metal-cation transporters, and the existence of mutant strains lacking their own transport systems provided an efficient tool for a molecular study of alkali-metal-cation transporters from higher eukaryotes upon their expression in yeast cells.

Mycopathologia, 2004 Feb, 157(2), 163 - 9
Fluconazole and itraconazole susceptibility of vaginal yeast isolates from Slovakia; Sojakova M et al.; Vulvovaginal candidiasis is a common mucosal infection caused by opportunistic yeasts of the Candida genus . In this study, we isolated and identified the yeast species in the vagina of patients treated in the gynecology clinic and tested in vitro activities of fluconazole and itraconazole against 227 clinical yeast isolates by the NCCLS microdilution method . C . albicans (87.6%) was the most frequently identified species followed by C . glabrata (6.2%) and C . krusei (2.2%) . Almost thirteen percent of yeast strains were resistant to fluconazole and 18.5% were resistant to itraconazole . Cross-resistance analyses of C . albicans isolates revealed that fluconazole resistance and itraconazole resistance were also associated with decreased susceptibilities to other azole derivatives mainly to ketoconazole and miconazole . At the same time no cross-resistance to polyene antibiotics amphotericin B and nystatin was observed . These results support the notion that antifungal agents used to treat vaginitis may be contributing to the drug resistance problem by promoting cross-resistance to a range of clinically used antifungals.

Mol Genet Genomics, 2004 Jun, 271(5), 616 - 26 Epub 2004 Apr 30.
The mitochondrial IMP peptidase of yeast: functional analysis of domains and identification of Gut2 as a new natural substrate; Esser K et al.; The mitochondrial inner membrane peptidase IMP of Saccharomyces cerevisiae is required for proteolytic processing of certain mitochondrially and nucleus-encoded proteins during their export from the matrix into the inner membrane or the intermembrane space . The membrane-associated signal peptidase complex is composed of the two catalytic subunits, Imp1 and Imp2, and the Som1 protein . The IMP subunits are thought to function in membrane association, interaction and stabilisation of subunits, substrate specificity, and proteolysis . We have analysed inner membrane peptidase mutants and substrates to gain more insight into the functions of various domains and investigate the basis of substrate recognition . The results suggest that certain conserved glycine residues in the second and third conserved regions of Imp1 and Imp2 are important for stabilisation of the Imp complex and for the proteolytic activity of the subunits, respectively . The non-conserved C-terminal parts of the Imp subunits are important for their proteolytic activities . The C-terminal region of Imp2, comprising a predicted second transmembrane segment, is dispensable for the stability of Imp2 and Imp1, and cannot functionally substitute for the C-terminal segment of Imp1 . Alteration of the Imp2 cleavage site in cytochrome c(1) (from A/M to N/D) reveals the specificity of the Imp2 peptidase . In addition, we have identified Gut2, the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase, as a new substrate for Imp1 . Failure to cleave the Gut2 precursor may contribute to the pet phenotype of certain imp mutants . Gut2 is associated with the inner membrane, and is essential for growth on glycerol-containing medium . Suggested functions of the analysed residues and domains of the IMP subunits, characteristics of the cleavage sites of substrates and implications for the phenotypes of imp mutants are discussed.

Biosci Biotechnol Biochem, 2004 Apr, 68(4), 909 - 19
RSC Nucleosome-remodeling complex plays prominent roles in transcriptional regulation throughout budding yeast gametogenesis; Koyama H et al.; RSC is a nucleosome-remodeling complex of Saccharomyces cerevisiae essential for growth that can alter histone-DNA interaction by using the energy of ATP hydrolysis . Nps1p/Sth1p is an ATPase subunit of RSC . A mutation in the conserved ATPase domain of Nps1p causes a sporulation defect with decreased expression of early meiotic genes, especially IME2 . This defect is partially suppressed by the overexpression of either IME1 or IME2 . A homozygous diploid of a novel temperature-sensitive nps1 mutation, nps1-13, harboring amino acid substitutions within the bromodomain, was unable to sporulate . Overexpression of IME, IME2, or both of these genes allowed the completion of meiosis I and meiosis II in nps1-13 but not the formation of mature asci . In nps1-13 carrying YEpIME1, the expression of a group of sporulation-specific genes, which express at the middle stages of sporulation and are required for spore-wall formation, notably diminished, and several late sporulation genes expressed at the early stages of sporulation . These results suggest that Nps1p/RSC plays important roles during the spore development process by controlling gene expression for initiating both meiosis and spore morphogenesis, and ensures proper expression timing of late meiotic genes.

Nucleic Acids Res, 2004 Apr 28, 32(8), 2378 - 85 Print 2004.
Meiosis-specific yeast Hop1 protein promotes synapsis of double-stranded DNA helices via the formation of guanine quartets; Anuradha S et al.; In most eukaryotes, genetic exchange between paired homologs occurs in the context of a tripartite proteinaceous structure called the synaptonemal complex (SC) . Genetic analyses have revealed that the genes encoding SC proteins are vital for meiotic chromosome pairing and recombination . However, the number, nature and/or the mechanism used by SC proteins to align chromosomes are yet to be clearly defined . Here, we show that Saccharomyces cerevisiae Hop1, a component of SC, was able to promote pairing of double-stranded DNA helices containing arrays of mismatched G/G sequences . Significantly, pairing was rapid and robust, independent of homology in the arms flanking the central G/G region, and required four contiguous guanine residues . Furthermore, data from truncated DNA double helices showed that 20 bp on either side of the 8 bp mismatched G/G region was essential for efficient synapsis . Methylation interference indicated that pairing between the two DNA double helices involves G quartets . These results suggest that Hop1 is likely to play a direct role in meiotic chromosome pairing and recombination by its ability to promote synapsis between double-stranded DNA helices containing arrays of G residues . To our knowledge, Hop1 is the first protein shown to promote synapsis of DNA double helices from yeast or any other organism.

Br Poult Sci, 2004 Feb, 45(1), 133 - 8
Uptake and distribution of astaxanthin in several tissues and plasma lipoproteins in male broiler chickens fed a yeast (Phaffia rhodozyma) with a high concentration of astaxanthin; Takahashi K et al.; 1 . The experiments were conducted to evaluate astaxanthin (Ax) uptake in several tissues and plasma lipoproteins of male broiler chickens fed on Phaffia rhodozyma containing a high concentration of Ax . 2 . Male broiler chicks (5 weeks of age) fasted for 16h were given 0 or 45 mg Ax as Phaffia rhodozyma through the crop and blood was collected over the following 24 h . Ax appeared in the plasma at 2 h after administration into the crop . Most (more than 70%) of the Ax was contained in the high density lipoprotein (HDL) fraction in the plasma irrespective of blood sampling times and administration procedure of Ax . 3 . Male broiler chicks (2 weeks of age) were fed on a diet containing 0, 50 or 100 mg/kg of yeast Ax for 2 weeks . Of the tissues examined, Ax concentration in the small intestine was highest, followed by subcutaneous fat, abdominal fat, spleen, liver, heart, kidney and skin . The lowest concentration was in the muscles . Ax concentration in the small intestine, subcutaneous fat, abdominal fat, liver and skin rose as dietary content increased, but this was not the case for the spleen, heart, kidney and muscles except for M . pecloralis superficialis . 4 . Over 50% of Ax deposited in liver tissues was detected in the microsomal fraction and 15% was in the mitochondrial fraction . In muscles, both fractions of mitochondria and sarcoplasmic reticulum contained Ax.

Protein J, 2004 Jan, 23(1), 33 - 8
Sequence-altered peptide adopts optimum conformation for modification-dependent binding of the yeast tRNAPhe anticodon domain; Mucha P et al.; Amino acid contributions to protein recognition of naturally modified RNAs are not understood . Circular dichroism spectra and predictive software suggested that peptide tF2 (S1ISPW5GFSGL10 LRWSY15), selected from a phage display library to bind the modified anticodon domain of yeast tRNAPhe (ASL), adopted a beta-sheet structure . Ala residues incorporated at positions Pro4 and Gly6, both predicted to be involved in a turn, did not alter the peptide binding affinity for the ASLPhe, although major changes in the peptide's CD spectra were observed . Substitutions at three positions Pro4, Gly6, and Gly9, the latter not predicted to be in a turn, reduced the peptide's binding affinity to 4% of that of the unsubstituted tF2 and strongly influenced the peptide's secondary structure . The results suggest that peptides with different conformations, but similar affinities, adopt the optimal binding conformation, indicative of a structurally adaptive model of binding in which the modified RNA serves as a scaffold.

Eksp Onkol, 2004 Mar, 26(1), 15 - 9
Identification of a novel binding partners for tumor suppressor PTEN by a yeast two-hybrid approach; Gorbenko O et al.; AIM: To identify novel PTEN-binding partners . METHODS: The technique of yeast two-hybrid screening was used in this study . A panel of bait constructs was created, containing the C-terminal domain of PTEN, full length PTEN, activated and phosphatase-dead mutants . The expression of LexA-fused baits, their nuclear localization and autoactivation potential were tested according to the standard protocol of Duplex A system . CDNA libraries from Colon Cancer, HeLa and Mouse Embryo were screened with two selected bait constructs . Isolated positive clones were further analysed by mating assay and identified by automated DNA sequencing and database searching . RESULTS: Extensive screening of cDNA libraries with the full length and the C-terminal domain of PTEN led to the identification of 43 positive clones, which were confirmed in mating assay . Sequence analysis indicated that two clones encode AEBP1 (Adipocyte Enhancer Binding Protein 1) . CONCLUSION: Our data indicate that the interaction between PTEN and AEBP1 is mediated by their C-terminal and N-terminal domains, respectively . The functional importance of PTEN-AEBP1 interaction is currently under investigation.

Plant Cell Physiol, 2004 Apr, 45(4), 496 - 500
Functional analysis of a rice putative voltage-dependent Ca2+ channel, OsTPC1, expressed in yeast cells lacking its homologous gene CCH1; Hashimoto K et al.; We isolated a cDNA (OsTPC1) from rice that was homologous to AtTPC1, a putative voltage-dependent Ca(2+) channel (VDCC) gene of Arabidopsis thaliana . The hydropathy profile of its deduced amino acid sequence showed significant structural features of the alpha 1-subunit of animal VDCCs . Functional analysis using a heterologous yeast expression system showed that OsTPC1 facilitates Ca(2+) permeation . The K(m) value for Ca(2+) of OsTPC1, 47.5 micro M, was comparable to that of intrinsic CCH1, a candidate VDCC in yeast . Ca(2+) permeation by OsTPC1 was inhibited by verapamil, a VDCC blocker . These findings indicate for the first time that OsTPC1 is a putative VDCC in rice.

J Biol Chem, 2004 Jun 25, 279(26), 27116 - 23 Epub 2004 Apr 24.
Cbf1p is required for chromatin remodeling at promoter-proximal CACGTG motifs in yeast; Kent NA et al.; Cbf1p is a basic-helix-loop-helix-zipper protein of Saccharomyces cerevisiae required for the function of centromeres and MET gene promoters, where it binds DNA via the consensus core motif CACRTG (R = A or G) . At MET genes Cbf1p appears to function in both activator recruitment and chromatin-remodeling . Cbf1p has been implicated in the regulation of other genes, and CACRTG motifs are common in potential gene regulatory DNA . A recent genome-wide location analysis showed that the majority of intergenic CACGTG palindromes are bound by Cbf1p . Here we tested whether all potential Cbf1p binding motifs in the yeast genome are likely to be bound by Cbf1p using chromatin immunoprecipitation . We also tested which of the motifs are actually functional by assaying for Cbf1p-dependent chromatin remodeling . We show that Cbf1p binding and activity is restricted to palindromic CACGTG motifs in promoter-proximal regions . Cbf1p does not function through CACGTG motifs that occur in promoter-distal locations within coding regions nor where CACATG motifs occur alone except at centromeres . Cbf1p can be made to function at promoter-distal CACGTG motifs by overexpression, suggesting that the concentration of Cbf1p is normally limiting for binding and is biased to gene regulatory DNA by interactions with other factors . We conclude that Cbf1p is required for normal nucleosome positioning wherever the CACGTG motif occurs in gene regulatory DNA . Cbf1p has been shown to interact with the chromatin-remodeling ATPase Isw1p . Here we show that recruitment of Isw1p by Cbf1p is likely to be general but that Isw1p is only partially required for Cbf1p-dependent chromatin structures.

Biophys J, 2004 May, 86(5), 2740 - 7
Elastic instability in growing yeast colonies; Nguyen B et al.; The differential adhesion between cells is believed to be the major driving force behind the formation of tissues . The idea is that an aggregate of cells minimizes the overall adhesive energy between cell surfaces . We demonstrate in a model experimental system that there exist conditions where a slowly growing tissue does not minimize this adhesive energy . A mathematical model demonstrates that the instability of a spherical shape is caused by the competition between elastic and surface energies.

Biophys Chem, 2004 Jun 1, 109(3), 413 - 26
Model evaluation for glycolytic oscillations in yeast biotransformations of xenobiotics; Brusch L et al.; Anaerobic glycolysis in yeast perturbed by the reduction of xenobiotic ketones is studied numerically in two models which possess the same topology but different levels of complexity . By comparing both models' predictions for concentrations and fluxes as well as steady or oscillatory temporal behavior we answer the question what phenomena require what kind of minimum model abstraction . While mean concentrations and fluxes are predicted in agreement by both models we observe different domains of oscillatory behavior in parameter space . Generic properties of the glycolytic response to ketones are discussed .

Biochem Biophys Res Commun, 2004 May 21, 318(1), 198 - 203
In vivo interaction between the human dehydrodolichyl diphosphate synthase and the Niemann-Pick C2 protein revealed by a yeast two-hybrid system; Kharel Y et al.; Dehydrodolichyl diphosphate (DedolPP) synthase catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize DedolPP, a biosynthetic precursor for dolichol which plays an important role as a sugar-carrier lipid in the biosynthesis of glycoprotein in eukaryotic cells . During certain pathological processes like Alzheimer's disease or some neurological disorders, dolichol has been shown to accumulate in human brain . In order to understand the regulatory mechanism of dolichol in eukaryotes, we performed a yeast two-hybrid screen using full length human DedolPP synthase gene {Endo et al . BBA 1625 (2003) 291} as a bait to find some proteins specifically interacting with the enzyme . We identified Niemann-Pick Type C2 protein (NPC2) to show a specific interaction with human DedolPP synthase . This interaction was further confirmed by in vitro co-immunoprecipitation experiment, indicating the possible physiological interaction between NPC2 and DedolPP synthase proteins in human.

Biochim Biophys Acta, 2004 May 3, 1672(2), 86 - 92
Functional co-expression of xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 and UDP-glucuronosyltransferase 1A6, in yeast microsomes; Ikushiro S et al.; Xenobiotic Phase I and Phase II reactions in hepatocytes occur sequentially and cooperatively during the metabolism of various chemical compounds including drugs . In order to investigate the sequential metabolism of 7-ethoxycoumarin (7EC) as model substrate in vitro, xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 (P450 1A1) and UDP-glucuronosyltransferase 1A6 (UGT1A6) were co-expressed in Saccharomyces cerevisiae AH22 . Rat P450 1A1 and yeast NADPH-P450 reductase were expressed on a multicopy plasmid (pGYR1) in the yeast . Rat UGT1A6 cDNA with a yeast alcohol dehydrogenase I promoter and terminator was integrated into yeast chromosomal DNA to achieve the stable expression . Co-expression of P450 1A1 and UGT1A6 in yeast microsomes was confirmed by immunoblot analysis . Protease treatment of the microsomes showed the correct topological orientation of UGT to the membranes . The metabolism of 7EC to 7-hydroxycoumarin (7HC) and its glucuronide in yeast microsomes was analyzed by reverse phase HPLC . In a co-expression system containing 7EC, NADPH and UDP-glucuronic acid, glucuronide formation was detected after a lag phase, following the accumulation of 7HC . In the case of P450 1A1 and UGT1A6, efficient coupling of hydroxylation and glucuronidation in 7EC metabolism was not observed in the co-expression system . This P450 and UGT co-expression system in yeast allows the sequential biotransformation of xenobiotics to be simulated in vitro.

FEMS Microbiol Lett, 2004 Apr 15, 233(2), 187 - 92
Experimental studies on ploidy evolution in yeast; Zeyl C; Variation in the prominence of haploidy and diploidy is a striking feature of eukaryote life cycles that has not been explained from an evolutionary point of view . the ease with which ploidy and other variables of population genetics may be manipulated in yeast make Saccharomyces cerevisiae an excellent subject for experiments on the fitness effects of ploidy . Several hypotheses have been advanced to explain the emphasis on diploidy in plants and animals, and yeast experiments have been particularly informative for a few . Evidence suggests that diploids may enjoy an immediate advantage over haploids in masking harmful mutations, avoiding the fitness cost such mutations impose on haploids . A convincing longer-term advantage for diploidy has proven elusive, and different evolutionary explanations for the origin and for the subsequent maintenance of diploidy may be required.

Cell Cycle, 2004 May, 3(5), 648 - 54 Epub 2004 May 15.
The yeast kinase Swe1 is required for proper entry into cell cycle after arrest due to ribosome biogenesis and protein synthesis defects; Saracino F et al.; Sda1 is an essential protein required for cell cycle progression in Saccharomyces cerevisiae . Here, we show that the sda1-1 mutation causes a defect in the formation and nuclear export of 60S ribosomal subunits . Moreover, the sda1-1, but also other mutants defective in ribosome biogenesis (e.g., rix1-1 and tif6Delta), exhibit a G1 arrest, which could be the consequence of impaired ribosome biogenesis . Interestingly, additional deletion of the non-essential Swe1 kinase, the homolog of S . pombe Wee1, causes a pronounced delay in entering a new cell cycle in sda1-1, rix1-1 and tif6Delta cells, when shifted back from restrictive to permissive conditions . However, such a prolonged delay is independent of the Tyr19 phosphorylation in Cdc28 . Moreover, the lack of Swe1 causes delay in budding and DNA replication in cells released from the G1 arrest due to the block of protein synthesis . Our data suggest that Swe1 is required for timely entry into cell cycle after a G1 arrest caused by impairment in pre-60S biogenesis and in protein synthesis . Therefore we propose that Swe1, which is required for coordination of cell growth and cell division in G2/M, also has a role in the beginning of the cell cycle.

