Mol Cell Biol, 1999 May, 19(5), 3312 - 27 NDD1, a high-dosage suppressor of cdc28-1N, is essential for expression of a subset of late-S-phase-specific genes in Saccharomyces cerevisiae; Loy CJ et al.; cdc28-1N mutants progress through the G1 and S phases normally at the restrictive temperature but fail to undergo nuclear division . We have isolated a gene, NDD1, which at a high dosage suppresses the nuclear-division defect of cdc28-1N . NDD1 (nuclear division defective) is an essential gene . Its expression during the cell cycle is tightly regulated such that NDD1 RNA is most abundant during the S phase . Cells lacking the NDD1 gene arrest with an elongated bud, a short mitotic spindle, 2N DNA content, and an undivided nucleus, suggesting that its function is required for some aspect of nuclear division . We show that overexpression of Ndd1 results in the upregulation of both CLB1 and CLB2 transcription, suggesting that the suppression of cdc28-1N by NDD1 may be due to an accumulation of these cyclins . Overproduction of Ndd1 also enhances the expression of SWI5, whose transcription, like that of CLB1 and CLB2, is activated in the late S phase . Ndd1 is essential for the expression of CLB1, CLB2, and SWI5, since none of these genes are transcribed in its absence . Both CLB2 expression and its upregulation by NDD1 are mediated by a 240-bp promoter sequence that contains four MCM1-binding sites . However, Ndd1 does not appear to be a component of any of the protein complexes assembled on this DNA fragment, as indicated by gel mobility shift assays . Instead, overexpression of NDD1 prevents the formation of one of the complexes whose appearance correlates with the termination of CLB2 expression in G1 . The inability of GAL1 promoter-driven CLB2 to suppress the lethality of NDD1 null mutant suggests that, in addition to CLB1 and CLB2, NDD1 may also be required for the transcription of other genes whose functions are necessary for G2/M transition. Mol Cell Biol, 1999 May, 19(5), 3237 - 45 Glycogen synthase phosphatase interacts with heat shock factor to activate CUP1 gene transcription in Saccharomyces cerevisiae; Lin JT et al.; Upon heat shock, transcription of many stress-inducible genes is rapidly and dramatically stimulated by heat shock factor (HSF) . A central region of the yeast HSF (designated HSFrr for "repression region") was previously identified and proposed to be involved in repressing the activation domain under non-heat-shock conditions . Here, we used the phage display system to isolate proteins that interact with HSFrr . This should identify factors that modulate HSF activity or directly participate in HSF-mediated transcriptional activation . We constructed a randomly sheared yeast genomic library to express yeast proteins on the surface of lambda phage . HSFrr binding phages were selected by cycles of affinity chromatography . DNA sequencing identified an HSFrr-interacting phage that contains the GAC1 gene . The GAC1 gene encodes the regulatory subunit for a type 1 serine/threonine phosphoprotein phosphatase, Glc7 . Both gac1 and glc7 mutations had little effect on HSF activation of gene transcription of two heat shock genes, SSA4 and HSP82 . In contrast, heat shock induction of CUP1 gene expression was completely abolished in a glc7 mutant and reduced in a gac1 mutant . The results demonstrate that the Glc7 phosphatase and its Gac1 regulatory subunit play positive roles in HSF activation of CUP1 transcription. J Biol Chem, 1999 Apr 23, 274(17), 11713 - 20 A new family of human histone deacetylases related to Saccharomyces cerevisiae HDA1p; Fischle W et al.; Histone deacetylases are the catalytic subunits of multiprotein complexes that are targeted to specific promoters through their interaction with sequence-specific DNA-binding factors . We have cloned and characterized a new human cDNA, HDAC-A, with homology to the yeast HDA1 family of histone deacetylases . Analysis of the predicted amino acid sequence of HDAC-A revealed an open reading frame of 967 amino acids containing two domains: a NH2-terminal domain with no homology to known proteins and a COOH-terminal domain with homology to known histone deacetylases (42% similarity to RPD3, 60% similarity to HDA1) . Three additional human cDNAs with high homology to HDAC-A were identified in sequence data bases, indicating that HDAC-A itself is a member of a new family of human histone deacetylases . The mRNA encoding HDAC-A was differentially expressed in a variety of human tissues . The expressed protein, HDAC-Ap, exhibited