Methods Mol Biol, 2004, 282, 243 - 54
The yeast three-hybrid system as a tool to study caspases; van Criekinge W et al.; Caspases are cysteine proteases that play an essential role during apoptotic cell death and inflammation . They are synthesized as catalytically dormant proenzymes, containing an N-terminal prodomain, a large subunit (p20) containing the active site cysteine, and a small subunit (p10) . The active enzymes function as tetramers, consisting of two p20/p10 subunit heterodimers . Both subunits contribute residues that are essential for substrate recognition . Activation of caspases culminates in the cleavage of a set of cellular proteins, resulting in disassembly of the cell or proinflammatory cytokine production . Inappropriate caspase activation contributes to or accounts for several diseases . The identification of caspase-interacting proteins that might act as activators, substrates, or inhibitors is therefore an attractive step in the development of novel therapeutics . However, caspase substrates and other proteins that bind specifically with the active heterodimeric p20/p10 form of caspases will escape detection in a classical two-hybrid approach with an unprocessed caspase precursor as bait . Alternatively, a number of so-called three-hybrid systems to analyze more complex macromolecular interactions have been developed . We describe the use of a three-hybrid approach adapted to the needs of caspases to detect and analyze the interaction of mature heteromeric caspases with protein substrates or inhibitors.

Methods Mol Biol, 2004, 282, 223 - 41
Yeast two-hybrid screening for proteins interacting with the anti-apoptotic protein A20; Heyninck K et al.; The yeast two-hybrid system is a powerful technique for identifying proteins that interact with a specific protein of interest . The rationale of the yeast two-hybrid system relies on the physical separation of the DNA-binding domain from the transcriptional activation domain of several transcription factors . Therefore, the protein of interest (bait) is fused to a DNA-binding domain, and complimentary DNA (cDNA) library-encoded proteins are fused to a transcriptional activation domain . When a protein encoded by the cDNA library binds to the bait, both activities of the transcription factor are rejoined and transcription from a reporter gene is started . Here, we will give a comprehensive guide for the GAL4-based two-hybrid system, exemplified by the detection of binding partners for the zinc finger protein A20 . The latter is an inducible cellular inhibitor of tumor necrosis factor (TNF)-induced apoptosis and nuclear factor (NF)-kappaB-dependent gene expression . Yeast two-hybrid screening with A20 as bait revealed several A20-binding proteins, including A20 itself, members of the 14-3-3 family, as well as three novel proteins ABIN-1, ABIN-2, and TXBP151 . The latter protein was subsequently shown to mediate at least part of the anti-apoptotic activities of A20, whereas ABIN-1 and -2 are more likely to be involved in the NF-kappaB inhibitory effects of A20.

J Biol Chem, 2004 Jun 25, 279(26), 27138 - 47 Epub 2004 Apr 22.
Akr1p-dependent palmitoylation of Yck2p yeast casein kinase 1 is necessary and sufficient for plasma membrane targeting; Babu P et al.; The Yck2 protein is a plasma membrane-associated casein kinase 1 isoform that attaches to membranes via palmitoylation of its C terminus . We have demonstrated that Yck2p traffics to the plasma membrane on secretory vesicles . Because Akr1p, the palmitoyl transferase for Yck2p, is located on Golgi membranes, it is likely that Yck2p first associates with Golgi membranes, and then is somehow recruited to budding plasma membrane-destined vesicles . We show here that residues 499-546 are sufficient for minimal Yck2p palmitoylation and plasma membrane localization . We previously described normal plasma membrane targeting of a Yck2p construct with the final five amino acids of Ras2p substituting for the final two Cys residues of Yck2p . This Yck2p variant no longer requires Akr1p for membrane association, but targets normally . We have generated the C-terminal deletions previously shown to affect Yck2p membrane association in this variant to determine which residues are important for targeting and/or modification . We find that all of the sequences previously identified as important for plasma membrane association are required only for Akr1p-dependent modification . Furthermore, palmitoylation is sufficient for specific association of Yck2p with secretory vesicles destined for the plasma membrane . Finally, both C-terminal Cys residues are palmitoylated, and dual acylation is required for efficient membrane association.

Antimicrob Agents Chemother, 2004 May, 48(5), 1600 - 13
Functional genomic analysis of fluconazole susceptibility in the pathogenic yeast Candida glabrata: roles of calcium signaling and mitochondria; Kaur R et al.; The pathogenic yeast Candida glabrata exhibits innate resistance to fluconazole, the most commonly used antifungal agent . By screening a library of 9,216 random insertion mutants, we identified a set of 27 genes which upon mutation, confer altered fluconazole susceptibility in C . glabrata . Homologues of three of these genes have been implicated in azole and/or drug resistance in Saccharomyces cerevisiae: two of these belong to the family of ABC transporters (PDR5 and PDR16), and one is involved in retrograde signaling from mitochondria to nucleus (RTG2) . The remaining 24 genes are involved in diverse cellular functions, including ribosomal biogenesis and mitochondrial function, activation of RNA polymerase II transcription, nuclear ubiquitin ligase function, cell wall biosynthesis, and calcium homeostasis . We characterized two sets of mutants in more detail . Strains defective in a putative plasma membrane calcium channel (Cch1-Mid1) were modestly more susceptible to fluconazole but showed a significant loss of viability upon prolonged fluconazole exposure, suggesting that calcium signaling is required for survival of azole stress in C . glabrata . These mutants were defective in calcium uptake in response to fluconazole exposure . The combined results suggest that, in the absence of Ca(2+) signaling, fluconazole has a fungicidal rather than a fungistatic effect on C . glabrata . The second set of mutants characterized in detail were defective in mitochondrial assembly and organization, and these exhibited very high levels of fluconazole resistance . Further analysis of these mutants indicated that in C . glabrata a mechanism exists for reversible loss of mitochondrial function that does not involve loss of mitochondrial genome and that C . glabrata can switch between states of mitochondrial competence and incompetence in response to fluconazole exposure.

Biotechnol Lett, 2004 Mar, 26(5), 409 - 13
Application of a yeast method for DNA extraction associated with database interrogations for the characterization of various filamentous fungi from diseased hop; Phalip V et al.; Filamentous fungi were collected from diseased hop (Humulus lupulus; L.) and DNA was prepared from 19 isolates . The critical step of cell lysis was carried out using zymolyase for fungal cell wall digestion . DNA was successfully prepared for all isolates and allowed ribosomal DNA region amplifications by PCR . The amplicons were sequenced and sequences were compared with nucleotides databases . These analyses allowed identification of each fungus and gave a precise view of the ecosystem . Seven different genera were found among the 19 isolates . A phylogenetic tree was constructed and revealed the diversity of the hop plant environment.

Antonie Van Leeuwenhoek, 2004 Jul, 86(1), 27 - 32
Candida sergipensis, a new asexual yeast species isolated from frozen pulps of tropical fruits; Trindade RC et al.; Sixteen strains of the new yeast species Candida sergipensis have been isolated from frozen pulps of the tropical fruits umbu ( Spondias tuberosa Avr . Cam.) and mangaba ( Hancornia speciosa Gom.) . Candida sergipensis was one of the prevalent species in the yeast community of these substrates . The new asexual ascomycetous yeast is phylogenetically related to Candida spandovensis and Candida sorbophila, species belonging to the Wickerhamiella clade, as evidenced by the sequences of the D1/D2 domains of their large subunit ribosomal DNAs . The species C . sergipensis and C . spandovensis can be separated on the basis of growth on 50% glucose agar, xylose and succinate, negative for the first species and positive for the second . The type culture is strain UFMG-R188 (CBS 9567).

BMC Biotechnol . 2004 Apr 21;4(1):8.
Rapid isolation of yeast genomic DNA: Bust n' Grab; Harju S et al.; BACKGROUND: Mutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants . Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall . Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed . We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates . RESULTS: Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA . Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA . Two hundred ng--3 microg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony . Samples were either resuspended directly in a restriction enzyme/RNase cocktail mixture for Southern blot hybridization or used for several PCR reactions . We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human beta-globin locus YAC . CONCLUSION: An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed . This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.

Org Lett, 2004 Apr 29, 6(9), 1409 - 12
Facile synthesis of cids: biotinylated estrone oximes efficiently heterodimerize estrogen receptor and streptavidin proteins in yeast three hybrid systems; Muddana SS et al.; {structure: see text} We synthesized estrone oximes as chemical inducers of protein heterodimerization (CIDs) . Estrone-17-(O-carboxymethyl)oxime coupled to biotinamidocaproic acid via N,N'-dimethylhexane-1,6-diamine efficiently heterodimerizes estrogen receptors (ERs) and streptavidin Y43A in yeast three hybrid systems, activating gene expression over 100-fold at 10 microM . Related hexane-1,6-diamine and estradiol-6-(O-carboxymethyl)oxime derivatives were ineffective CIDs due to low affinity for ERs when bound to streptavidin . Estrone oximes bind ERs with submicromolar affinity and effectively display small molecules to target proteins expressed in yeast.

Proc R Soc Lond B Biol Sci, 2004 Feb 7, 271 Suppl 3, S25 - 6
The Prisoner's Dilemma and polymorphism in yeast SUC genes; Greig D et al.; The SUC multigene family of the single-celled yeast Saccharomyces cerevisiae is polymorphic, with genes varying both in number and activity . All of the genes encode invertase, an enzyme that is secreted to digest sucrose outside of the cell . This communal endeavour creates the potential for individual cells to defect (cheat) by stealing the sugar digested by their neighbours without contributing the enzyme themselves . We measured the fitness of a defector, with a deleted suc2 gene, relative to an otherwise isogenic cooperator, with a functional SUC2 gene . We manipulated the level of social interaction within the community by varying the population density and found that the defector is less fit than the cooperator at low levels of sociality but more fit in dense communities . We propose that selection for antisocial cheating causes SUC polymorphism in nature . The infamous Prisoner's Dilemma game shows that social behaviour is generally unstable, and the success of both cooperation and defection can vary continuously in time and space . The variation in SUC genes reflects constant adaptation to an ever-changing biotic environment that is a consequence of the instability of cooperation . It is interesting that social interactions can have a direct effect on molecular evolution, even in an organism as simple as yeast.

RNA, 2004 May, 10(5), 863 - 79
Kinetic analysis of ribozyme-substrate complex formation in yeast; Yadava RS et al.; Many RNA-mediated reactions in transcription, translation, RNA processing, and transport require assembly of RNA complexes, yet assembly pathways remain poorly understood . Assembly mechanisms can be difficult to assess in a biological context because many components interact in complex pathways and individual steps are difficult to isolate experimentally . Our previous studies of self-cleaving hairpin ribozymes showed that kinetic and equilibrium parameters measured in yeast agree well with parameters measured in vitro under ionic conditions that mimic the intracellular environment . We now report studies of intermolecular reactions with ribozyme and target sequences expressed in yeast as separate chimeric U3 snoRNAs . In this system, intracellular cleavage rates reflect the kinetics of ribozyme-substrate complex formation through annealing of base-paired helices . Second-order rate constants increased with increasing helix length for in vitro reactions with 2 mM MgCl(2) and 150 mM NaCl and in vivo but not in reactions with 10 mM MgCl(2) . Thus, efficient RNA complex formation required a larger extent of complementarity in vivo than in vitro under conditions with high concentrations of divalent cations . The most efficient intracellular cleavage reactions exhibited second-order rate constants that were 15- to 30-fold below rate constants for cleavage of oligonucleotides in vitro . Careful analysis of structural features that influence cleavage efficiency points to substrate binding as the rate-determining step in the intracellular cleavage pathway . Second-order rate constants for intermolecular cleavage agree well with diffusion coefficients reported for U3 snoRNPs in vivo suggesting that complex formation between chimeric ribozyme and substrate snoRNPs in yeast nuclei is diffusion limited.

RNA, 2004 May, 10(5), 813 - 27
Role of the yeast Rrp1 protein in the dynamics of pre-ribosome maturation; Horsey EW et al.; The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necessary for biogenesis of 60S ribosomal subunits . Processing of 27S pre-ribosomal RNA to mature 25S rRNA is blocked and 60S subunits are deficient in the temperature-sensitive rrp1-1 mutant . We have used recent advances in proteomic analysis to examine in more detail the function of Rrp1p in ribosome biogenesis . We show that Rrp1p is a nucleolar protein associated with several distinct 66S pre-ribosomal particles . These pre-ribosomes contain ribosomal proteins plus at least 28 nonribosomal proteins necessary for production of 60S ribosomal subunits . Inactivation of Rrp1p inhibits processing of 27SA(3) to 27SB(S) pre-rRNA and of 27SB pre-rRNA to 7S plus 25.5S pre-rRNA . Thus, in the rrp1-1 mutant, 66S pre-ribosomal particles accumulate that contain 27SA(3) and 27SB(L) pre-ribosomal RNAs.

J Immunol Methods, 2004 Apr, 287(1-2), 147 - 58
Domain-level antibody epitope mapping through yeast surface display of epidermal growth factor receptor fragments; Cochran JR et al.; Individual domains from extracellular proteins are potential reagents for biochemical characterization of ligand/receptor interactions and antibody binding sites . Here, we describe an approach for the identification and characterization of stable protein domains with cell surface display in Saccharomyces cerevesiae, using the epidermal growth factor receptor (EGFR) as a model system . Fragments of the EGFR were successfully expressed on the yeast cell surface . The yeast-displayed EGFR fragments were properly folded, as assayed with conformationally specific EGFR antibodies . Heat denaturation of yeast-displayed EGFR proteins distinguished between linear and conformational antibody epitopes . In addition, EGFR-specific antibodies were categorized based on their ability to compete ligand binding, which has been shown to have therapeutic implications . Overlapping EGFR antibody epitopes were determined based on a fluorescent competitive binding assay . Yeast surface display is a useful method for identifying stable folded protein domains from multidomain extracellular receptors, as well as characterizing antibody binding epitopes, without the need for soluble protein expression and purification.

Biochem Biophys Res Commun, 2004 May 14, 317(4), 1138 - 43
The fission yeast ptr1+ gene involved in nuclear mRNA export encodes a putative ubiquitin ligase; Andoh T et al.; Fission yeast ptr1-1 is one of the mRNA transport mutants that accumulate poly(A)+ RNA in the nuclei at the nonpermissive temperature . We found that the ptr1+ gene encodes a homolog of Saccharomyces cerevisiae Tom1p, a hect type ubiquitin ligase . In ptr1-1, a conserved amino acid in the hect domain of Ptr1p is mutated . The ptr1+ gene is essential for growth and its mutation did not affect nuclear protein export . A ptr1-1 rae1-167 double mutant showed a synthetic effect on a growth defect, indicating that Ptr1p functionally interacts with an essential mRNA export factor Rae1p . We also isolated a multi-copy suppressor for ptr1-1 and found that it is the mpd2+ gene isolated as a multi-copy suppressor of cdc7-PD1.

Biochem Biophys Res Commun, 2004 May 14, 317(4), 1017 - 22
Self-association of the hepatitis B virus X protein in the yeast two-hybrid system; Reddi HV et al.; Self-association of the transactivator HBx protein of hepatitis B virus was investigated using the yeast two-hybrid system . Expression vectors for the full-length HBx (X0) and its truncated mutants (X15 and X16) were constructed by separately ligating the DNA-binding (BD) and transactivation domains (AD) of Gal4 . Co-transformants of the BD and AD constructs of HBx were selected using defined minimal medium and analyzed for the reconstitution of beta-galactosidase activity . No two-hybrid interaction was observed either between the full-length HBx molecules or its highly truncated mutant X16 . However, a strong functional interaction between X0 and X15, X0 and X16, and X15 and X16 suggested that HBx could self-associate in a cellular environment through its carboxy-terminal region.

FEBS Lett, 2004 Apr 23, 564(1-2), 24 - 34
Yeast mating for combinatorial Fab library generation and surface display; Weaver-Feldhaus JM et al.; Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single chain Fv antibodies for increased affinity and thermal stability, and more recently for the display and screening of a non-immune library . In this paper, we describe an elegant and simple method for constructing large combinatorial Fab libraries for display on the surface of Saccharomyces cerevisiae, from modestly sized, and easily constructed, heavy and light chain libraries . To this end, we have constructed a set of yeast strains and a two vector system for heavy chain and light chain surface display of Fab fragments with free native amino termini . Through yeast mating of the haploid libraries, a very large heterodimeric immune Fab library was displayed on the diploids and high affinity antigen specific Fabs were isolated from the library.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 May 5, 804(1), 25 - 30
Separation and determination of astaxanthin from microalgal and yeast samples by molecularly imprinted microspheres; Lai JP et al.; In this work, molecularly imprinted microspheres (MIMs) were synthesized by aqueous microsuspension polymerization using astaxanthin (3,3'-dihydroxy-beta,beta'-carotene-4,4'-dione) as imprinting molecule . The MIMs obtained were subsequently packed into the stainless steel column and the chromatographic characterization of the column was investigated . The effects of pH and composition of the mobile phase on the retention factor (k') were investigated in detail . The mixture of methanol and dichloromethane (DCM) (8:2, v/v) was used as mobile phase A while the mixture of methanol and water (5:5, v/v) as mobile phase B . The separation of astaxanthin and zeaxanthin (3,3'-dihydroxyl-beta-carotene) was obtained when the concentration of mobile phase B was higher than 30% (v/v) due to their strong lipophilicity . The method developed was successfully applied to separate astaxanthin in the saponified samples of the microalga Haematococcus pluvialis and the yeast Phaffia rhodozyma . The recovery of adding 40 mg astaxanthin to 1.0 g microalgal sample was 95.5% with an R.S.D . (n =5) of 5.3% . The results of determination of astaxanthin in the microalga and the yeast were 3.7% (R.S.D (n = 1.5%, n = 9) and 0.041% (R.S.D n= 7.3%, n = 9), respectively.

Environ Pollut, 2000 Jul, 109(1), 43 - 52
A new sensitive test based on yeast cells for studying environmental pollution; Terziyska A et al.; Different tests based on yeast cells were developed for determination of mutagenic/carcinogenic action; however, they all showed lower sensitivity compared to bacterial tests, the main reason for this being the limited permeability of yeast cells . We found that general permeability of Saccharomyces cerevisiae cells can be increased by mutation and on this basis we developed a more sensitive test . The aim of this study was to prove the applicability of our test, called D7ts1, in environmental studies . Soil, water and air samples were taken during 1998 from regions in Bulgaria with declared low, average or high pollution levels and investigated for presence of mutagenic/carcinogenic activities in the bacterial test of Ames, the yeast D7 test of Zimmermann and our new D7ts1 test . Results obtained evidenced the following conclusions: (1) the usage of D7ts1 test instead of D7 test permits a clearer measurement of positive samples and detects mutagenic/carcinogenic activities undetectable by D7 test; (2) all samples with positive Ames test were positive in the D7ts1 test; however, some samples, clearly positive in the D7ts1, were negative in the Ames test; therefore, the simultaneous usage of D7ts1 and Ames tests in environmental studies is advantageous because it detects dangers for the human health activities to which bacterial cells do not respond; and (3) regions in Bulgaria declared clean were found to be polluted; particularly troubled are the whole-year positive data in the three tests for air samples from a 'clean' region.

Environ Pollut, 1996, 91(2), 169 - 75
Comparison of the effects of seleno-l-methionine, seleno-dl-methionine, and selenized yeast on reproduction of mallards; Heinz GH et al.; The toxicities of seleno-l-methionine, seleno-dl-methionine, and selenized yeast were compared . Ten pairs of mallards were fed a control diet and 15 pairs were fed diets containing 10 ppm selenium as seleno-dl-methionine, seleno-l-methionine, or selenized yeast . Hatching of fertile eggs was significantly lower for females fed 10 ppm selenium as seleno-dl-methionine (7.6%) and seleno-l-methionine (6.4%) than for controls (41.3%) . Survival of ducklings was lower when their parents had been fed 10 ppm selenium as seleno-l-methionine (20.0%) than for controls (98.4%) . The number of 6-day-old ducklings produced per female was significantly lower for mallards fed 10 ppm selenium as seleno-dl-methionine (0.47) or selenized yeast (2.67) than for controls (6.10), and was significantly lower for mallards fed seleno-l-methionine (0.13) than for mallards fed selenized yeast . The eighth eggs of females fed the dl or l forms of selenomethionine contained means of 9.2 and 8.9 ppm selenium, wet weight; these means were higher than the mean (6.6 ppm) for females fed selenized yeast . Among embryos that died at 7 days of age or older, the percentage of embryos that were deformed was 1.3% for controls, 24.6% for seleno-dl-methionine, 28.2% for seleno-l-methionine, and 11.0% for selenized yeast . The results suggested that seleno-dl-methionine and seleno-l-methionine were of similar toxicity and were both more toxic than selenium from selenized yeast.

Mol Biol Cell, 2004 Jul, 15(7), 3015 - 30 Epub 2004 Apr 16.
Cell cycle-dependent nuclear localization of yeast RNase III is required for efficient cell division; Catala M et al.; Members of the double-stranded RNA-specific ribonuclease III (RNase III) family were shown to affect cell division and chromosome segregation, presumably through an RNA interference-dependent mechanism . Here, we show that in Saccharomyces cerevisiae, where the RNA interference machinery is not conserved, an orthologue of RNase III (Rnt1p) is required for progression of the cell cycle and nuclear division . The deletion of Rnt1p delayed cells in both G1 and G2/M phases of the cell cycle . Nuclear division and positioning at the bud neck were also impaired in Deltarnt1 cells . The cell cycle defects were restored by the expression of catalytically inactive Rnt1p, indicating that RNA cleavage is not essential for cell cycle progression . Rnt1p was found to exit from the nucleolus to the nucleoplasm in the G2/M phase, and perturbation of its localization pattern delayed the progression of cell division . A single mutation in the Rnt1p N-terminal domain prevented its accumulation in the nucleoplasm and slowed exit from mitosis without any detectable effects on RNA processing . Together, the data reveal a new role for a class II RNase III in the cell cycle and suggest that at least some members of the RNase III family possess catalysis-independent functions.

J Biol Chem, 2004 Jul 9, 279(28), 29469 - 77 Epub 2004 Apr 16.
Cell surface expression of the prion protein in yeast does not alter copper utilization phenotypes; Li A et al.; Prion diseases are fatal neurodegenerative disorders that result from conversion of a normal, cell surface glycoprotein (PrP(C)) into a conformationally altered isoform (PrP(Sc)) that is thought to be infectious . Although a great deal is known about the role of PrP(Sc) in the disease process, the physiological function of PrP(C) has remained enigmatic . In this report, we have used the yeast Saccharomyces cerevisiae to test one hypothesized function of PrP(C), as a receptor for the uptake or efflux of copper ions . We first modified the PrP signal peptide by replacing its hydrophobic core with the signal sequence from the yeast protein dipeptidyl aminopeptidase B, so that the resulting protein was targeted cotranslationally to the secretory pathway when synthesized in yeast . PrP molecules with the modified signal peptide were efficiently glycosylated, glycolipid-anchored, and localized to the plasma membrane . We then tested whether PrP expression altered the growth deficiency phenotypes of yeast strains harboring deletions in genes that encode key components of copper utilization pathways, including transporters, chaperones, pumps, reductases, and cuproenzymes . We found that PrP did not rescue any of these mutant phenotypes, arguing against a direct role for the protein in copper utilization . Our results provide further clarification of the physiological function of PrP(C), and lay the groundwork for using PrP-expressing yeast to study other aspects of prion biology.

Assay Drug Dev Technol, 2003 Feb, 1(1 Pt 2), 147 - 60
Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases; Hanley K et al.; Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult . The activity assays are labor-intensive and not amenable to parallel screening . Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity . Plant virus gene expression systems have been utilized for a diverse group of organisms . In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity . Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression . Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves . No phenotype was detected on negative control constructs . YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods . The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library . A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants . All YlALK gene constructs scored positive in the randomized screen . Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis . This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.

Bioinformatics, 2004 Jun 12, 20(9), 1413 - 5 Epub 2004 Apr 15.
PINdb: a database of nuclear protein complexes from human and yeast; Luc PV et al.; SUMMARY: Proteins Interacting in the Nucleus database (PINdb) is a database of protein complexes purified from the nucleus of human and yeast cells . It is compiled from the published literature and existing databases . Currently, PINdb contains mostly protein complexes that may be involved in gene transcription . To facilitate comparative analyses and identification of protein complexes, the compositional information is integrated with standardized gene nomenclature, annotation and protein sequences from public databases . The PINdb web interface provides a number of tools for (1) comparison of protein complexes, (2) search for a protein complex by its published name or by a partial list of its components and (3) browsing specific subsets or a functional classification of the complexes . Availablity: http://pin.mskcc.org

J Immunol Methods, 2004 Mar, 286(1-2), 141 - 53
High efficiency recovery and epitope-specific sorting of an scFv yeast display library; Siegel RW et al.; In order to more productively utilize the rich source of antigen-specific reagents present in the previously described non-immune single chain fragment variable (scFv) yeast display library, one must be able to efficiently isolate and characterize clones within the library . To this end, we have developed and validated a magnetic bead sorting technique utilizing the Miltenyi Macs system to recover greater than 90% of the antigen-specific clones present in the library . In combination with flow cytometry, we rapidly reduced diversity and enriched for antigen-specific clones in three rounds of selection . Furthermore, we demonstrate the use of pre-existing monoclonal antibodies (mAbs) for antigen labeling and subsequent flow cytometric sorting and characterization of epitope-specific scFv . Combining these two improvements in library screening allowed isolation and characterization of three epitope-specific scFv, including a previously uncharacterized epitope to a 6-kDa protein, epidermal growth factor.

Traffic, 2004 Mar, 5(3), 165 - 80
The internalization of yeast Ste6p follows an ordered series of events involving phosphorylation, ubiquitination, recognition and endocytosis; Kelm KB et al.; A general pathway for the internalization of plasma membrane proteins that involves phosphorylation, ubiquitination, recognition and endocytosis has recently emerged from multiple studies in yeast . We refer to this series of events as the PURE pathway . Here we investigate whether the yeast a-factor transporter Ste6p, an ATP-binding cassette protein, utilizes the PURE pathway . Deletion of a 52-amino acid sequence (the 'A box') within the linker region of Ste6p has previously been shown to block ubiquitination and endocytosis (Kolling R, Losko S . EMBO J 1997; 16:2251-2261) . Using wild-type and mutant forms of GFP-tagged Ste6p, we identified two residues (T(613) and S(623)) within the A box as likely sites of Ste6p phosphorylation important for internalization . Mutation of these residues to alanine blocked ubiquitination and endocytosis of Ste6p, similar to the effect of deleting the entire A box, while substitution with glutamic acid (to mimic phosphorylation) suppressed the ubiquitination and endocytic defects . Importantly, a translational fusion of monoubiquitin to the C-terminus of Ste6p-T(613)A, S(623)A or Ste6p-DeltaA restored endocytosis, providing strong evidence that the role of phosphorylation is to direct ubiquitination, which in turn is a critical signal for Ste6p internalization . We also identified multiple (five) lysine residues in the linker that are important for Ste6p ubiquitination . Our results demonstrate that Ste6p follows the PURE pathway and that GFP-tagged Ste6p provides a powerful model protein for studies of endocytosis and post-endocytic events in yeast.

J AOAC Int, 2004 Jan-Feb, 87(1), 225 - 32
Speciation and bioavailability of selenium in yeast-based intervention agents used in cancer chemoprevention studies; Larsen EH et al.; This study investigated the speciation and bioavailability of selenium in yeast-based intervention agents from multiple manufacturers from several time points . Sources of selenized yeast included Nutrition 21 (San Diego, CA), which supplied the Nutritional Prevention of Cancer (NPC) Trial from 1981-1996; Cypress Systems (Fresno, CA; 1997-1999); and Pharma Nord (Vejle, Denmark; 1999-2000), which supplied the Prevention of Cancer by Intervention by Selenium (PRECISE) Trial pilot studies . The low-molecular-selenium species were liberated from the samples by proteolytic hydrolysis followed by separation by ion exchange liquid chromatography and detection by inductively coupled plasma-mass spectrometry . The results for the NPC tablets showed that selenomethionine, together with 3 unidentified selenium compounds, were predominant in the sample hydrolysates . The relative amounts of the 4 selenium species varied (p < 0.05) among several of the 7 tablet batches used during the course of the NPC Trial . In comparison, 5 batches of more recently produced selenized yeasts, which were used as a source of selenium in the PRECISE and other trials, contained less of the unknown compounds and more selenomethionine at 54-60% of the total selenium in the yeasts . One batch of yeast, however (from 1985), which originated from the same producer as the yeast used in the NPC tablets, contained only 27% of selenium in the sample as selenomethionine . Human subjects receiving 200 microg selenium/day in the UK PRECISE Pilot Trial showed a higher concentration (p < 0.01) and higher increase from baseline in plasma selenium than did the same dosage used in the NPC Trial . Differences in intake, speciation, or bioavailability of selenium from the yeast-based supplements in the population groups studied may explain this . Furthermore, the selenium concentration in whole blood from the Danish PRECISE Pilot Trial was higher (p < 0.001) than that obtained with synthetic L-selenomethionine in a comparable group of Danes, both groups having been treated with 300 microg selenium/day.

Mol Cell Biol, 2004 May, 24(9), 4019 - 31
Anatomy and dynamics of DNA replication fork movement in yeast telomeric regions; Makovets S et al.; Replication initiation and replication fork movement in the subtelomeric and telomeric DNA of native Y' telomeres of yeast were analyzed using two-dimensional gel electrophoresis techniques . Replication origins (ARSs) at internal Y' elements were found to fire in early-mid-S phase, while ARSs at the terminal Y' elements were confirmed to fire late . An unfired Y' ARS, an inserted foreign (bacterial) sequence, and, as previously reported, telomeric DNA each were shown to impose a replication fork pause, and pausing is relieved by the Rrm3p helicase . The pause at telomeric sequence TG(1-3) repeats was stronger at the terminal tract than at the internal TG(1-3) sequences located between tandem Y' elements . We show that the telomeric replication fork pause associated with the terminal TG(1-3) tracts begins approximately 100 bp upstream of the telomeric repeat tract sequence . Telomeric pause strength was dependent upon telomere length per se and did not require the presence of a variety of factors implicated in telomere metabolism and/or known to cause telomere shortening . The telomeric replication fork pause was specific to yeast telomeric sequence and was independent of the Sir and Rif proteins, major known components of yeast telomeric heterochromatin.

Mol Cell Biol, 2004 May, 24(9), 3907 - 17
Structural features of nucleosomes reorganized by yeast FACT and its HMG box component, Nhp6; Rhoades AR et al.; The Saccharomyces cerevisiae Spt16/Cdc68, Pob3, and Nhp6 proteins (SPN or yFACT) bind to and alter nucleosomes in vitro, providing a potential explanation for their importance in both transcription and replication in vivo . We show that nucleosomes bound by either Nhp6 alone or the yFACT complex remain largely intact and immobile but are significantly reorganized, as indicated by changes in the pattern of sensitivity to DNase I and enhanced digestion by some restriction endonucleases . In contrast, yFACT enhanced access to exonuclease III only at very high levels of enzyme, suggesting that the DNA near the entry and exit sites of nucleosomes is largely unperturbed and that the position of the histone octamers relative to the DNA is not altered during reorganization . DNase I sensitivity was enhanced at sites clustered near the center of the nucleosomal DNA, away from the entry and exit points, and the pattern of nuclease sensitivity was only mildly affected by the configuration of linker extensions, further indicating that linkers play only a minor role in the reorganization of nucleosomes by yFACT . The DNA in contact with H2A-H2B dimers is therefore the region whose nuclease sensitivity was the least affected by yFACT reorganization . The most dramatic changes in nucleosome structure occurred when Spt16-Pob3 and the HMG box protein Nhp6 were both present, but Nhp6 alone altered DNase I sensitivity at some specific sites, supporting an independent role for this class of proteins in the general management of chromatin properties . yFACT activity does not require ATP hydrolysis and does not alter the position of nucleosomes, indicating that it acts through a mechanism distinct from chromatin remodeling . The results presented here suggest instead that yFACT promotes polymerase progression by reorganizing nucleosome cores into a less inhibitory conformation in which the properties of DNA sequences near the center of the nucleosomes are altered.

Mol Cell Biol, 2004 May, 24(9), 3660 - 9
A novel protein with similarities to Rb binding protein 2 compensates for loss of Chk1 function and affects histone modification in fission yeast; Ahmed S et al.; The conserved protein kinase Chk1 mediates cell cycle progression and consequently the ability of cells to survive when exposed to DNA damaging agents . Cells deficient in Chk1 are hypersensitive to such agents and enter mitosis in the presence of damaged DNA, whereas checkpoint-proficient cells delay mitotic entry to permit time for DNA repair . In a search for proteins that can improve the survival of Chk1-deficient cells exposed to DNA damage, we identified fission yeast Msc1, which is homologous to a mammalian protein that binds to the tumor suppressor Rb (RBP2) . Msc1 and RBP2 each possess three PHD fingers, domains commonly found in proteins that influence the structure of chromatin . Msc1 is chromatin associated and coprecipitates a histone deacetylase activity, a property that requires the PHD fingers . Cells lacking Msc1 have a dramatically altered histone acetylation pattern, exhibit a 20-fold increase in global acetylation of histone H3 tails, and are readily killed by trichostatin A, an inhibitor of histone deacetylases . We postulate that Msc1 plays an important role in regulating chromatin structure and that this function modulates the cellular response to DNA damage.

Mol Cell Biol, 2004 May, 24(9), 3648 - 59
Phosphorylation of the yeast heat shock transcription factor is implicated in gene-specific activation dependent on the architecture of the heat shock element; Hashikawa N et al.; Heat shock transcription factor (HSF) binds to the heat shock element (HSE) and regulates transcription, where the divergence of HSE architecture provides gene- and stress-specific responses . The phosphorylation state of HSF, regulated by stress, is involved in the activation and inactivation of the transcription activation function . A domain designated as CTM (C-terminal modulator) of the Saccharomyces cerevisiae HSF is required for the activation of genes containing atypical HSE but not typical HSE . Here, we demonstrate that CTM function is conserved among yeast HSFs and is necessary not only for HSE-specific activation but also for the hyperphosphorylation of HSF upon heat shock . Moreover, both transcription and phosphorylation defects due to CTM mutations were restored concomitantly by a set of intragenic suppressor mutations . Therefore, the hyperphosphorylation of HSF is correlated with the activation of genes with atypical HSE but is not involved in that of genes with typical HSE . The function of CTM was circumvented in an HSF derivative lacking CE2, a yeast-specific repression domain . Taken together, we suggest that CTM alleviates repression by CE2, which allows HSF to be heat-inducibly phosphorylated and presume that phosphorylation is a prerequisite for the activator function of HSF when it binds to an atypical HSE.