histone deacetylase activity and this activity mapped to the COOH-terminal region (amino acids 495-967) with homology to HDA1p . In immunoprecipitation experiments, HDAC-A interacted specifically with several cellular proteins, indicating that it might be part of a larger multiprotein complex. J Biol Chem, 1999 Apr 23, 274(17), 11653 - 9 Cloning of murine translation initiation factor 6 and functional analysis of the homologous sequence YPR016c in Saccharomyces cerevisiae; Wood LC et al.; The cDNA sequence of a murine gene whose expression was up-regulated after epidermal injury was cloned utilizing differential display . The full-length cDNA was isolated by 3' and 5' rapid amplification of cDNA ends from mouse liver . The predicted protein is >97% identical to the human sequence for eukaryotic translation initiation factor (eIF) 6, thus identifying the gene as murine eIF6 . Functional studies of the yeast eIF6 homolog, YPR016c, were initiated in Saccharomyces cerevisiae to determine the cellular role(s) of eIF6 . Complete deletion of the YPR016c coding sequence was lethal . Viability was restored in the presence of either YPR016c or murine eIF6, when either was expressed as amino-terminal green fluorescent protein fusion protein . Moreover, both fusion proteins localized to nuclear/perinuclear compartments in their respective yeast strains . When the expression of YPR016c-green fluorescent protein was repressed, there was a dramatic reduction in the 60 S ribosomal subunit and polysome content and decreased 80S monosome content . Additionally, the YPR016c-depleted cells arrested in G1 . These studies show that YPR016c, which encodes yeast eIF6, is necessary for maximal polysome formation and plays an important role in determining free 60 S ribosomal subunit content. Microbiology, 1999 Jan, 145 ( Pt 1), 249 - 58 KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genes; Martin H et al.; The KNR4 gene, originally isolated by complementation of a K9 killer-toxin-resistant mutant displaying reduced levels of both 1,3-beta-glucan and 1,3-beta-glucan synthase activity, was recloned from a YCp50 genomic library as a suppressor of Saccharomyces cerevisiae calcofluor-white-hypersensitive (cwh) mutants . In these mutants, which were characterized by increased chitin levels, the suppressor effect of KNR4 resulted, for some of them, in a lowering of polymer content to close to wild-type level, with no effect on the contents of beta-glucan and mannan . In all cases, this effect was accompanied by a strong reduction in mRNA levels corresponding to CHS1, CHS2 and CHS3, encoding chitin synthases, without affecting expression of FKS1 and RHO1, two genes encoding the catalytic subunit and a regulatory component of 1,3-beta-glucan synthase, respectively . Overexpression of KNR4 also inhibited expression of CHS genes in wild-type strains and in two other cwh mutants, whose sensitivity to calcofluor white was not suppressed by this gene . The physiological relevance of the KNR4 transcriptional effect was addressed in two different ways . In a wild-type strain exposed to alpha-factor, overexpression of this gene inhibited CHS1 induction and delayed shmoo formation, two events which are triggered in response to the pheromone, whereas it did not affect bud formation and cell growth in a chs1 chs2 double mutant . A chimeric protein made by fusing green fluorescent protein to the C terminus of Knr4p which fully complemented a knr4delta mutation was found to localize in patches at presumptive bud sites in unbudded cells and at the incipient bud site during bud emergence . Taken together, these results demonstrate that KNR4 has a regulatory role in chitin deposition and in cell wall assembly . A mechanism by which this gene affects expression of CHS genes is proposed. Microbiology, 1999 Jan, 145 ( Pt 1), 231 - 9 Stress induction of HSP30, the plasma membrane heat shock protein gene of Saccharomyces cerevisiae, appears not to use known stress-regulated transcription factors; Seymour IJ et al.; More than one transcription factor contributes to the Saccharomyces cerevisiae heat shock response . Many genes are induced through the activation of heat shock factor (Hsf1), a protein that is constitutively bound to heat shock promoter elements (HSEs) . Other genes are switched on by Msn2/Msn4-dependent activation of a quite separate promoter element (the stress response element, STRE) . While Hsf directs gene activation mainly in response to heat stress, STRE-directed transcription is stimulated not only by heat but also by several other stresses, starvation included . HSP30, encoding the plasma membrane heat shock protein, is shown in this study to be activated by several stresses . It is most strongly induced with heat shock, ethanol and weak organic acid exposure . The HSP30 promoter has no good agreement to the HSE consensus and its stress activation is unaffected by a mutation (hsf1-m3) that causes defective heat shock activation of Hsf1-dependent genes . Activation of HSP30 occurs with some, but not all, STRE-inducing stresses and is largely unaffected either by loss of the Msn2/Msn4 transcription factors or with mutation of all STRE-like consensus sequences of the promoter . Stress activation of HSP30 appears therefore to involve as yet unidentified components of the yeast transcriptional apparatus. J Photochem Photobiol B, 1999 Jan, 48(1), 57 - 62 Fungicidal properties of meso-arylglycosylporphyrins: influence of sugar substituents on photoinduced damage in the yeast Saccharomyces cerevisiae; Carre V et al.; A series of neutral meso-arylglycosylporphyrins has been tested in order to evaluate their potency as antifungal agents against the yeast Saccharomyces cerevisiae . Photodynamic activity of these molecules results in intracellular damage as evidenced by the loss of clonogenicity and DNA fragmentation . The ability of these photosensitizers to permeate yeast cells is determined by microspectrofluorimetry and is correlated with their antifungal potency . Amphiphilic porphyrin derivatives are shown to exhibit the more pronounced photoactivity. Genetika, 1998 Dec, 34(12), 1603 - 9 {Cryptic heterokaryons in Saccharomyces cerevisiae strains . Model experiments}; Nevzgliadova OV et al.; Heterokaryons capable of segregating clones of different phenotypes were obtained in saccharomycetes yeast . Clones expressing phenotypic traits encoded by the nuclear genome of one parent and the mitochondrial genome of another were shown to contain cells that carried the second phenotypically silent nucleus . The cryptic nucleus can be uncovered by growing these "cytoductants" on the corresponding medium . The use of strains carrying an insertion in the URA3 gene and the endogenic plasmid, 2 microns DNA, made it possible to detect this nucleus by blot hybridization. Genetika, 1998 Dec, 34(12), 1597 - 602 {Cryptic heterokaryons in Saccharomyces cerevisiae strains: pseudopleiotropic suppression in the multiple marked YPH857 strain}; Nevzgliadova OV et al.; The yeast strain YPH857 carrying multiple genetic markers was shown to segregate clones that had the pleiotropic suppression phenotype . The phenotype was designated Ppsu+ . This suppression involves deletion alleles of the TRP1 and HIS3 genes and an insertion in the URA3 gene . Unlike the original YPH857 culture that carries an unidentified mutation of resistance to cycloheximide, the Ppsu+ clones exhibited a decreased level of resistance to this inhibitor of protein synthesis . In addition, they have a lower mating ability and can produce asci on a standard medium for sporulation . A comparative analysis of total DNA from the YPH857 strain and Ppsu+ segregants by Southern blotting provided evidence for the presence of an extraneous nucleus in these segregants . Ppsu+ strains were shown to contain wild-type alleles, apart from deletion and insertional alleles typical for the YPH857 strain . Moreover, they contain the 2 microns DNA of the Scp3 type with deletion of one of the two EcoRI and HpaI recognition sites (whereas the 2 microns DNA of YPH857 belongs to the Scp1 type) and exhibit heterogeneity with respect to the presence of one EcoR1 recognition site in the gene of 5S ribosomal RNA . It was supposed that the "cryptic" nucleus belongs to a strain of low viability and can survive as an unexpressed DNA in a small fraction of cells . Nuclei from the cryptic and YPH857 strains can be fused at a low rate to yield Ppsu+ cells capable of sporulation . In certain cases, a Ppsu+ clone may be heterogeneous: some of its cells contain nuclei in an unfused heterokaryotic state . This assumption has been confirmed by selecting Cyhr colonies with the original YPH857 genome among Ppsu+ clones on the cycloheximide-containing medium. Nucleic Acids Res, 1999 May 1, 27(9), 1978 - 84 The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G-G paired DNAs; Sun H et al.; The Saccharomyces cerevisiae Sgs1p helicase localizes to the nucleolus and is required to maintain the integrity of the rDNA repeats . Sgs1p is a member of the RecQ DNA helicase family, which also includes Schizo-saccharomyces