Mol Cell Biol, 2004 May, 24(9), 3596 - 606
High-level activation of transcription of the yeast U6 snRNA gene in chromatin by the basal RNA polymerase III transcription factor TFIIIC; Shivaswamy S et al.; Transcription of the U6 snRNA gene (SNR6) in Saccharomyces cerevisiae by RNA polymerase III (pol III) requires TFIIIC and its box A and B binding sites . In contrast, TFIIIC has little or no effect on SNR6 transcription with purified components in vitro due to direct recognition of the SNR6 TATA box by TFIIIB . When SNR6 was assembled into chromatin in vitro by use of the Drosophila melanogaster S-190 extract, transcription of these templates with highly purified yeast pol III, TFIIIC, and TFIIIB displayed a near-absolute requirement for TFIIIC but yielded a 5- to 15-fold-higher level of transcription relative to naked DNA (>100-fold activation over repressed chromatin) . Analysis of chromatin structure demonstrated that TFIIIC binding leads to remodeling of U6 gene chromatin, resulting in positioning of a nucleosome between boxes A and B . The resulting folding of the intervening DNA into the nucleosome could bring the suboptimally spaced SNR6 box A and B elements into greater proximity and thus facilitate activation of transcription . In the absence of ATP, however, the binding of TFIIIC to box B in chromatin was not accompanied by remodeling and the transcription activation was approximately 35% of that seen in its presence, implying that both TFIIIC binding and ATP-dependent chromatin remodeling were required for the full activation of the gene . Our results suggest that TFIIIC, which is a basal transcription factor of pol III, also plays a direct role in remodeling chromatin on the SNR6 gene.

Mutat Res, 2004 May, 566(3), 231 - 48
New insights into the mechanism of homologous recombination in yeast; Aylon Y et al.; Genome stability is of primary importance for the survival and proper functioning of all organisms . Double-strand breaks (DSBs) arise spontaneously during growth, or can be created by external insults . Repair of DSBs by homologous recombination provides an efficient and fruitful pathway to restore chromosomal integrity . Exciting new work in yeast has lately provided insights into this complex process . Many of the proteins involved in recombination have been isolated and the details of the repair mechanism are now being unraveled at the molecular level . In this review, we focus on recent studies which dissect the recombinational repair of a single broken chromosome . After DSB formation, a decision is made regarding the mechanism of repair (recombination or non-homologous end-joining) . This decision is under genetic control . Once committed to the recombination pathway, the broken chromosomal ends are resected by a still unclear mechanism in which the DNA damage checkpoint protein Rad24 participates . At this stage several proteins are recruited to the broken ends, including Rad51p, Rad52p, Rad55p, Rad57p, and possibly Rad54p . A genomic search for homology ensues, followed by strand invasion, promoted by the Rad51 filament with the participation of Rad55p, Rad57p and Rad54p . DNA synthesis then takes place, restoring the resected ends . Crossing-over formation depends on the length of the homologous recombining sequences, and is usually counteracted by the activity of the mismatch repair system . Given the conservation of the repair mechanisms and genes throughout evolution, these studies have profound implications for other eukaryotic organisms.

Arch Biochem Biophys, 2004 May 1, 425(1), 14 - 24
Glutathione and thioredoxin peroxidases mediate susceptibility of yeast mitochondria to Ca(2+)-induced damage; Monteiro G et al.; The effect of thioredoxin peroxidases on the protection of Ca(2+)-induced inner mitochondrial membrane permeabilization was studied in the yeast Saccharomyces cerevisiae using null mutants for these genes . Since deletion of a gene can promote several other effects besides the absence of the respective protein, characterizations of the redox state of the mutant strains were performed . Whole cellular extracts from all the mutants presented lower capacity to decompose H(2)O(2) and lower GSH/GSSG ratios, as expected for strains deficient for peroxide-removing enzymes . Interestingly, when glutathione contents in mitochondrial pools were analyzed, all mutants presented lower GSH/GSSG ratios than wild-type cells, with the exception of DeltacTPxI strain (cells in which cytosolic thioredoxin peroxidase I gene was disrupted) that presented higher GSH/GSSG ratio . Low GSH/GSSG ratios in mitochondria increased the susceptibility of yeast to damage induced by Ca(2+) as determined by membrane potential and oxygen consumption experiments . However, H(2)O(2) removal activity appears also to be important for mitochondria protection against permeabilization because exogenously added catalase strongly inhibited loss of mitochondrial potential . Moreover, exogenously added recombinant peroxiredoxins prevented inner mitochondrial membrane permeabilization . GSH/GSSG ratios decreased after Ca(2+) addition, suggesting that reactive oxygen species (ROS) probably mediate this process . Taken together our results indicate that both mitochondrial glutathione pools and peroxide-removing enzymes are key components for the protection of yeast mitochondria against Ca(2+)-induced damage.

J Biol Chem, 2004 Jun 18, 279(25), 25986 - 94 Epub 2004 Apr 12.
A mutational study in the transmembrane domain of Ccc2p, the yeast Cu(I)-ATPase, shows different roles for each Cys-Pro-Cys cysteine; Lowe J et al.; Ccc2p is homologous to the human Menkes and Wilson copper ATPases and is herein studied as a model for human copper transport . Most studies to date have sought to understand how mutations in the human Menkes or Wilson genes impair copper homeostasis and induce disease . Here we analyze whether eight conserved amino acids of the transmembrane domain are important for copper transport . Wild-type Ccc2p and variants were expressed in a ccc2-Delta yeast strain to check whether they were able to restore copper transport by complementation . Wild-type Ccc2p and variants were also expressed in Sf9 cells using baculovirus to study their enzymatic properties on membrane preparations . The latter system allowed us to measure a copper-activated ATPase activity of about 20 nmol/mg/min for the wild-type Ccc2p at 37 degrees C . None of the variants was as efficient as the wild type in restoring copper homeostasis . The mutation of each cysteine of the (583)CPC(585) motif into a serine resulted in nonfunctional proteins that could not restore copper homeostasis in yeast and had no ATPase activity . Phosphorylation by ATP was still possible with the C583S variant, although it was not possible with the C585S variant, suggesting that the cysteines of the CPC motif have a different role in copper transport . Cys(583) would be necessary for copper dissociation and/or enzyme dephosphorylation and Cys(585) would be necessary for ATP phosphorylation, suggesting a role in copper binding.

J Biol Chem, 2004 Jun 11, 279(24), 25353 - 8 Epub 2004 Apr 12.
Ebp2p, the yeast homolog of Epstein-Barr virus nuclear antigen 1-binding protein 2, interacts with factors of both the 60 S and the 40 s ribosomal subunit assembly; Shirai C et al.; Ebp2p, the yeast homolog of human Epstein-Barr virus nuclear antigen 1-binding protein 2, is essential for biogenesis of the 60 S ribosomal subunit . Two-hybrid screening exhibited that, in addition to factors necessary for assembly of the 60 S subunit, Ebp2p interacts with Rps16p, ribosomal protein S16, and the 40 S ribosomal subunit assembly factor, Utp11p, as well as Yil019w, the function of which was previously uncharacterized . Depletion of Yil019w resulted in reduction in levels of both of 18 S rRNA and 40 S ribosomal subunit without affecting levels of 25 S rRNA and 60 S ribosomal subunits . 35 S pre-rRNA and aberrant 23 S RNA accumulated, indicating that pre-rRNA processing at sites A(0)-A(2) is inhibited when Yil019w is depleted . Each combination from Yil019w, Utp11p, and Rps16p showed two-hybrid interaction.

Mycoses, 2004 Apr, 47(3-4), 143 - 9
Comparative serological evaluation of 10 Blastomyces dermatitidis yeast phase lysate antigens from different sources; Abuodeh RO et al.; Yeast phase lysate antigens from 10 isolates of Blastomyces dermatitidis (dog, ERC-2 and T-58; T-27, polar bear; woodpile, ER-3; bat lung, 48938; human, B5929, B5895, B5896, B5931, and CAPP) from different geographical regions, in addition to a Histoplasma capsulatum (G217B) lysate preparation were compared with respect to their reactivity against serum specimens from dogs, rabbits and humans positive for blastomycosis using an indirect enzyme-linked immunosorbent assay . In addition, the lysate antigens were also assayed against histoplasmosis-positive human serum samples to study their cross-reactivity . Variable results were obtained with T-58 and T-27 exhibiting the greatest reactivity . We also noticed that the lysate did not react consistently to serum samples across species with lesser reactivity evidenced when testing dog sera . Finally, T-58 gave the highest cross-reactivity with histoplasmosis-positive sera . The study may prove valuable in the development of antigen candidates for blastomycosis serodiagnosis.

Eukaryot Cell, 2004 Apr, 3(2), 264 - 76
Plasmodium falciparum histone acetyltransferase, a yeast GCN5 homologue involved in chromatin remodeling; Fan Q et al.; The yeast transcriptional coactivator GCN5 (yGCN5), a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcriptional activation . Like other eukaryotes, the malaria parasite DNA is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes . Here we show that GCN5 is conserved in Plasmodium species and that the most homologous regions are within the HAT domain and the bromodomain . The Plasmodium falciparum GCN5 homologue (PfGCN5) is spliced with three introns, encoding a protein of 1,464 residues . Mapping of the ends of the PfGCN5 transcript suggests that the mRNA is 5.2 to 5.4 kb, consistent with the result from Northern analysis . Using free core histones, we determined that recombinant PfGCN5 proteins have conserved HAT activity with a substrate preference for histone H3 . Using substrate-specific antibodies, we determined that both Lys-8 and -14 of H3 were acetylated by the recombinant PfGCN5 . In eukaryotes, GCN5 homologues interact with yeast ADA2 homologues and form large multiprotein HAT complexes . We have identified an ADA2 homologue in P . falciparum, PfADA2 . Yeast two-hybrid and in vitro binding assays verified the interactions between PfGCN5 and PfADA2, suggesting that they may be associated with each other in vivo . The conserved function of the HAT domain in PfGCN5 was further illustrated with yeast complementation experiments, which showed that the PfGCN5 region corresponding to the full-length yGCN5 could partially complement the yGCN5 deletion mutation . Furthermore, a chimera comprising the PfGCN5 HAT domain fused to the remainder of yeast GCN5 (yGCN5) fully rescued the yGCN5 deletion mutant . These data demonstrate that PfGCN5 is an authentic GCN5 family member and may exist in chromatin-remodeling complexes to regulate gene expression in P . falciparum.

Microbiology, 2004 Apr, 150(Pt 4), 929 - 34
Overexpression of HAP4 in glucose-derepressed yeast cells reveals respiratory control of glucose-regulated genes; Lascaris R et al.; A link between control of respiration and glucose repression in yeast is reported . The HAP4 gene was overexpressed in a Delta mig1 deletion background, generating a mutant in which respiratory function is stimulated and glucose repression is diminished . Although this combination does not result in derepression of genes encoding proteins involved in respiratory function, it nevertheless generates resistance against 2-deoxyglucose and hence contributes to more derepressed growth characteristics . Unexpectedly, overexpression of HAP4 in the Delta mig1 deletion strain causes strong repression of several target genes of the Mig1p repressor . Repression is not restricted to glucose growth conditions and does not require the glucose repressors Mig2p or Hxk2p . It was observed that expression of the SUC2 gene is transiently repressed after glucose is added to respiratory-growing Delta mig1 cells . Additional overexpression of HAP4 prevents release from this novel repressed state . The data presented show that respiratory function controls transcription of genes required for the metabolism of alternative sugars . This respiratory feedback control is suggested to regulate the feed into glycolysis in derepressed conditions.

J Exp Bot, 2004 May, 55(399), 1149 - 52 Epub 2004 Apr 08.
Molecular characterization of OsRAD21-1, a rice homologue of yeast RAD21 essential for mitotic chromosome cohesion; Zhang LR et al.; Rad21/Rec8 is an important component and key regulator of cohesins . A RAD21-like gene from rice (Oryza sativa L . ssp . japonica) has been cloned and termed OsRAD21-1 . OsRAD21-1 is a single-copy gene in the rice genome and is expressed in the entire plant . OsRad21-1 consists of 1055 amino acid residues and is the largest of the Rad21/Rec8 family identified to date . Based on sequence similarity comparison with other members of this family and gene expression patterns, it is concluded that OsRad21 is a rice orthologue of yeast Rad21.

EMBO Rep, 2004 May, 5(5), 497 - 502 Epub 2004 Apr 08.
The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange; Prado F et al.; Histone chaperone Asf1 participates in heterochromatin silencing, DNA repair and regulation of gene expression, and promotes the assembly of DNA into chromatin in vitro . To determine the influence of Asf1 on genetic stability, we have analysed the effect of asf1Delta on homologous recombination . In accordance with a defect in nucleosome assembly, asf1Delta leads to a loss of negative supercoiling in plasmids . Importantly, asf1Delta increases spontaneous recombination between inverted DNA sequences . This increase correlates with an accumulation of double-strand breaks (DSBs) as determined by immunodetection of phosphorylated histone H2A and fluorescent detection of Rad52-YFP foci during S and G2/M phases . In addition, asf1Delta shows high levels of sister chromatid exchange (SCE) and is proficient in DSB-induced SCE as determined by physical analysis . Our results suggest that defective chromatin assembly caused by asf1Delta leads to DSBs that can be repaired by SCE, affecting genetic stability.

Mol Biol Evol, 2004 Jul, 21(7), 1171 - 6 Epub 2004 Apr 07.
Functional evolution of the yeast protein interaction network; Kunin V et al.; Protein interactions are central to most biological processes . We investigated the dynamics of emergence of the protein interaction network of Saccharomyces cerevisiae by mapping origins of proteins on an evolutionary tree . We demonstrate that evolutionary periods are characterized by distinct connectivity levels of the emerging proteins . We found that the most-connected group of proteins dates to the eukaryotic radiation, and the more ancient group of pre-eukaryotic proteins is less connected . We show that functional classes have different average connectivity levels and that the time of emergence of these functional classes parallels the observed connectivity variation in evolution . We take these findings as evidence that the evolution of function might be the reason for the differences in connectivity throughout evolutionary time . We propose that the understanding of the mechanisms that generate the scale-free protein interaction network, and possibly other biological networks, requires consideration of protein function.

Proc Natl Acad Sci U S A, 2004 Mar 30, 101(13), 4525 - 30 Epub 2004 Mar 15.
Yeast genome-wide drug-induced haploinsufficiency screen to determine drug mode of action; Baetz K et al.; Methods to systematically test drugs against all possible proteins in a cell are needed to identify the targets underlying their therapeutic action and unwanted effects . Here, we show that a genome-wide drug-induced haploinsufficiency screen by using yeast can reveal drug mode of action in yeast and can be used to predict drug mode of action in human cells . We demonstrate that dihydromotuporamine C, a compound in preclinical development that inhibits angiogenesis and metastasis by an unknown mechanism, targets sphingolipid metabolism . The systematic, unbiased and genome-wide nature of this technique makes it attractive as a general approach to identify cellular pathways affected by drugs.

Proc Natl Acad Sci U S A, 2004 Mar 30, 101(13), 4441 - 6 Epub 2004 Mar 22.
Evolving potassium channels by means of yeast selection reveals structural elements important for selectivity; Bichet D et al.; Potassium channels are widely distributed . To serve their physiological functions, such as neuronal signaling, control of insulin release, and regulation of heart rate and blood flow, it is essential that K+ channels allow K+ but not the smaller and more abundant Na+ ions to go through . The narrowest part of the channel pore, the selectivity filter formed by backbone carbonyls of the GYG-containing K+ channel signature sequence, approximates the hydration shell of K+ ions . However, the K+ channel signature sequence is not sufficient for K+ selectivity . To identify structural elements important for K+ selectivity, we randomly mutagenized the G protein-coupled inwardly rectifying potassium channel 3.2 (GIRK2) bearing the S177W mutation on the second transmembrane segment . This mutation confers constitutive channel activity but abolishes K+ selectivity and hence the channel's ability to complement the K+ transport deficiency of Deltatrk1Deltatrk2 mutant yeast . S177W-containing GIRK2 mutants that support yeast growth in low-K+ medium contain multiple suppressors, each partially restoring K+ selectivity to S177W-containing double mutants . These suppressors include mutations in the first transmembrane segment and the pore helix, likely exerting long-range actions to restore K+ selectivity, as well as a mutation of a second transmembrane segment residue facing the cytoplasmic half of the pore, below the selectivity filter . Some of these suppressors also affected channel gating (channel open time and opening frequency determined in single-channel analyses), revealing intriguing interplay between ion permeation and channel gating.

J Basic Microbiol, 2004, 44(2), 114 - 21
Isolation and characterization of cerebrosides of the hydrocarbon-assimilating yeast Yarrowia lipolytica; Rupcic J et al.; Yarrowia lipolytica yeast was grown batchwise on n-hexadecane as the carbon and energy source . Two cerebroside species were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative high-performance thin-layer chromatography . The cerebroside content accounted for 1.3% of the total cell lipids . Glucose was detected as the sole sugar constituent in cerebrosides . The fatty acid composition of cerebrosides was characterized by a high proportion of hydroxylated long-chain saturated fatty acids . The major fatty acids were h16:0 and 16:0 . The long-chain bases composition shows a preponderance of trihydroxy bases and a small amount of dihydroxy bases . The striking finding was a high proportion of 19-phytosphingosine .