pombe Rqh1, and the human BLM and WRN genes . These genes encode proteins which are essential to maintenance of genomic integrity and which share a highly conserved helicase domain . Here we show that recombinant Sgs1p helicase efficiently unwinds guanine-guanine (G-G) paired DNA . Unwinding of G-G paired DNA is ATP- and Mg2+-dependent and requires a short 3' single-stranded tail . Strikingly, Sgs1p unwinds G-G paired substrates more efficiently than duplex DNAs, as measured either in direct assays or by competition experiments . Sgs1p efficiently unwinds G-G paired telomeric sequences, suggesting that one function of Sgs1p may be to prevent telomere-telomere interactions which can lead to chromosome non-disjunction . The rDNA is G-rich and has considerable potential for G-G pairing . Diminished ability to unwind G-G paired regions may also explain the deleterious effect of mutation of Sgs1 on rDNA stability, and the accelerated aging characteristic of yeast strains that lack Sgs1 as well as humans deficient in the related WRN helicase. Mol Biol Cell, 1999 Apr, 10(4), 1147 - 61 Kinase activity-dependent nuclear export opposes stress-induced nuclear accumulation and retention of Hog1 mitogen-activated protein kinase in the budding yeast Saccharomyces cerevisiae; Reiser V et al.; Budding yeast adjusts to increases in external osmolarity via a specific mitogen-activated protein kinase signal pathway, the high-osmolarity glycerol response (HOG) pathway . Studies with a functional Hog1-green fluorescent protein (GFP) fusion reveal that even under nonstress conditions the mitogen-activated protein kinase Hog1 cycles between cytoplasmic and nuclear compartments . The basal distribution of the protein seems independent of its activator, Pbs2, and independent of its phosphorylation status . Upon osmotic challenge, the Hog1-GFP fusion becomes rapidly concentrated in the nucleus from which it is reexported after return to an iso-osmotic environment or after adaptation to high osmolarity . The preconditions and kinetics of increased nuclear localization correlate with those found for the dual phosphorylation of Hog1-GFP . The duration of Hog1 nuclear residence is modulated by the presence of the general stress activators Msn2 and Msn4 . Reexport of Hog1 to the cytoplasm does not require de novo protein synthesis but depends on Hog1 kinase activity . Thus, at least three different mechanisms contribute to the intracellular distribution pattern of Hog1: phosphorylation-dependent nuclear accumulation, retention by nuclear targets, and a kinase-induced export. J Bacteriol, 1999 Apr, 181(8), 2640 - 2 Cyclic AMP can decrease expression of genes subject to catabolite repression in Saccharomyces cerevisiae; Zaragoza O et al.; External cyclic AMP (cAMP) hindered the derepression of gluconeogenic enzymes in a pde2 mutant of Saccharomyces cerevisiae, but it did not prevent invertase derepression . cAMP reduced nearly 20-fold the transcription driven by upstream activation sequence (UAS1FBP1) from FBP1, encoding fructose-1,6-bisphosphatase; it decreased 2-fold the activation of transcription by UAS2FBP1 . Nuclear extracts from cells derepressed in the presence of cAMP were impaired in the formation of specific UASFBP1-protein complexes in band shift experiments . cAMP does not appear to act through the repressing protein Mig1 . Control of FBP1 transcription through cAMP is redundant with other regulatory mechanisms. J Bacteriol, 1999 Apr, 181(8), 2555 - 63 Clathrin and two components of the COPII complex, Sec23p and Sec24p, could be involved in endocytosis of the Saccharomyces cerevisiae maltose transporter; Penalver E et al.; The Saccharomyces cerevisiae maltose transporter is a 12-transmembrane segment protein that under certain physiological conditions is degraded in the vacuole after internalization by endocytosis . Previous studies showed that endocytosis of this protein is dependent on the actin network, is independent of microtubules, and requires the binding of ubiquitin . In this work, we attempted to determine which coat proteins are involved in this endocytosis . Using mutants defective in the heavy chain of clathrin and in several subunits of the COPI and the COPII complexes, we found that clathrin, as well as two cytosolic subunits of COPII, Sec23p and Sec24p, could be involved in internalization of the yeast maltose transporter . The results also indicate that endocytosis of the maltose transporter and of the alpha-factor receptor could have different requirements. J Bacteriol, 1999 Apr, 181(8), 