Appl Environ Microbiol, 2004 Apr, 70(4), 2279 - 88
Characterization of azo reduction activity in a novel ascomycete yeast strain; Ramalho PA et al.; Several model azo dyes are reductively cleaved by growing cultures of an ascomycete yeast species, Issatchenkia occidentalis . In liquid media containing 0.2 mM dye and 2% glucose in a mineral salts base, more than 80% of the dyes are removed in 15 h, essentially under microaerophilic conditions . Under anoxic conditions, decolorization does not occur, even in the presence of pregrown cells . Kinetic assays of azo reduction activities in quasi-resting cells demonstrated the following: (i) while the optimum pH depends on dye structure, the optimum pH range was observed in the acidic range; (ii) the maximum decolorizing activity occurs in the late exponential phase; and (iii) the temperature profile approaches the typical bell-shaped curve . These results indirectly suggest the involvement of an enzyme activity in azo dye reduction . The decolorizing activity of I . occidentalis is still observed, although at a lower level, when the cells switch to aerobic respiration at the expense of ethanol after glucose exhaustion in the culture medium . Decolorization ceased when all the ethanol was consumed; this observation, along with other lines of evidence, suggests that azo dye reduction depends on cell growth . Anthraquinone-2-sulfonate, a redox mediator, enhances the reduction rates of the N,N-dimethylaniline-based dyes and reduces those of the 2-naphthol-based dyes, an effect which seems to be compatible with a thermodynamic factor . The dye reduction products were tested as carbon and nitrogen sources . 1-Amino-2-naphthol was used as a carbon and nitrogen source, and N,N-dimethyl-p-phenylenediamine was used only as a nitrogen source . Sulfanilic and metanilic acids did not support growth either as a carbon or nitrogen source.

Mutat Res, 2004 Apr 11, 559(1-2), 199 - 210
Effects of 3H-1,2-dithiole-3-thione, 1,4-phenylenebis(methylene)selenocyanate, and selenium-enriched yeast individually and in combination on benzo{a}pyrene-induced mutagenesis in oral tissue and esophagus in lacZ mice; Guttenplan JB et al.; We have studied the effects of three chemopreventive agents alone or in binary combinations on benzo{a}pyrene (BaP)-induced mutagenesis in the oral cavity and esophagus of lacZ mice using galE(-) selection . The mice were fed diets supplemented with 1,4-phenylenebis(methylene)selenocyanate (p-XSC) at 2.5 and 10 ppm Se, selenium-enriched yeast (SeY) at 2.5 and 10 ppm Se, and 3H-1,2-dithiole-3-thione (D3T) at 65 and 250 ppm, for 6 weeks . Two weeks after the start of the dietary regimen, mice were gavaged with five doses of 125 mg/kg BaP over 2 weeks, and the experiment was terminated 2 weeks later . Mutagenesis was measured in tongue, other pooled oral tissues (OTs), and esophagus . In mice treated with BaP alone, mutagenesis in the above tissues was in the range of 21-32 mutants/10(5)pfu (ca . 6-10 background levels for the corresponding tissues) . p-XSC modestly inhibited mutagenesis (10-33% inhibition) in all tissues, but statistical significance was only observed at the low dose in esophagus, and pooled OT . SeY was not inhibitory alone . Greater inhibitory effects were observed with D3T, and inhibition was statistically significant at the high dose in tongue and esophagus (ca . 33%) . Two combinations of low doses of the inhibitors were tested, and the D3T + SeY mix was most effective, leading to statistically significant inhibition in all three tissues (ca . 30-40% inhibition) . The mixture D3T + p-XSC was of similar effectiveness as the low dose of D3T alone . This study combined with those previously done in our laboratory demonstrates effectiveness of D3T and to a lesser extent, p-XSC in the inhibition of mutagenesis, and provides support for the use of certain combinations of inhibitors as a means to increase effectiveness and reduce the dose of chemopreventive agents.

J Mol Biol, 2004 Apr 23, 338(2), 401 - 18
Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p; Lamontagne B et al.; Members of the double-stranded RNA (dsRNA) specific RNase III family are known to use a conserved dsRNA-binding domain (dsRBD) to distinguish RNA A-form helices from DNA B-form ones, however, the basis of this selectivity and its effect on cleavage specificity remain unknown . Here, we directly examine the molecular requirements for dsRNA recognition and cleavage by the budding yeast RNase III (Rnt1p), and compare it to both bacterial RNase III and fission yeast RNase III (Pac1) . We synthesized substrates with either chemically modified nucleotides near the cleavage sites, or with different DNA/RNA combinations, and investigated their binding and cleavage by Rnt1p . Substitution for the ribonucleotide vicinal to the scissile phosphodiester linkage with 2'-deoxy-2'-fluoro-beta-d-ribose (2' F-RNA), a deoxyribonucleotide, or a 2'-O-methylribonucleotide permitted cleavage by Rnt1p, while the introduction of a 2', 5'-phosphodiester linkage permitted binding, but not cleavage . This indicates that the position of the phosphodiester link with respect to the nuclease domain, and not the 2'-OH group, is critical for cleavage by Rnt1p . Surprisingly, Rnt1p bound to a DNA helix capped with an NGNN tetraribonucleotide loop indicating that the binding of at least one member of the RNase III family is not restricted to RNA . The results also suggest that the dsRBD may accommodate B-form DNA duplexes . Interestingly, Rnt1p, but not Pac1 nor bacterial RNase III, cleaved the DNA strand of a DNA/RNA hybrid, indicating that A-form RNA helix is not essential for cleavage by Rnt1p . In contrast, RNA/DNA hybrids bound to, but were not cleaved by Rnt1p, underscoring the critical role for the nucleotide located at 3' end of the tetraloop and suggesting an asymmetrical mode of substrate recognition . In cell extracts, the native enzyme effectively cleaved the DNA/RNA hybrid, indicating much broader Rnt1p substrate specificity than previously thought . The discovery of this novel RNA-dependent deoxyribonuclease activity has potential implications in devising new antiviral strategies that target actively transcribed DNA.

Methods Mol Biol, 2004, 261, 297 - 312
The split-ubiquitin membrane-based yeast two-hybrid system; Thaminy S et al.; Protein-protein interactions are essential in almost all biological processes, extending from the formation of cellular macromolecular structures and enzymatic complexes to the regulation of signal transduction pathways . It is assumed that approximately one-third of all proteins in eukaryotic cells are membrane associated . Because of their hydrophobic nature, the analysis of membrane-protein interactions is difficult to be studied in a conventional two-hybrid assay . We described here a new genetic method for in vivo detection of membrane-protein interactions in the budding yeast Saccharomyces cerevisiae . The system uses the split-ubiquitin approach based on the detection of the in vivo processing of a reconstituted split ubiquitin . On interaction of X and Y proteins, ubiquitin reconstitution occurs and leads to the proteolytic cleavage and subsequent release of a transcription factor that triggers the activation of a reporter system enabling easy detection . In this manner, and in contrast to the conventional yeast-two hybrid system in which interactions occur in the nucleus, the membrane-based yeast two-hybrid system represents an in vivo system that detects interactions between membrane proteins in their natural environment.

Methods Mol Biol, 2004, 261, 263 - 96
Analysis of protein-protein interactions utilizing dual bait yeast two-hybrid system; Serebriiskii IG et al.; To characterize a protein's function, it is often advantageous to identify other proteins with which it interacts . The yeast two-hybrid system is one of the most versatile methods available for detection and characterization of protein-protein interactions, and in the recent years it has become a mature and robust technology . A further improvement to this technique is the ability to examine and distinguish more than one interaction simultaneously . This is achieved in the Dual Bait, which has successfully been used to detect proteins and peptides that target specific motifs in larger proteins, to facilitate rapid identification of specific interactors from a pool of putative interacting proteins obtained in a library screen, and to score specific drug-mediated disruption of protein-protein interaction.

Methods Mol Biol, 2004, 261, 247 - 62
Using the yeast two-hybrid system to identify interacting proteins; Miller J et al.; The yeast two-hybrid system is a powerful technique for studying protein-protein interactions . Two proteins are separately fused to the independent DNA-binding and transcriptional activation domains of the Gal4p transcription factor . If the proteins interact, they reconstitute a functional Gal4p that activates expression of reporter gene(s) . In this way, two individual proteins may be tested for their ability to interact, and a transcriptional readout can be measured to detect this interaction . Furthermore, novel interacting partners can be found by screening a single protein or domain against a library of other proteins using this system . It is this latter feature-the ability to search for interacting proteins without any prior knowledge of the identity of such proteins-that is the most powerful application of the two-hybrid technique.

Nucleic Acids Res, 2004 Apr 02, 32(6), 2031 - 8 Print 2004.
Ded1p, a conserved DExD/H-box translation factor, can promote yeast L-A virus negative-strand RNA synthesis in vitro; Chong JL et al.; Viruses are intracellular parasites that must use the host machinery to multiply . Identification of the host factors that perform essential functions in viral replication is thus of crucial importance to the understanding of virus-host interactions . Here we describe Ded1p, a highly conserved DExD/H-box translation factor, as a possible host factor recruited by the yeast L-A double-stranded RNA (dsRNA) virus . We found that Ded1p interacts specifically and strongly with Gag, the L-A virus coat protein . Further analysis revealed that Ded1p interacts with the L-A virus in an RNA-independent manner and, as a result, L-A particles can be affinity purified via this interaction . The affinity-purified L-A particles are functional, as they are capable of synthesizing RNA in vitro . Critically, using purified L-A particles, we demonstrated that Ded1p specifically promotes L-A dsRNA replication by accelerating the rate of negative-strand RNA synthesis in vitro . In light of these data, we suggest that Ded1p may be a part of the long sought after activity shown to promote yeast viral dsRNA replication . This and the fact that Ded1p is also required for translating brome mosaic virus RNA2 in yeast thus raise the intriguing possibility that Ded1p is one of the key host factors favored by several evolutionarily related RNA viruses, including the human hepatitis C virus.

J Biotechnol, 2004 Apr 8, 109(1-2), 179 - 91
Impact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells; Fogolin MB et al.; Recently, we demonstrated that a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to partially reconstitute the missing link between glycolysis and TCA, increasing the flux of glucose into the TCA and achieving higher yields of recombinant erythropoietin . In the present study, a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor was used to evaluate the impact of PYC2 expression and reduced culture temperature . Temperature reduction from 37 to 33 degrees C revealed a reduced growth rate, a prolonged stationary phase and a 2.1-fold increase of the cell specific rhGM-CSF production rate for CHO-K1-hGM-CSF cells . The PYC2-expressing cell clones showed a decreased cell growth and a lower maximum cell concentration compared to the control expressing rhGM-CSF but no PYC2 . However, only 65% lactate were produced in PYC2-expressing cells and the product yield was 200% higher compared to the control . The results obtained for CHO cells compared to BHK cells reported previously, indicated that the PYC2 expression dominantly reduced the lactate formation and increased the yield of the recombinant protein to be produced . Finally, the growth and productivity of PYC2-expressing CHO-K1-hGM-CSF cells under both temperature conditions were investigated . The average cell specific rhGM-CSF production increased by 3.2-fold under reduced temperature conditions . The results revealed that the expression of PYC2 and a reduced culture temperature have an additive effect on the cell specific productivity of CHO-K1-hGM-CSF cells.

J Biotechnol, 2004 Apr 8, 109(1-2), 63 - 81
Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review; Madzak C et al.; The production of heterologous proteins is a research field of high interest, with both academic and commercial applications . Yeasts offer a number of advantages as host systems, and, among them, Yarrowia lipolytica appears as one of the most attractive . This non-conventional dimorphic yeast exhibits a remarkable regularity of performance in the efficient secretion of various heterologous proteins . This review presents the main characteristics of Y . lipolytica, and the genetic and molecular tools available in this yeast . A particular emphasis is given to newly developed tools such as efficient promoters, a non-homologous integration method, and an amplification system using defective selection markers . A table recapitulates the 42 heterologous proteins produced until now in Y . lipolytica . A few relevant examples are exposed in more detail, in order to illustrate some peculiar points of the Y . lipolytica physiology, and to offer a comparison with other production systems . This amount of data demonstrates the global reliability and versatility of Y . lipolytica as a host for heterologous production.

Proc Natl Acad Sci U S A, 2004 Apr 13, 101(15), 5506 - 11 Epub 2004 Apr 01.
Metalloregulation of yeast membrane steroid receptor homologs; Lyons TJ et al.; Zinc is an essential micronutrient that can also be toxic . An intricate mechanism exists in yeast that maintains cellular zinc within an optimal range . The centerpiece of this mechanism is the Zap1p protein, a transcription factor that senses zinc deficiency and responds by up-regulating genes involved in zinc metabolism . A microarray screen for novel Zap1p target genes suggested a role in zinc homeostasis for four homologous yeast genes . The expression of two of these genes, YDR492w and YOL002c, suggested direct regulation by Zap1p, whereas the expression of YOL002c and a third homologous gene, YOL101c, was induced by high zinc . YDR492w and YOL002c are confirmed to be direct Zap1p target genes . The induction of YOL002c and YOL101c by toxic metal ion exposure is shown to be mediated by the Mga2p hypoxia sensor . Furthermore, YOL101c is induced by deletion of the Aft1p iron-responsive transcription factor . These three genes, along with a fourth yeast homolog, YLR023c, have phenotypic effects on zinc tolerance and Zap1p activity . Because of their metalloregulation, zinc-related phenotypes, and highly conserved motifs containing potential metal-binding residues, this family has been renamed the IZH gene family (Implicated in Zinc Homeostasis) . Furthermore, these genes are regulated by exogenous fatty acids, suggesting a dual role in lipid metabolism . The IZH genes encode membrane proteins that belong to a ubiquitous protein family that includes hemolysin III and vertebrate membrane steroid receptors . We propose that the IZH genes affect zinc homeostasis either directly or indirectly by altering sterol metabolism.

Mol Cell Biol, 2004 Apr, 24(8), 3307 - 23
Evidence of a new role for the high-osmolarity glycerol mitogen-activated protein kinase pathway in yeast: regulating adaptation to citric acid stress; Lawrence CL et al.; Screening the Saccharomyces cerevisiae disruptome, profiling transcripts, and determining changes in protein expression have identified an important new role for the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway in the regulation of adaptation to citric acid stress . Deletion of HOG1, SSK1, PBS2, PTC2, PTP2, and PTP3 resulted in sensitivity to citric acid . Furthermore, citric acid resulted in the dual phosphorylation, and thus activation, of Hog1p . Despite minor activation of glycerol biosynthesis, the inhibitory effect of citric acid was not due to an osmotic shock . HOG1 negatively regulated the expression of a number of proteins in response to citric acid stress, including Bmh1p . Evidence suggests that BMH1 is induced by citric acid to counteract the effect of amino acid starvation . In addition, deletion of BMH2 rendered cells sensitive to citric acid . Deletion of the transcription factor MSN4, which is known to be regulated by Bmh1p and Hog1p, had a similar effect . HOG1 was also required for citric acid-induced up-regulation of Ssa1p and Eno2p . To counteract the cation chelating activity of citric acid, the plasma membrane Ca(2+) channel, CCH1, and a functional vacuolar membrane H(+)-ATPase were found to be essential for optimal adaptation . Also, the transcriptional regulator CYC8, which mediates glucose derepression, was required for adaptation to citric acid to allow cells to metabolize excess citrate via the tricarboxylic acid (TCA) cycle . Supporting this, Mdh1p and Idh1p, both TCA cycle enzymes, were up-regulated in response to citric acid.

Mol Cell Biol, 2004 Apr, 24(8), 3180 - 7
Stress induction and mitochondrial localization of Oxr1 proteins in yeast and humans; Elliott NA et al.; Reactive oxygen species (ROS) are critical molecules produced as a consequence of aerobic respiration . It is essential for cells to control the production and activity of such molecules in order to protect the genome and regulate cellular processes such as stress response and apoptosis . Mitochondria are the major source of ROS within the cell, and as a result, numerous proteins have evolved to prevent or repair oxidative damage in this organelle . The recently discovered OXR1 gene family represents a set of conserved eukaryotic genes . Previous studies of the yeast OXR1 gene indicate that it functions to protect cells from oxidative damage . In this report, we show that human and yeast OXR1 genes are induced by heat and oxidative stress and that their proteins localize to the mitochondria and function to protect against oxidative damage . We also demonstrate that mitochondrial localization is required for Oxr1 protein to prevent oxidative damage.

Lett Appl Microbiol, 2004, 38(5), 388 - 92
Yeast differentiation using histone promoter sequences; Bell PJ; AIMS: To evaluate a new platform for yeast differentiation based on histone promoter regions . METHODS AND RESULTS: The histone gene amino acid sequences of a wide phylogenetic range of organisms were aligned, and primers designed that were capable of amplifying the divergent promoters of the H3-H4 and H2a-H2b loci from yeast . Analysis indicated that the promoter regions were variable in length between species and represented rapidly changing sequences flanked by highly conserved sequences . CONCLUSIONS: The histone promoter regions in yeast provide an excellent locus for the rapid and accurate identification of yeast species . SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes an alternative platform to the ribosomal internal transcribed spacer (ITS) sequences for the identification of yeast species.

Curr Genet, 2004 Jul, 46(1), 1 - 9 Epub 2004 Apr 01.
The yeast Pho80-Pho85 cyclin-CDK complex has multiple substrates; Waters NC et al.; The Pho85-Pho80 cyclin-CDK (cyclin-dependent protein kinase) complex of Saccharomyces cerevisiae functions as a key regulator of the phosphate-repressible acid phosphatase system . We have further characterized the Pho85-Pho80 kinase complex and identified the Pho80 cyclin subunit and the Pho81 CDK inhibitor as substrates of the Pho85 protein kinase . The phosphorylation sites within Pho80 have been identified at Ser234 and Ser267 . Of the two sites, phosphorylation of Ser234 is required for Pho80 function, to form an active kinase complex and repress acid phosphatase expression . Evidence suggests that the activity of Pho81 is regulated by a post-translational modification and therefore that Pho85-mediated phosphorylation of Pho81 may alter its ability to function as a CDK inhibitor . Thus, the control of acid phosphatase expression involves the phosphorylation of several of the regulatory components of the system.

Biosci Biotechnol Biochem, 2004 Mar, 68(3), 676 - 84
Syntheses of (+)- and (-)-dihydropinidine and (+)- and (-)-epidihydropinidine by using yeast reduction of methyl (2-oxocyclohexyl)acetate; Yamauchi S et al.; (+) and (-)-Dihydropinidine and (+)- and (-)-epidihydropinidine were synthesized from hydroxy esters 1 and 2 which had been prepared by yeast reduction of methyl (2-oxocyclohexyl)acetate . The enantiomeric excess at the C-1 positions of 1 and 2 were both determined as more than 99% ee.

Biochim Biophys Acta, 2004 Apr 7, 1672(1), 21 - 6
Concanavalin A induced activity change in yeast PM-bound NADH-HCF(III) oxidoreductase; Awasthi D et al.; The activity of plasma membrane bound redox enzyme, NADH-HCF(III) oxidoreductase, in wild and mutant strains of the yeast Saccharomyces cerevisiae is modulated by Con A in a dose-dependent manner . The solubilized activity is enhanced at lower concentration and inhibited at higher concentration of Con A . The enzyme in mutant strain is more sensitive to inhibition . The activation of enzyme by Con A is suppressed in the presence of either alpha-methyl-D-mannoside or 2-deoxy-D-glucose, indicating the glycoproteic nature of enzyme as well as the resulting conformational change due to interaction with Con A as the factor for modulated activities . This was supported by recording the decrease in K(m) value of enzyme with respect to substrate NADH in the presence of lower concentration of Con A . The purified enzyme was more sensitive to lectin stimulation and, on the basis of comparative stimulatory effects of Con A and PSA on activity, it is likely that mannosyl moiety in enzyme is involved in binding the lectins to cause enzymic activation.

Mol Cell, 2004 Mar 26, 13(6), 771 - 81
Yeast adapt to near-freezing temperatures by STRE/Msn2,4-dependent induction of trehalose synthesis and certain molecular chaperones; Kandror O et al.; Virtually nothing is known about the biochemical adaptations in eukaryotic cells that may enhance survival at low temperatures or upon freezing . Here we demonstrate an adaptive response in yeast that is activated below 10 degrees C and increases tolerance to low temperatures and freezing . This response involves a dramatic accumulation of the chemical chaperone trehalose and induction of trehalose-synthesizing enzymes (Tps1, Tps2) and certain heat shock proteins (Hsp104, Hsp42, Hsp12, Ssa4) . mRNAs for these proteins increase dramatically below 10 degrees C and even at 0 degrees C . Their expression requires Msn2,4 transcription factors but also involves marked mRNA stabilization . Upon return to 30 degrees C, TPS1, TPS2, and HSP104 mRNAs, trehalose levels and tolerance to freezing fall dramatically within minutes . Mutants lacking trehalose or Msn2,4 die more rapidly at 0 degrees C and upon freezing . Thus, below 10 degrees C, yeast show an adaptive response that sustains viability at low or freezing temperatures, which are commonly encountered in natural environments and laboratory refrigerators.

Anal Chem, 2004 Apr 1, 76(7), 2112 - 7
Denaturation and renaturation of self-assembled yeast iso-1-cytochrome c on Au; Chah S et al.; We have made surface plasmon resonance (SPR) measurements of yeast iso-1-cytochrome c (Cyt c) on a gold surface . Angle-resolved SPR curves are recorded as a function of urea concentration before and after self-assembly of the Cyt c . Exposure to a urea solution causes denaturation of Cyt c, which shifts the minimum in the SPR curve to a larger angle and decreases the signal amplitude . The Gibbs free energy change for denaturation of the protein on Au is calculated from the change of the SPR signal amplitude with urea concentration . We find that (1) Cyt c can be reversibly denatured and renatured, depending on the urea concentration, and (2) the Gibbs free energy change for denaturation of Cyt c on Au surface in water, DeltaG degrees (water), is 1.5 kcal/mol, which is approximately 4 times less than that in bulk solution.

J Agric Food Chem, 2004 Apr 7, 52(7), 2047 - 51
Effects of asparagine, fructose, and baking conditions on acrylamide content in yeast-leavened wheat bread; Surdyk N et al.; A repeatable procedure for studying the effects of internal and external factors on acrylamide content in yeast-leavened wheat bread has been developed . The dough contained wheat endosperm flour with a low content of precursors for acrylamide formation (asparagine and reducing sugars), dry yeast, salt, and water . The effects of asparagine and fructose, added to the dough, were studied in an experiment with a full factorial design . More than 99% of the acrylamide was found in the crust . Added asparagine dramatically increased the content of acrylamide in crusts dry matter (from about 80 microg/kg to between 600 and 6000 microg/kg) while added fructose did not influence the content . The effects of temperature and time of baking were studied in another experiment using a circumscribed central composite design . Mainly temperature (above 200 degrees C) but also time increased the acrylamide content in crust dry matter (from below 10 to 1900 microg/kg), and a significant interaction was found between these two factors . When baked at different conditions with the same ingredients, a highly significant relationship (P < 0.001) between color and acrylamide content in crust was found . Added asparagine, however, did not increase color, showing that mainly other amino compounds are involved in the browning reactions.

Biochem Cell Biol, 2004 Feb, 82(1), 62 - 70
Phospholipid synthesis in yeast: regulation by phosphorylation; Carman GM et al.; The yeast Saccharomyces cerevisiae is a model eukaryotic organism for the study of the regulation of phospholipid synthesis . The major phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine) are synthesized by complementary (CDP-diacylglycerol and Kennedy) pathways . The regulation of these pathways is complex and is controlled by genetic and biochemical mechanisms . Inositol plays a major role in the regulation of phospholipid synthesis . Inositol-mediated regulation involves the expression of genes and the modulation of enzyme activities . Phosphorylation is a major mechanism by which enzymes and transcription factors are regulated, and indeed, key phospholipid biosynthetic enzymes have been identified as targets of phosphorylation . Protein kinase A phosphorylates CTP synthetase, choline kinase, Mg2+-dependent phosphatidate phosphatase, phosphatidylserine synthase, and the transcription factor Opi1p . CTP synthetase and Opi1p are also phosphorylated by protein kinase C . The phosphorylation of these proteins plays a role in regulating their activities and (or) function in phospholipid synthesis.

Biochem Cell Biol, 2004 Feb, 82(1), 45 - 61
Yeast sphingolipid metabolism: clues and connections; Sims KJ et al.; This review of sphingolipid metabolism in the budding yeast Saccharomyces cerevisiae contains information on the enzymes and the genes that encode them, as well as connections to other metabolic pathways . Particular attention is given to yeast homologs, domains, and motifs in the sequence, cellular localization of enzymes, and possible protein-protein interactions . Also included are genetic interactions of special interest that provide clues to the cellular biological roles of particular sphingolipid metabolic pathways and specific sphingolipids.

J Biol Chem, 2004 May 28, 279(22), 23207 - 13 Epub 2004 Mar 29.
Peroxiredoxin-null yeast cells are hypersensitive to oxidative stress and are genomically unstable; Wong CM et al.; Peroxiredoxins are a family of abundant peroxidases found in all organisms . Although these antioxidant enzymes are thought to be critically involved in cellular defense and redox signaling, their exact physiological roles are largely unknown . In this study, we took a genetic approach to address the functions of peroxiredoxins in budding yeast . We generated and characterized a yeast mutant lacking all five peroxiredoxins . The quintuple peroxiredoxin-null mutant was still viable, though the growth rate was lower under normal aerobic conditions . Although peroxiredoxins are not essential for cell viability, peroxiredoxin-null yeast cells were more susceptible to oxidative and nitrosative stress . In the complete absence of peroxiredoxins, the expression of other antioxidant proteins including glutathione peroxidase and glutathione reductase was induced . In addition, the quintuple mutant was hypersensitive to glutathione depletion . Thus, the glutathione system might cooperate with other antioxidant enzymes to compensate for peroxiredoxin deficiency . Interestingly, the peroxiredoxinnull yeast cells displayed an increased rate of spontaneous mutations that conferred resistance to canavanine . This mutator phenotype was rescued by yeast peroxiredoxin Tsa1p, but not by its active-site mutant defective for peroxidase activity . Our findings suggest that the antioxidant function of peroxiredoxins is important for maintaining genome stability in eukaryotic cells.

J Mol Biol, 2004 Apr 16, 338(1), 139 - 48
Two homologous domains of similar structure but different stability in the yeast linker histone, Hho1p; Ali T et al.; The Saccharomyces cerevisiae homologue of the linker histone H1, Hho1p, has two domains that are similar in sequence to the globular domain of H1 (and variants such as H5) . It is an open question whether both domains are functional and whether they play similar structural roles . Preliminary structural studies showed that the two isolated domains, GI and GII, differ significantly in stability . In 10 mM sodium phosphate (pH 7), the GI domain, like the globular domains of H1 and H5, GH1 and GH5, was stably folded, whereas GII was largely unstructured . However, at high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate), which might mimic the charge-screening effects of DNA phosphate groups, GII was folded . In view of the potential significance of these observations in relation to the role of Hho1p, we have now determined the structures of its GI and GII domains by NMR spectroscopy under conditions in which GII (like GI) is folded . The backbone r.m.s.d . over the ordered residues is 0.43 A for GI and 0.97 A for GII . Both structures show the "winged-helix" fold typical of GH1 and GH5 and are very similar to each other, with an r.m.s.d . over the structured regions of 1.3 A, although there are distinct differences . The potential for GII to adopt a structure similar to that of GI when Hho1p is bound to chromatin in vivo suggests that both globular domains might be functional . Whether Hho1p performs a structural role by bridging two nucleosomes remains to be determined.

Biochemistry, 2004 Apr 6, 43(13), 3987 - 95
The tricalbin C2 domains: lipid-binding properties of a novel, synaptotagmin-like yeast protein family; Schulz TA et al.; The tricalbins are a recently discovered family of Saccharomyces cerevisae proteins containing a predicted N-terminal transmembrane domain and at least three C2 domains . They are thought to be yeast homologues of synaptotagmin, a hypothesis supported by structural similarities and past studies that implicated tricalbins in processes of membrane trafficking and sorting . We expressed and purified constructs consisting of single tricalbin C2 domains, and assayed their ability to bind lipids in response to calcium . Protein-lipid overlay assays indicated that the C-terminal C2 domains (C2C) of tricalbins 1 and 3 bind numerous species of acidic phospholipid, including phosphatidylserine and several phosphoinositides, and the amount of protein bound was greatly enhanced in the presence of 1 mM calcium . Sedimentation assays using mixed phosphatidylserine/phosphatidylcholine (PS/PC) vesicles confirmed that the C2C domains of tricalbin 1 and 3 bind membranes in a calcium-responsive manner and showed that they are more sensitive to calcium than the C2A domain of synaptotagmin I . Both assays revealed that all of the C2 domains of tricalbin 2 are insensitive to calcium . Fluorimetric assays exploiting the position of naturally occurring tryptophans in tricalbin 1 C2C and tricalbin 3 C2C confirmed that these domains are capable of binding calcium and that this is coupled to the binding of acidic phospholipid . Combining this with past protein-protein interaction data, we theorize that the calcium-insensitive tricalbin 2 mediates the creation of hetero-oligomeric tricalbin complexes in which tricalbin 1 or 3 or both supply a calcium-dependent membrane binding activity.

Biochemistry, 2004 Apr 6, 43(13), 3862 - 9
The ligand specificity of yeast Rad53 FHA domains at the +3 position is determined by nonconserved residues; Yongkiettrakul S et al.; On the basis of the results from our laboratory and others, we recently suggested that the ligand specificity of forkhead-associated (FHA) domains is controlled by variations in three major factors: (i) residues interacting with pThr, (ii) residues recognizing the +1 to +3 residues from pThr, and (iii) an extended binding surface . While the first factor has been well established by several solution and crystal structures of FHA-phosphopeptide complexes, the structural bases of the second and third factors are not well understood and are likely to vary greatly between different FHA domains . In this work, we proposed and tested the hypothesis that nonconserved residues G133 and G135 of FHA1 and I681 and D683 of FHA2, located outside of the core FHA region of yeast Rad53 FHA domains, contribute to the specific recognition of the +3 position of different phosphopeptides . By rational mutagenesis of these residues, the specificity of FHA1 has been changed from predominantly pTXXD to be equally acceptable for pTXXD, pTXXL, and pYXL, which are similar to the specificities of the FHA2 domain of Rad53 . Conversely, the +3 position specificity of FHA2 has been engineered to be more like FHA1 with the I681A mutation . These results were based on library screening as well as binding analyses of specific phosphopeptides . Furthermore, results of structural analyses by NMR indicate that some of these residues are also important for the structural integrity of the loops.

Mol Genet Genomics, 2004 May, 271(4), 437 - 46 Epub 2004 Mar 27.
Targeted DNA integration within different functional gene domains in yeast reveals ORF sequences as recombinational cold-spots; Gjuracic K et al.; The efficiency of gene targeting within different segments of genes in yeast was estimated by transforming yeast cells with double-stranded integrative plasmids, bearing functional gene domains {promoter (P), ORF (O) and terminator (T)} derived from the common genetic markers HIS3, LEU2, TRP1 and URA3 . Transformation experiments with circular plasmids carrying a single gene domain demonstrated that the 5' and 3' flanking DNA regions (P and T) of the HIS3 and URA3 genes are preferred as sites for plasmid integration by several fold over the corresponding ORFs . Moreover, when plasmids bearing combinations of two or three regions were linearized to target them to a specific site of integration, three of the ORFs were found to be less preferred as sites for plasmid integration than their corresponding flanking regions . Surprisingly, in up to 50% of the transformants obtained with plasmids that had been linearized within coding sequences, the DNA actually integrated into neighbouring regions . Almost the same frequencies of ORF mis-targeting were obtained with plasmid vectors containing only two functional domains ("PO" or "OT") of the gene URA3, demonstrating that this event is not the consequence of competition between homologous DNA regions distal to the ORF . Therefore, we suggest that coding sequences could be considered to be "cold spots" for plasmid integration in yeast.

Mol Biol Cell, 2004 Jun, 15(6), 2920 - 31 Epub 2004 Mar 26.
Loss of Apm1, the micro1 subunit of the clathrin-associated adaptor-protein-1 complex, causes distinct phenotypes and synthetic lethality with calcineurin deletion in fission yeast; Kita A et al.; Calcineurin is a highly conserved regulator of Ca(2+) signaling in eukaryotes . In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl(-) homeostasis . In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1(+) gene that encodes a homolog of the mammalian micro1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex . The cis1-1/apm1-1 mutant as well as the apm1-deleted (Deltaapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion . Electron micrographs revealed that Deltaapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles . Deltaapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase . These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment . Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region . These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events.

Nucleic Acids Res, 2004 Mar 26, 32(6), 1917 - 27 Print 2004.
Humanized telomeres and an attempt to express a functional human telomerase in yeast; Bah A et al.; The maintenance of telomeric repeat DNA depends on an evolutionarily conserved reverse trans criptase called telomerase . In vitro, only the catalytic subunit and a telomerase-associated RNA are required for the synthesis of species-specific repeat DNA . In an attempt to establish a heterologous system for the study of the human telomerase enzyme, we expressed the two core components and predicted regulatory subunits in the yeast Saccharomyces cerevisiae . We show that adequate substrates for human telomerase can be generated; the expressed enzyme was localized in the nucleus and it had the capacity to synthesize human-specific repeats in vitro . However, there was no evidence for human telomerase activity at yeast telomeres in vivo . Therefore functional replacement of the yeast telomerase by the human enzyme may require additional human-specific components . We also replaced the template region of the yeast telomerase RNA with one that dictates the synthesis of vertebrate repeats and performed a detailed molecular analysis of the composition of the telomeres upon outgrowth of such strains . The results suggest that vertebrate repeats on yeast telomeres are subject to a very high degree of repeat turnover and show that an innermost tract of 50 bp of yeast repeats are resistant to replacement.

Mol Cell Proteomics, 2004 Jul, 3(7), 625 - 59 Epub 2004 Mar 28.
Depth of proteome issues: a yeast isotope-coded affinity tag reagent study; Parker KC et al.; As a test case for optimizing how to perform proteomics experiments, we chose a yeast model system in which the UPF1 gene, a protein involved in nonsense-mediated mRNA decay, was knocked out by homologous recombination . The results from five complete isotope-coded affinity tag (ICAT) experiments were combined, two using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) and three using electrospray MS/MS . We sought to assess the reproducibility of peptide identification and to develop an informatics structure that characterizes the identification process as well as possible, especially with regard to tenuous identifications . The cleavable form of the ICAT reagent system was used for quantification . Most proteins did not change significantly in expression as a consequence of the upf1 knockout . As expected, the Upf1 protein itself was down-regulated, and there were reproducible increases in expression of proteins involved in arginine biosynthesis . Initially, it seemed that about 10% of the proteins had changed in expression level, but after more thorough examination of the data it turned out that most of these apparent changes could be explained by artifacts of quantification caused by overlapping heavy/light pairs . About 700 proteins altogether were identified with high confidence and quantified . Many peptides with chemical modifications were identified, as well as peptides with noncanonical tryptic termini . Nearly all of these modified peptides corresponded to the most abundant yeast proteins, and some would otherwise have been attributed to "single hit" proteins at low confidence . To improve our confidence in the identifications, in MALDI experiments, the parent masses for the peptides were calibrated against nearby components . In addition, five novel parameters reflecting different aspects of identification were collected for each spectrum in addition to the Mascot score that was originally used . The interrelationship between these scoring parameters and confidence in protein identification is discussed.