2527 - 34 The extracellular domain of the Saccharomyces cerevisiae Sln1p membrane osmolarity sensor is necessary for kinase activity; Ostrander DB et al.; The function of the extracellular domain (ECD) of Sln1p, a plasma membrane two-transmembrane domain (TMD) sensor of the high-osmolarity glycerol (HOG) response pathway, has been studied in the yeast Saccharomyces cerevisiae . Truncations of SLN1 that retain an intact kinase domain are capable of complementing the lethality of an sln1Delta strain . By observing levels of Hog1p phosphorylation as well as the phosphorylation state of Sln1p, the kinase activities of various SLN1 constructions were determined . In derivatives that do not contain the first TMD, Sln1p activity was no longer dependent on medium osmolarity but appeared to be constitutively active even under conditions of high osmolarity . Removal of the first TMD (DeltaTMD1 construct) gave a protein that was strongly phosphorylated whereas Hog1p was largely dephosphorylated, as expected if the active form of Sln1p is phosphorylated . When both TMDs as well as the ECD were deleted, so that the kinase domain is cytosolic, Sln1p was not phosphorylated whereas Hog1p became constitutively hyperphosphorylated . Surprisingly, this hyperactivity of the HOG mitogen-activated protein kinase signaling pathway was not sufficient to result in cell lethality . When the ECD of the DeltaTMD1 construct was replaced with a leucine zipper motif, Sln1p was hyperactive, so that Hog1p became mostly unphosphorylated . In contrast, when the Sln1p/leucine zipper construct was crippled by a mutation of one of the internal leucines, the Sln1 kinase was inactive . These experiments are consistent with the hypothesis that the ECD of Sln1p functions as a dimerization and activation domain but that osmotic regulation of activity requires the presence of the first TMD. J Bacteriol, 1999 Apr, 181(8), 2472 - 6 Control of nitrogen catabolite repression is not affected by the tRNAGln-CUU mutation, which results in constitutive pseudohyphal growth of Saccharomyces cerevisiae; Beeser AE et al.; Saccharomyces cerevisiae responds to nitrogen availability in several ways . (i) The cell is able to distinguish good nitrogen sources from poor ones through a process designated nitrogen catabolite repression (NCR) . Good and poor nitrogen sources do not demonstrably affect the cell cycle other than to influence the cell's doubling time . (ii) Nitrogen starvation promotes the initiation of sporulation and pseudohyphal growth . (iii) Nitrogen starvation strongly affects the cell cycle; nitrogen-starved cells arrest in G1 . A specific allele of the SUP70/CDC65 tRNAGln gene (sup70-65) has been reported to be defective in nitrogen signaling associated with pseudohyphal formation, sporulation, and NCR . Our data confirm that pseudohyphal growth occurs gratuitously in sup70-65 mutants cultured in nitrogen-rich medium at 30 degrees C . However, we find neither any defect in NCR in the sup70-65 mutant nor any alteration in the control of YVH1 expression, which has been previously shown to be specifically induced by nitrogen starvation. J Biotechnol, 1999 Feb 19, 68(2-3), 197 - 212 Investigation of the impact of MIG1 and MIG2 on the physiology of Saccharomyces cerevisiae; Klein CJ et al.; The gene functions of MIG1 and MIG2 are well known for their role in glucose control in Saccharomyces cerevisiae . A prototrophic mig1 disruptant (T468) and mig1mig2 double disruptant (T475) as well as their congenic wild-type strain (CEN.PK 113-7D) were analysed for changes in their peripheral metabolism (batch cultivations on sugar mixtures) and central metabolism (batch and continuous cultivations as well as acceleratostats) . Sucrose metabolism was alleviated of glucose control in the mig1 disruptant, and even more so in the mig1mig2 disruptant compared with their wild-type strain . The lag phase in a batch cultivation grown on a glucose-galactose mixture was reduced by 50% in either disruptant, i.e . additional disruption of MIG2 in a mig1 background did not further alleviate galactose metabolism from glucose control . In contrast, both disruptants exhibited a more stringent glucose control of maltose metabolism compared with the wild-type strain . Growing on glucose, the mig1mig2 double disruptant exhibited a 12% higher specific growth rate than the wild-type strain, as well as a significantly higher respiratory capacity. J Cell Sci, 1999 May, 112 ( Pt 9), 1375 - 83 NH4+-induced down-regulation of the Saccharomyces cerevisiae Gap1p permease