J Mol Biol, 2004 Apr 9, 337(5), 1123 - 35
Distinct properties of the two putative "globular domains" of the yeast linker histone, Hho1p; Ali T et al.; The putative linker histone in Saccharomyces cerevisiae, Hho1p, has two regions of sequence (GI and GII) that are homologous to the single globular domains of linker histones H1 and H5 in higher eukaryotes . However, the two Hho1p "domains" differ with respect to the conservation of basic residues corresponding to the two putative DNA-binding sites (sites I and II) on opposite faces of the H5 globular domain . We find that GI can protect chromatosome-length DNA, like the globular domains of H1 and H5 (GH1 and GH5), but GII does not protect . However, GII, like GH1 and GH5, binds preferentially (and with higher affinity than GI) to four-way DNA junctions in the presence of excess linear DNA competitor, and binds more tightly than GI to linker-histone-depleted chromatin . Surprisingly, in 10 mM sodium phosphate (pH 7.0), GII is largely unfolded, whereas GI, like GH1 and GH5, is structured, with a high alpha-helical content . However, in the presence of high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate) GII is also folded; the anions presumably mimic DNA in screening the positive charge . This raises the possibility that chromatin-bound Hho1p may be bifunctional, with two folded nucleosome-binding domains.

PLoS Biol . 2004 Apr;2(4):E86 . Epub 2004 Mar 23.
Dissection and design of yeast prions; Osherovich LZ et al.; Many proteins can misfold into beta-sheet-rich, self-seeding polymers (amyloids) . Prions are exceptional among such aggregates in that they are also infectious . In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication . We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance . In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates . Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate . Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine.

Science, 2004 Mar 26, 303(5666), 2026 - 9
Structure-based assembly of protein complexes in yeast; Aloy P et al.; Images of entire cells are preceding atomic structures of the separate molecular machines that they contain . The resulting gap in knowledge can be partly bridged by protein-protein interactions, bioinformatics, and electron microscopy . Here we use interactions of known three-dimensional structure to model a large set of yeast complexes, which we also screen by electron microscopy . For 54 of 102 complexes, we obtain at least partial models of interacting subunits . For 29, including the exosome, the chaperonin containing TCP-1, a 3'-messenger RNA degradation complex, and RNA polymerase II, the process suggests atomic details not easily seen by homology, involving the combination of two or more known structures . We also consider interactions between complexes (cross-talk) and use these to construct a structure-based network of molecular machines in the cell.

J Biol Chem, 2004 May 21, 279(21), 22183 - 9 Epub 2004 Mar 24.
A basic helix-loop-helix transcription factor essential for cytochrome p450 induction in response to alkanes in yeast Yarrowia lipolytica; Yamagami S et al.; When the alkane-assimilating yeast Yarrowia lipolytica is cultivated on n-alkanes, it changes cellular metabolism for adaptation by inducing cytochrome p450 and other genes . From a comparative analysis of promoters of alkane-inducible genes, we identified a cis-acting element, ARE1 (alkane responsive element 1), which provides transcription induction in response to n-alkanes . In a genetic selection for mutants that were defective in ARE1-mediated transcription induction in the presence of n-alkanes, we found that the YAS1 (yeast alkane signaling) gene is essential for alkane response . The YAS1 gene encodes a basic helix-loop-helix (bHLH) family protein . Loss of Yas1p causes defects in n-alkane-dependent transcription induction of the p450 gene and growth on n-alkanes . Yas1p localizes to nuclei and binds to promoters containing ARE1 . Yas1p also binds to its own promoter, and the expression of YAS1 is induced by n-alkanes . These features suggest that Yas1p is a novel transcription factor mediating alkane signaling and that it provides an autoregulatory loop.

J Biol Chem, 2004 Jun 4, 279(23), 23988 - 95 Epub 2004 Mar 23.
Mouse CAF1 can function as a processive deadenylase/3'-5'-exonuclease in vitro but in yeast the deadenylase function of CAF1 is not required for mRNA poly(A) removal; Viswanathan P et al.; The mouse CAF1 (mCAF1) is an ortholog of the yeast (y) CAF1 protein, which is a component of the CCR4-NOT complex, the major cytoplasmic deadenylase of Saccharomyces cerevisiae . Although CAF1 protein belongs to the DEDDh family of RNases, CCR4 appears to be the principle deadenylase of the CCR4-NOT complex . Here, we present evidence that mCAF1 is a processive, 3'-5'-RNase with a preference for poly(A) substrates . Like CCR4, increased length of RNA substrates converted mCAF1 into a processive enzyme . In contrast to two other DEDD family members, PAN2 and PARN, mCAF1 was not activated either by PAB1 or capped RNA substrates . The rate of deadenylation in vitro by yCCR4 and mCAF1 were both strongly influenced by secondary structures present in sequences adjacent to the poly(A) tail, suggesting that the ability of both enzymes to deadenylate might be affected by the context of the mRNA 3'-untranslated region sequences . The ability of mCAF1 to complement a ycaf1 deletion in yeast, however, did not require the RNase function of mCAF1 . Importantly, yCAF1 mutations, which have been shown to block its RNase activity in vitro, did not inactivate yCAF1 in vivo, and mRNAs were deadenylated in vivo at nearly the same rate as found for wild type yCAF1 . These results indicate that at least in yeast the CAF1 RNase activity is not required for its in vivo function.

J Biol Chem, 2004 Jun 4, 279(23), 24024 - 33 Epub 2004 Mar 25.
Neuropathy target esterase and its yeast homologue degrade phosphatidylcholine to glycerophosphocholine in living cells; Zaccheo O et al.; Eukaryotic cells control the levels of their major membrane lipid, phosphatidylcholine (PtdCho), by balancing synthesis with degradation via deacylation to glycerophosphocholine (GroPCho) . Here we present evidence that in both yeast and mammalian cells this deacylation is catalyzed by neuropathy target esterase (NTE), a protein originally identified by its reaction with organophosphates, which cause nerve axon degeneration . YML059c, a Saccharomyces cerevisiae protein with sequence homology to NTE, had similar catalytic properties to the mammalian enzyme in assays of microsome preparations and, like NTE, was localized to the endoplasmic reticulum . Yeast lacking YML059c were viable under all conditions examined but, unlike the wild-type strain, did not convert PtdCho to GroPCho . Despite the absence of the deacylation pathway, the net rate of {(14)C}choline incorporation into PtdCho in YML059c-null yeast was not greater than that in the wild type; this was because, in the null strain diminished net uptake of extracellular choline and decreased formation of the rate-limiting intermediate, CDP-choline, resulted in a reduced rate of PtdCho synthesis . In {(14)C}choline labeling experiments with cultured mammalian cell lines, production of {(14)C}GroPCho was enhanced by overexpression of catalytically active NTE and was diminished by reduction of endogenous NTE activity mediated either by RNA interference or organophosphate treatment . We conclude that NTE and its homologues play a central role in membrane lipid homeostasis.

Bioinformatics, 2004 Aug 12, 20(12), 1914 - 27 Epub 2004 Mar 25.
Quantitative characterization of the transcriptional regulatory network in the yeast cell cycle; Chen HC et al.; MOTIVATION: Genome-wide gene expression programs have been monitored and analyzed in the yeast Saccharomyces cerevisiae, but how cells regulate global gene expression programs in response to environmental changes is still far from being understood . We present a systematic approach to quantitatively characterize the transcriptional regulatory network of the yeast cell cycle . For the interpretative purpose, 20 target genes were selected because their expression patterns fluctuated in a periodic manner concurrent with the cell cycle and peaked at different phases . In addition to the most significant five possible regulators of each specific target gene, the expression pattern of each target gene affected by synergy of the regulators during the cell cycle was characterized . Our first step includes modeling the dynamics of gene expression and extracting the transcription rate from a time-course microarray data . The second step embraces finding the regulators that possess a high correlation with the transcription rate of the target gene, and quantifying the regulatory abilities of the identified regulators . RESULTS: Our network discerns not only the role of the activator or repressor for each specific regulator, but also the regulatory ability of the regulator to the transcription rate of the target gene . The highly coordinated regulatory network has identified a group of significant regulators responsible for the gene expression program through the cell cycle progress . This approach may be useful for computing the regulatory ability of the transcriptional regulatory networks in more diverse conditions and in more complex eukaryotes . SUPPLEMENTARY INFORMATION: Matlab code and test data are available at http://www.ee.nthu.edu.tw/~bschen/quantitative/regulatory_network.htm

Mol Genet Genomics, 2004 May, 271(4), 490 - 8 Epub 2004 Mar 20.
A novel small-molecule inhibitor of the chromosome segregation process in yeast; Yoon HJ; The centromeres of budding yeasts contain specific and essential DNA sequences, in contrast to the regional heterochromatic centromeres found in higher organisms . Small molecules that perturb centromere function in budding yeast could be valuable candidates for identifying yeast-specific growth inhibitors . A combination of two in vivo assays, one based on transcription blockade of a reporter gene by the centromeric DNA-protein complex, the other on a test for mitotic minichromosome stability, was used to identify small molecules that affect the process of chromosome segregation . One compound, here named incentrom A, leads to a minichromosome loss phenotype, and is cytotoxic to the budding yeast Saccharomyces cerevisiae . Furthermore, whereas cells carrying a conditional mutation in a gene for an essential kinetochore protein, skp1-4, are hypersensitive to incentrom A, cells that overexpress the SKP1 gene are resistant to the compound . Incentrom A also results in mitotic loss of a centromere-bearing plasmid, and inhibits the growth of the pathogenic yeast Candida glabrata . Incentrom A will therefore be a useful tool for studying the molecular basis of yeast chromosome segregation and could form the basis for the development of novel antifungal drugs.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Mar, 24(3), 273 - 6
{Construction and identification of yeast expression vectors containing human platelet-derived growth factor B-chain gene}; Yang CL et al.; OBJECTIVE: To investigate the expression efficiency of human platelet-derived growth factor B-chain (PDGF-B) gene in yeast and assess the activity of the expressed product . METHODS: A full-length complementary DNA of human PDGF-B gene was amplified from the total RNA extracted from human vascular endothelial cells using reverse transcription (RT)-PCR and then cloned into pGEM-T vector . The PCR products with specific primers were recombined into yeast expression plasmids pMETB or pMETalphaA, followed by identification with restriction endonuclease . RESULTS: The 578 bp fragment encoding mature PDGF-BB peptide with signal peptide and the 340 bp fragment without signal peptide were identified and verified by restriction endonuclease mapping and sequencing, and two yeast expression vectors pMETB-PDGFB(1) and pMETalphaA-PDGFB(2) were reconstructed . CONCLUSION: The yeast expression vectors containing human PDGF-B gene have been successfully constructed for further studies of gene expression in yeast.

FEMS Yeast Res, 2004 Mar, 4(6), 633 - 41
Yeast cells display a regulatory mechanism in response to methylglyoxal; Aguilera J et al.; Methylglyoxal (MG), a glycolytic by-product, is an extremely toxic compound . This fact suggests that its synthesis and degradation should be tightly controlled . However, little is known about the mechanisms that protect yeast cells against MG toxicity . Here, we show that in Saccharomyces cerevisiae, MG exposure increased the internal MG content and activated the expression of GLO1 and GRE3, two genes involved in MG detoxification; GPD1, the gene for glycerol synthesis; and TPS1 and TPS2, the trehalose pathway genes . This response was specific as demonstrated by the analysis of marker genes and effectors of the general stress response . Physiological experiments with MG-treated cells showed that this compound triggers the overproduction of glycerol . Furthermore, a gpd1 gpd2 double mutant showed enhanced MG contents compared with the wild-type . Overall, these results appeared to indicate that up-variations in the intracellular content of the toxic compound are perceived by the cell as a primary signal to trigger the transcriptional response . In agreement with this, MG-instigated GPD1 activation was enhanced in strains lacking GLO1, and this effect correlated with the internal MG content . Finally, induction of GPD1, TPS1 and GRE3, and enhanced MG contents were also observed in low-glucose-growing cells subjected to a sudden increase in glucose availability . The implications of this regulatory mechanism on protection against MG are discussed.

FEMS Yeast Res, 2004 Mar, 4(6), 579 - 86
Four new yeast species of the genus Sporobolomyces from plant leaves; Wang QM et al.; Among the ballistoconidium-forming yeast strains isolated from various plant leaves collected in North and Northeast China, 12 strains forming orange to orange-red colored colonies were revealed to represent four novel species of the genus Sporobolomyces by conventional, chemotaxonomic and molecular phylogenetic studies, based on the 26S-rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequences . Sporobolomyces beijingensis sp . nov., represented by eight strains (type strain CB 80T = AS 2.2365T = CBS 9730T), and Sporobolomyces jilinensis sp . nov., represented by two strains (type strain CB 118T = AS 2.2301T = CBS 9728T), clustered in the Johnsonii clade of the Sporidiobolus lineage . Sporobolomyces clavatus sp . nov., represented by strain CB 281T (= AS 2.2318T = CBS 9729T), belonged to the Agaricostilbum lineage and showed a close relationship to Sporobolomyces ruber and Sporobolomyces dracophylli . Sporobolomyces symmetricus sp . nov., represented by strain CB 64T(= AS 2.2299T = CBS 9727T), formed nearly symmetrical ballistoconidia and was closely related with Sporobolomyces vermiculatus and Sporobolomyces gracilis in the Gracilis clade of the Erythrobasidium lineage.

J Biol Chem, 2004 May 28, 279(22), 23030 - 7 Epub 2004 Mar 23.
A Gcn4p homolog is essential for the induction of a ribosomal protein L41 variant responsible for cycloheximide resistance in the yeast Candida maltosa; Takaku H et al.; Cycloheximide (CYH) resistance in the yeast Candida maltosa is based on the inducible expression of genes encoding a variant of ribosomal protein L41-Q, with glutamine at position 56 instead of the proline found in normal L41 . The promoter of L41-Q2a, one of the L41-Q gene alleles encoding L41-Q, has an element similar to the Gcn4p-responsive element of Saccharomyces cerevisiae . In a previous study, this element was shown to be essential for the induction of L41-Q by CYH . In the present study, a C . maltosa GCN4 homolog, C-GCN4, was cloned . It had a long 5'-leader region with three upstream open reading frames . Enhanced expression of the C-GCN4 reporter fusion gene upon the addition of 3-aminotriazole or by mutations in start codons of all three upstream open reading frames indicates that C-GCN4 expression is under translation repression as was seen with GCN4 . The C-GCN4-depleted mutant was unable to grow in a nutrient medium containing CYH and did not express L41-Q genes . Recombinant C-Gcn4p bound to the consensus DNA element for Gcn4p, 5'-(G/A)TGACTCAT-3', located upstream of L41-Q2a . Thus, C-Gcn4p, which likely functions in the general control of amino acid biosynthesis, is essential for the expression of L41-Q genes.

Methods, 2004 May, 33(1), 59 - 67
Purification and nucleosome mapping analysis of native yeast plasmid chromatin; Kim Y et al.; There is much evidence indicating the importance in gene regulation of the positions of nucleosomes with respect to DNA sequence . Low resolution chromatin structures have been described for many genes, but there is a dearth of detailed high resolution chromatin structures . In the cases where they are available, high resolution maps have revealed much more complex chromatin structures, with multiple alternative nucleosome positions . The discovery that ATP-dependent chromatin remodelling machines are recruited to genes, with their ability to mobilise nucleosomes on DNA and to alter nucleosomal conformation, emphasises the necessity for obtaining high resolution nucleosome maps, so that the details of these remodelling reactions can be defined in vivo . Here, we describe protocols for purifying plasmid chromatin from cells of the yeast Saccharomyces cerevisiae and for mapping nucleosome positions on the plasmid using the monomer extension mapping method . This method requires purified chromatin, but is capable of mapping relatively long stretches of chromatin in great detail . Typically, it reveals very complex chromatin structures.

Environ Monit Assess, 2004 Mar, 92(1-3), 253 - 72
A yeast DNA microarray for the evaluation of toxicity in environmental water containing burned ash; Kim HJ et al.; Numerous studies on the hazard assessment and epidemiological health responses to burned ash have been reported . However, there is little information on the potential toxicity of unknown chemical complexes in burned ash . For an overall evaluation of the multiple toxicities of burned ash, a DNA microarray was used in this study, as a new attempt to assess these toxicities . Using the global gene expression on yeast DNA chip to reflect the changes in mRNA levels, our study discovered a lot of evidences for the action of cell homeostasis and stress response etc., against the toxic effects on yeast cells . On the genes of 5,117 open reading frames (ORFs), as valid spots in a microarray, 997 were up-regulated, 1,259 were down-regulated and 2,861 remained unchanged . A detailed analysis of the microarray revealed the genes that were dynamically correlated to the function of the subcellular localization, energy/metabolism, various stress responses/cell homeostasis and detoxification . Significantly, the toxicities, caused by reactive oxygen species (ROS), metals and the other xenobiotics, were indicated in burned ash . Also, the possibility of mutagenicity of the burned ash was suggested on the basis of the DNA repair related genes.

RNA, 2004 Apr, 10(4), 558 - 64
Mutations in the middle domain of yeast poly(A) polymerase affect interactions with RNA but not ATP; Zhelkovsky A et al.; The eukaryotic poly(A) polymerase (PAP) is responsible for the posttranscriptional extension of mRNA 3' ends by the addition of a poly(A) tract . The recently published three-dimensional structures of yeast and bovine PAPs have made a more directed biochemical analysis of this enzyme possible . Based on these structures, the middle domain of PAP was predicted to interact with ATP . However, in this study, we show that mutations of conserved residues in this domain of yeast PAP, Pap1, do not affect interaction with ATP, but instead disrupt the interaction with RNA and affect the enzyme's ability to process substrate lacking 2' hydroxyls at the 3' end . These results are most consistent with a model in which the middle domain of PAP interacts directly with the recently extended RNA and pyrophosphate byproduct.