involves its ubiquitination with lysine-63-linked chains; Springael JY et al.; Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces internalization of the general amino acid permease Gap1p and its subsequent degradation in the vacuole . An essential step in this down-regulation is Gap1p ubiquitination through a process requiring the Npi1p/Rsp5p ubiquitin ligase . We show in this report that NPI2, a second gene required for NH4+-induced down-regulation of Gap1p, codes for the ubiquitin hydrolase Doa4p/Ubp4p/Ssv7p and that NH4+-induced Gap1p ubiquitination is strongly reduced in npi2 cells . The npi2 mutation results in substitution of an aromatic amino acid located in a 33-residue sequence shared by some ubiquitin hydrolases of the Ubp family . In this mutant, as in doa4(delta) cells, the amount of free monomeric ubiquitin is at least four times lower than in wild-type cells . Both ubiquitination and down-regulation of the permease can be restored in npi2 cells by over-expression of ubiquitin . In proline-grown wild-type and npi2/doa4 cells overproducing ubiquitin, Gap1p appears to be mono-ubiquitinated at two lysine acceptor sites . Addition of NH4+ triggers rapid poly-ubiquitination of Gap1p, the poly-ubiquitin chains being specifically formed by linkage through the lysine 63 residue of ubiquitin . Gap1p is thus ubiquitinated differently from the proteins targeted by ubiquitination for proteolysis by the proteasome, but in the same manner as the uracil permease, also subject to ubiquitin-dependent endocytosis . When poly-ubiquitination through Lys63 is blocked, the Gap1p permease still undergoes NH4+-induced down-regulation, but to a lesser extent. Biochem J, 1999 Apr 15, 339 ( Pt 2), 407 - 11 Identification and characterization of Saccharomyces cerevisiae yapsin 3, a new member of the yapsin family of aspartic proteases encoded by the YPS3 gene; Olsen V et al.; A new aspartic protease from Saccharomyces cerevisiae, with a high degree of similarity with yapsin 1 and yapsin 2 and a specificity for basic residue cleavage sites of prohormones, has been cloned . This enzyme was named yapsin 3 . Expression of a C-terminally truncated non-membrane anchored yapsin 3 in yeast yielded a heterogeneous protein between 135-200 kDa which, upon treatment with endoglycosidase H, migrated as a 60 kDa form . Amino-acid analysis of the N-terminus of expressed yapsin 3 revealed two different N-terminal residues, serine-48 and phenylalanine-54, which followed a dibasic and a monobasic residue respectively . Cleavage of several prohormones by non-anchored yapsin 3 revealed a specificity distinct from that of yapsin 1. Biochem J, 1999 Apr 15, 339 ( Pt 2), 299 - 307 Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein; Kruckeberg AL et al.; The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP) . The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7 . Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein . We calculated from the fluorescence level and transport kinetics that induced cells had 1.4x10(5) Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 degrees C . Expression of Hxt2-GFP was induced by growth at low concentrations of glucose . Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane . In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm . When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h . When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose. Mol Genet Metab, 1999 Apr, 66(4), 314 - 9 Investigation of Batten disease with the yeast Saccharomyces cerevisiae; Pearce DA et al.; The CLN3 gene, which encodes the protein whose absence is responsible for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995 . The function of the protein, Cln3p, still remains elusive . We previously cloned the Saccharomyces cerevisiae homolog to the human CLN3 gene, designated BTN1, whose product is 39% identical and 59% similar to Cln3p . We report that yeast strains lacking Btn1p, btn1-Delta deletion yeast strains, are more resistant to d-(-)-threo-2-amino-1-{p-nitrophenyl}-1,3-propanediol (ANP), in a pH-dependent manner . This phenotype is complemented in yeast by the human CLN3 gene . In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced into BTN1, altered the degree of ANP resistance . Severity of Batten disease due to mutations in CLN3 and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared . These results indicate that yeast can be used as a model for the study of Batten disease . J Cell Biol, 1999 Apr 5, 145(1), 15 - 28 Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle; Hyland