Proc Natl Acad Sci U S A, 2004 Apr 6, 101(14), 4781 - 6 Epub 2004 Mar 22.
The yeast cell-cycle network is robustly designed; Li F et al.; The interactions between proteins, DNA, and RNA in living cells constitute molecular networks that govern various cellular functions . To investigate the global dynamical properties and stabilities of such networks, we studied the cell-cycle regulatory network of the budding yeast . With the use of a simple dynamical model, it was demonstrated that the cell-cycle network is extremely stable and robust for its function . The biological stationary state, the G1 state, is a global attractor of the dynamics . The biological pathway, the cell-cycle sequence of protein states, is a globally attracting trajectory of the dynamics . These properties are largely preserved with respect to small perturbations to the network . These results suggest that cellular regulatory networks are robustly designed for their functions.

J Biol Chem, 2004 Jun 4, 279(23), 23961 - 8 Epub 2004 Mar 22.
High osmolarity glycerol (HOG) pathway-induced phosphorylation and activation of 6-phosphofructo-2-kinase are essential for glycerol accumulation and yeast cell proliferation under hyperosmotic stress; Dihazi H et al.; In response to changes in the environment, yeast cells coordinate intracellular activities to optimize survival and proliferation . The transductions of diverse extracellular stimuli are exerted through multiple mitogen-activated protein kinase (MAPK) cascades . The high osmolarity glycerol (HOG) MAPK pathway is activated by increased environmental osmolarity and results in a rise of the cellular glycerol concentration to adapt the intracellular osmotic pressure . We studied the importance of the short time regulation of glycolysis under hyperosmotic stress for the survival and proliferation of yeast cells . A stimulation of the HOG-MAPK pathway by increasing the medium osmolarity through addition of salt or glucose to cultivated yeast leads to an activation of 6-phosphofructo-2-kinase (PFK2), which is accompanied by a complex phosphorylation pattern of the enzyme . An increase in medium osmolarity with 5% NaCl activates PFK2 3-fold over the initial value . This change in the activity is the result of a 4-fold phosphorylation of the enzyme mediated by protein kinases from the HOG-MAPK pathway . In the case of hyperosmolar glucose a 5-fold PFK2 activation was achieved by a single phosphorylation with protein kinase A near the carboxyl terminus of the protein on Ser(644) and an additional 5-fold phosphorylation within the same amino-terminal fragment as in the presence of salt . The effect of hyperosmolar glucose is the result of an activation of the Ras-cAMP pathway together with the HOG-MAPK pathway . The activation of PFK2 leads to an activation of the upper part of glycolysis, which is a precondition for glycerol accumulation . Yeast cells containing PFK2 accumulate three times more glycerol than cells lacking PFK2, which are not able to grow under hypertonic stress.

J Biol Chem, 2004 Jun 18, 279(25), 25947 - 54 Epub 2004 Mar 22.
A mutation in human topoisomerase II alpha whose expression is lethal in DNA repair-deficient yeast cells; Walker JV et al.; Type II DNA topoisomerases are ATP-dependent enzymes that catalyze alterations in DNA topology . These enzymes are important targets of a variety of anti-bacterial and anti-cancer agents . We identified a mutation in human topoisomerase II alpha, changing aspartic acid 48 to asparagine, that has the unique property of failing to transform yeast cells deficient in recombinational repair . In repair-proficient yeast strains, the Asp-48 --> Asn mutant can be expressed and complements a temperature-sensitive top2 mutation . Purified Asp-48 --> Asn Top2alpha has relaxation and decatenation activity similar to the wild type enzyme, but the purified protein exhibits several biochemical alterations compared with the wild type enzyme . The mutant enzyme binds both covalently closed and linear DNA with greater avidity than the wild type enzyme . hTop2alpha(Asp-48 --> Asn) also exhibited elevated levels of drug-independent cleavage compared with the wild type enzyme . The enzyme did not show altered sensitivity to bisdioxopiperazines nor did it form stable closed clamps in the absence of ATP, although the enzyme did form elevated levels of closed clamps in the presence of a non-hydrolyzable ATP analog compared with the wild type enzyme . We suggest that the lethality exhibited by the mutant is likely because of its enhanced drug-independent cleavage, and we propose that alterations in the ATP binding domain of the enzyme are capable of altering the interactions of the enzyme with DNA . This mutant enzyme also serves as a new model for understanding the action of drugs targeting topoisomerase II.

Mol Genet Genomics, 2004 May, 271(4), 479 - 89 Epub 2004 Mar 19.
Genetic analysis of RSC58, which encodes a component of a yeast chromatin remodeling complex, and interacts with the transcription factor Swi6; Taneda T et al.; Before transcription can begin, the chromatin structure must be rearranged at the nucleosome level . In yeast the nucleosome remodeling complex RSC is involved in this process and it is essential for growth . Recent analysis of the RSC by mass spectrometry has suggested that the product of YLR033w, an essential gene of unknown function, is a novel component of the complex, and the gene has been renamed RSC58 . Rsc58 is predicted to be 502 amino acids long . We have isolated five temperature-sensitive mutations in RSC58 and studied the cellular function of the gene . Our major findings are the following . (1) Two of the alleles have a frameshift mutation near the 3' end of the gene, in codons 482 and 485, respectively . The first mutation is associated with the more severe phenotype . This is compatible with the finding that removal of the C-terminal 25 residues of Rsc58 is lethal to cells . These results suggest that C-terminal region is essential for Rsc58 function . (2) RSC4, which codes for another member of the RSC, was found to be a multicopy suppressor of the phenotype of one of the temperature-sensitive mutants . (3) Two-hybrid analysis identified Swi6, a transcription factor, as a candidate interactor with Rsc58 . An interaction between Rsc58 and Swi6 is also suggested by the fact that rsc58-ts Deltaswi6 double mutants show a more severe growth defect than either mutation alone . These results suggest the possibility that Rsc58 mediates between nucleosome remodeling and the initiation of transcription.

Cell Cycle, 2004 May, 3(5), 629 - 33 Epub 2004 May 03.
Cycling without the cyclosome: modeling a yeast strain lacking the APC; Thornton BR et al.; The construction of viable Saccharomyces cerevisiae strains that lack the anaphase promoting complex (APC) was recently reported . The normally lethal deletions of APC genes were suppressed by the double deletion of the PDS1 and CLB5 genes in conjunction with the insertion of multiple copies of the SIC1 gene controlled by its endogenous promoter . It was proposed that cyclic expression and degradation of Sic1 results in oscillations of Clb/CDK activity necessary for the cell cycle . We have used an updated version of a mathematical model of the yeast cell cycle to model strains that lack the APC . With a few modifications, the model accurately simulates the viability of Apc- strains, as well as the phenotypes of 27 other previously characterized strains . We discuss a few minor inconsistencies between the model and experiment, and how these may inform future revisions to the model.

EMBO Rep, 2004 Apr, 5(4), 368 - 72 Epub 2004 Mar 12.
The Snf1 kinase controls glucose repression in yeast by modulating interactions between the Mig1 repressor and the Cyc8-Tup1 co-repressor; Papamichos-Chronakis M et al.; Among lower eukaryotes, glucose repression is a conserved, widely spread mechanism regulating carbon catabolism . The yeast Snf1 kinase, the Mig1 DNA-binding repressor and the Mig1-interacting co-repressor complex Cyc8(Ssn6)-Tup1 are central components of this pathway . Previous experiments suggested that cytoplasmic translocation of Mig1, upon its phosphorylation by Snf1 in the nucleus, is the key regulatory step for releasing glucose repression . In this report we re-evaluate this model . We establish the coordinated repressive action of Mig1 and Cyc8-Tup1 on GAL1 transcription, but we find that Cyc8-Tup1 is not tethered by Mig1 to the promoter DNA . We demonstrate that both negative regulators occupy GAL1 continuously under either repression or activation conditions, although the majority of the Mig1 is redistributed to the cytoplasm upon activation . We show that Snf1-dependent phosphorylation of Mig1 abolishes interaction with Cyc8-Tup1, and we propose that regulation of this interaction, not the Mig1 cytoplasmic localization, is the molecular switch that controls transcriptional repression/de-repression.

Antonie Van Leeuwenhoek, 2004 Apr, 85(3), 187 - 9
Variation in functional ascospore parts in the ascomycetous yeast Dipodascopsis uninucleata; Bareetseng AS et al.; A variation in functional ascospore morphology was detected using electron microscopy (EM) in two varieties of the yeast Dipodascopsis uninucleata, i.e., D . uninucleata var . uninucleata and D . uninucleata var . wickerhamii . It was found that the latter produces ascospores characterized by the absence of small surface hooks which have been implicated in the release and re-assembly of ascospores in D . uninucleata var . uninucleata . These varieties are closely related on the basis of their mode of sexual reproduction, ascospore morphology as observed under the light microscope, physiological characteristics as well as the extent of divergence in the variable D1/D2 domain of the large subunit 26S ribosomal DNA.

Antonie Van Leeuwenhoek, 2004 May, 85(4), 327 - 34
Morphology-associated expression of NADP-dependent glutamate dehydrogenases during yeast-mycelium transition of a dimorphic fungus Benjaminiella poitrasii; Amin A et al.; Benjaminiella poitrasii, a dimorphic zygomycetous fungus possesses three glutamate dehydrogenases, one requiring NAD while the other two use NADP as a coenzyme . In the activity staining after electrophoresis on native polyacrylamide gel NAD- dependent glutamate dehydrogenase revealed the presence of one enzyme that was expressed in both, yeast- and mycelium-form cells . While in case of NADP- dependent glutamate dehydrogenase two distinct activity bands that were differentially expressed in yeast- and mycelium-form cells were seen . Interestingly, during yeast-mycelium transition and reverse, quantitative changes in form-specific native NADP-dependent glutamate dehydrogenase activities were seen . The biochemical data on temperature and pH optima, thermostability, and kinetic properties confirmed the presence of two NADP-dependent proteins in B . poitrasii, parent strain . The monomorphic mutant (Y-5, yeast form) showed NADP- glutamate dehydrogenase similar to parent yeast-form enzyme . For the first time the significance of differential expression of these enzymes during morphological transition in B . poitrasii has been suggested.

Dev Cell, 2004 Mar, 6(3), 329 - 41
Dynamics of homologous chromosome pairing during meiotic prophase in fission yeast; Ding DQ et al.; Pairing of homologous chromosomes is important for homologous recombination and correct chromosome segregation during meiosis . It has been proposed that telomere clustering, nuclear oscillation, and recombination during meiotic prophase facilitate homologous chromosome pairing in fission yeast . Here we examined the contributions of these chromosomal events to homologous chromosome pairing, by directly observing the dynamics of chromosomal loci in living cells of fission yeast . Homologous loci exhibited a dynamic process of association and dissociation during the time course of meiotic prophase . Lack of nuclear oscillation reduced association frequency for both centromeric and arm regions of the chromosome . Lack of telomere clustering or recombination reduced association frequency at arm regions, but not significantly at centromeric regions . Our results indicate that homologous chromosomes are spatially aligned by oscillation of telomere-bundled chromosomes and physically linked by recombination at chromosome arm regions; this recombination is not required for association of homologous centromeres.

Eur J Biochem, 2004 Apr, 271(7), 1292 - 8
Disruption of the interaction between the Rieske iron-sulfur protein and cytochrome b in the yeast bc1 complex owing to a human disease-associated mutation within cytochrome b; Fisher N et al.; The mitochondrial cytochrome b missense mutation, G167E, has been reported in a patient with cardiomyopathy . The residue G167 is located in an extramembranous helix close to the hinge region of the iron-sulfur protein . In order to characterize the effects of the mutation on the structure and function of the bc(1) complex, we introduced G167E into the highly similar yeast cytochrome b . The mutation had a severe effect on the respiratory function, with the activity of the bc(1) complex decreased to a few per cent of the wild type . Analysis of the enzyme activity indicated that the mutation affected its stability, which could be the result of an altered binding of the iron-sulfur protein on the complex . G167E had no major effect on the interaction between the iron-sulfur protein headgroup and the quinol oxidation site, as judged by the electron paramagnetic resonance signal, and only a minor effect on the rate of cytochrome b reduction, but it severely reduced the rate of cytochrome c(1) reduction . This suggested that the mutation G167E could hinder the movement of the iron-sulfur protein, probably by distorting the structure of the hinge region . The function of bc(1) was partially restored by mutations (W164L and W166L) located close to the primary change, which reduced the steric hindrance caused by G167E . Taken together, these observations suggest that the protein-protein interaction between the n-sulfur protein hinge region and the cytochrome b extramembranous cd2 helix is important for maintaining the structure of the hinge region and, by consequence, the movement of the headgroup and the integrity of the enzyme.

Eur J Biochem, 2004 Apr, 271(7), 1284 - 91
SUT2 is a novel multicopy suppressor of low activity of the cAMP/protein kinase A pathway in yeast; Rutzler M et al.; SUT2 was found in a screen for multicopy suppressors of the synthetic slow growth phenotype of a Deltaras2Deltagpa2 double deletion mutant . It failed, however, to cure the lethal phenotype of a Deltaras1Deltaras2 mutant suggesting that it acts upstream of Ras or in a parallel pathway . By testing cAMP-dependent reactions including the accumulation of storage carbohydrates, pseudohyphal differentiation, entry of meiosis as well as the measurement of FLO11 reporter activity we show that Sut2p modulates the activity of protein kinase A (PKA) . Additionally, we demonstrate that cellular levels of Ras2p are affected by Sut2p and that Sut2-GFPp accumulates significantly in the nucleus . Based on the observed influence of high SUT2 gene dosage on PKA activity as well as Sut2p's homology to the presumptive transcription factor Sut1p, we suggest that Sut2p contributes to regulation of PKA activity at the level of transcription.

J Biol Chem, 2004 May 21, 279(21), 21992 - 2001 Epub 2004 Mar 17.
The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family; Shaner L et al.; The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family . Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain . However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them . To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast . Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional . Complementation ability correlated well with ATP binding assessed in vitro . The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active . Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis . In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans . These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.

Curr Biol, 2004 Mar 9, 14(5), 406 - 11
A novel transport pathway for a yeast plasma membrane protein encoded by a localized mRNA; Juschke C et al.; Generally, plasma membrane (PM) proteins are cotranslationally inserted into the endoplasmic reticulum (ER) and travel in vesicles via the Golgi apparatus to the PM . In the yeast Saccharomyces cerevisiae, the polytopic membrane protein Ist2p is encoded by an mRNA that is localized to the cortex of daughter cells . It has been suggested that IST2 mRNA localization leads to the accumulation of the protein at the PM of daughter cells . Since small- and medium-sized daughter cells only contain cortical, but not perinuclear ER, this implies the local translation of Ist2p specifically at the cortical ER . Here, we show that localization of constitutively expressed IST2 mRNA is required for delivery of Ist2p to the PM of daughter, but not mother cells and that it does not result in daughter-specific Ist2p accumulation . In contrast to a PM-located hexose transporter (Hxt1p) that follows the standard secretory pathway, the trafficking of Ist2p is independent of myosin-mediated vesicular transport . Furthermore, colocalization experiments in mutants of the secretory pathway demonstrate that trafficking of Ist2p does not require the classical secretory machinery . These data suggest the existence of a novel trafficking pathway connecting specialized domains of the ER with the PM.

Antioxid Redox Signal, 2004 Apr, 6(2), 259 - 67
Utilization of yeast to investigate the role of lipid oxidation in cell death; Manon S; The yeast Saccharomyces cerevisiae is a powerful tool to investigate several aspects of the molecular mechanisms underlying programmed cell death, namely, the events involving mitochondria . Yeast has allowed new information to be gained about crucial aspects of the interaction between proapoptotic proteins Bax and Bid and mitochondria, namely, their addressing and insertion into the mitochondrial outer membrane and their ability to induce the relocalization of cytochrome c . Going one step further, the prooxidant effect of Bax can also be studied in yeast . Bax expression induces both the accumulation of reactive oxygen species and lipid oxidation . Lipid oxidation is involved in Bax-induced cell death and may be required for optimal insertion of Bax in mitochondria . The easy modulation of lipid composition in yeast is a powerful tool to investigate this process further, and studies can be extended to other regulators of apoptosis, such as proapoptotic Bid or antiapoptotic Bcl-x(L) . Also, yeast is a model for the study of other types of cell death, such as autophagy-related forms of death, for which a role of lipid oxidation has also been evidenced.

Biochem J, 2004 Jul 1, 381(Pt 1), 321 - 8
SLI1 (YGR212W) is a major gene conferring resistance to the sphingolipid biosynthesis inhibitor ISP-1, and encodes an ISP-1 N-acetyltransferase in yeast; Momoi M et al.; ISP-1 (myriocin) is a potent inhibitor of serine palmitoyltransferase, the primary enzyme of sphingolipid biosynthesis, and is a useful tool for studying the biological functions of sphingolipids in both mammals and yeast (Saccharomyces cerevisiae) . In a previous study, we cloned yeast multicopy suppressor genes for ISP-1, and one of these, YPK1/SLI2, was shown to encode a serine/threonine kinase which is a yeast homologue of mammalian SGK1 (serum/glucocorticoid-regulated kinase 1) . In the present study, another gene, termed SLI1 (YGR212W; GenBank accession number CAA97239.1), was characterized . Sli1p has weak similarity to Atf1p and Atf2p, which are alcohol acetyltransferases . Although a sli1-null strain grew normally, the IC50 of ISP-1 for the growth of this strain was markedly decreased compared with that for the parental strain, indicating that Sli1p is a major contributor to ISP-1 resistance in yeast . On a sli1-null background, the increase in resistance to ISP-1 induced by YPK1 gene transfection was almost abolished . These data indicate that Sli1p co-operates with Ypk1p in mediating resistance to ISP-1 in yeast . Sli1p was found to convert ISP-1 into N-acetyl-ISP-1 in vitro . Furthermore, N-acetyl-ISP-1 did not share the ability of ISP-1 to inhibit the growth of yeast cells, and the serine palmitoyltransferase inhibitory activity of N-acetyl-ISP-1 was much lower than that of ISP-1 . These data suggest that Sli1p inactivates ISP-1 due to its N-acetyltransferase activity towards ISP-1.

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