KM et al.; A genetic synthetic dosage lethality (SDL) screen using CTF13 encoding a known kinetochore protein as the overexpressed reference gene identified two chromosome transmission fidelity (ctf) mutants, YCTF58 and YCTF26 . These mutant strains carry independent alleles of a novel gene, which we have designated CTF19 . In light of its potential role in kinetochore function, we have cloned and characterized the CTF19 gene in detail . CTF19 encodes a nonessential 369-amino acid protein . ctf19 mutant strains display a severe chromosome missegregation phenotype, are hypersensitive to benomyl, and accumulate at G2/M in cycling cells . CTF19 genetically interacts with kinetochore structural mutants and mitotic checkpoint mutants . In addition, ctf19 mutants show a defect in the ability of centromeres on minichromosomes to bind microtubules in an in vitro assay . In vivo cross-linking and chromatin immunoprecipitation demonstrates that Ctf19p specifically interacts with CEN DNA . Furthermore, Ctf19-HAp localizes to the nuclear face of the spindle pole body and genetically interacts with a spindle-associated protein . We propose that Ctf19p is part of a macromolecular kinetochore complex, which may function as a link between the kinetochore and the mitotic spindle. J Biol Chem, 1999 Apr 9, 274(15), 10523 - 32 Control of expression of one-carbon metabolism genes of Saccharomyces cerevisiae is mediated by a tetrahydrofolate-responsive protein binding to a glycine regulatory region including a core 5'-CTTCTT-3' motif; Hong SP et al.; Expression of yeast genes involved in one-carbon metabolism is controlled by glycine, by L-methionine, and by nitrogen sources . Here we report a novel control element containing a core CTTCTT motif mediating the glycine response, demonstrating that a protein binds this element, that binding is modulated by tetrahydrofolate, and that folate is required for the in vivo glycine response . In an heterologous CYC1 promoter the region needed for the glycine response of GCV2 (encoding the P-subunit of glycine decarboxylase) mediated repression that was relieved by glycine . It was also responsible for L-methionine control but not nitrogen repression . GCV1 and GCV3 have an homologous region in their promoters . The GCV1 region conferred a glycine response on an heterologous promoter acting as a repressor or activator depending on promoter context . A protein was identified that bound to the glycine regulatory regions of GCV1 and GCV2 only if the CTTCTT motif was intact . This protein protected a 17-base pair CATCN7CTTCTT region of GCV2 that is conserved between GCV1 and GCV2 . Protein binding was increased by tetrahydrofolate, and use of a fol1 deletion mutant indicated the involvement of a folate in the in vivo glycine response . Tetrahydrofolate or a derivative may act as a ligand for the transcription factor controlling expression of one-carbon metabolism genes. Mol Gen Genet, 1999 Mar, 261(2), 388 - 401 Lic4, a nuclear phosphoprotein that cooperates with calcineurin to regulate cation homeostasis in Saccharomyces cerevisiae; Hemenway CS et al.; The target of the immunosuppressants cyclosporin A(CsA) and FK506 is calcineurin, a highly conserved protein phosphatase that is required for T-cell activation and the regulation of ion homeostasis in yeast . Here we identify two genes, PMR2B and LIC4 which, when overexpressed, suppress the cation-sensitive phenotype of yeast cells lacking calcineurin . PMR2B encodes a Na+/Li+-specific plasma membrane pump and is similar to PMR2A, whose expression is known to be regulated by calcineurin . LIC4 (lithium comvertas) encodes a novel 33-kDa protein with no identity to known proteins . LIC4 overexpression suppresses the Li+-sensitive phenotype of calcineurin mutants but not the defect in recovery from pheromone arrest or viability of calcineurin dependent mutants, indicating a specific role in cation homeostasis . Similarly, lic4 mutations increase the Li+ sensitivity of both wild-type and calcineurin mutant strains, and reduce expression of pmr2A in calcineurin mutant strains, indicating that calcineurin and Lic4 may regulate parallel cation homeostatic pathways . lic4 mutations also exacerbate the Li+-sensitive phenotype of hal3 mutant strains, and overexpression of either Lic4 or Hal3 suppresses the salt sensitivity of mutant strains lacking calcineurin, Hal3, or Lic4, either singly or in combination . Taken together, these observations suggest that calcineurin, Hal3, and Lic4 cooperatively regulate the response of yeast cells to cation stress . Lic4 is phosphoprotein in vivo and a calcineurin substrate in vitro . By indirect and direct immunofluorescence detection of HA- and GFP-tagged proteins, Lic4 is localized in the nucleus in wild-type cells but predominantly cytoplasmic in cells lacking calcineurin . Taken together, our findings support a model in which calcineurin and Lic4 are components of signalling cascades that regulate cation stress responses in yeast. Genetics, 1999 Apr, 151(4), 1425 - 44 Relative dependence of different outputs of the Saccharomyces cerevisiae pheromone response pathway on the MAP kinase Fus3p; Farley FW et al.; Fus3p and Kss1p act at the end of a conserved signaling cascade that mediates numerous cellular responses for mating . To determine the role of Fus3p in different outputs, we isolated and characterized a series of partial-function fus3 point mutants for their ability to phosphorylate a substrate (Ste7p), activate Ste12p, undergo G1 arrest, form shmoos, select partners, mate, and recover . All the mutations lie in residues that are conserved among MAP kinases and are predicted to affect either enzyme activity or binding to Ste7p or substrates . The data argue that Fus3p regulates the various outputs assayed through the phosphorylation of multiple substrates . Different levels of Fus3p function are required for individual outputs, with the most function required for shmoo formation, the terminal output . The ability of Fus3p to promote shmoo formation strongly correlates with its ability to promote G1 arrest, suggesting that the two events are coupled . Fus3p promotes recovery through a mechanism that is distinct from its ability to promote G1 arrest and may involve a mechanism that does not require kinase activity . Moreover, catalytically inactive Fus3p inhibits the ability of active Fus3p to activate Ste12p and hastens recovery without blocking G1 arrest or shmoo formation . These results raise the possibility that in the absence of sustained activation of Fus3p, catalytically inactive Fus3p blocks further differentiation by restoring mitotic growth . Finally, suppression analysis argues that Kss1p contributes to the overall pheromone response in a wild-type strain, but that Fus3p is the critical kinase for all of the outputs tested. Genetics, 1999 Apr, 151(4), 1365 - 78 Specific components of the SAGA complex are required for Gcn4- and Gcr1-mediated activation of the his4-912delta promoter in Saccharomyces cerevisiae; Dudley AM et al.; Mutations selected as suppressors of Ty or solo delta insertion mutations in Saccharomyces cerevisiae have identified several genes, SPT3, SPT7, SPT8, and SPT20, that encode components of the SAGA complex . However, the mechanism by which SAGA activates transcription of specific RNA polymerase II-dependent genes is unknown . We have conducted a fine-structure mutagenesis of one widely used SAGA-dependent promoter, the delta element of his4-912delta, to identify sequence elements important for its promoter activity . Our analysis has characterized three delta regions necessary for full promoter activity and accurate start site selection: an upstream activating sequence, a TATA region, and an initiator region . In addition, we have shown that factors present at the adjacent UASHIS4 (Gcn4, Bas1, and Pho2) also activate the delta promoter in his4-912delta . Our results suggest a model in which the delta promoter in his4-912delta is primarily activated by two factors: Gcr1 acting at the UASdelta and Gcn4 acting at the UASHIS4 . Finally, we tested whether activation by either of these factors is dependent on components of the SAGA complex . Our results demonstrate that Spt3 and Spt20 are required for full delta promoter activity, but that Gcn5, another member of SAGA, is not required . Spt3 appears to be partially required for activation of his4-912delta by both Gcr1 and Gcn4 . Thus, our work suggests that SAGA exerts a large effect on delta promoter activity through a combination of smaller effects on multiple factors. Genetics, 1999 Apr, 151(4), 1353 - 63 Suppression of a nuclear aep2 mutation in Saccharomyces cerevisiae by a base substitution in the 5'-untranslated region of the mitochondrial oli1 gene encoding subunit 9 of ATP synthase; Ellis TP et al.; Mutations in the nuclear AEP2 gene of Saccharomyces generate greatly reduced levels of the mature form of mitochondrial oli1 mRNA, encoding subunit 9 of mitochondrial ATP synthase . A series of mutants was isolated in which the temperature-sensitive phenotype resulting from the aep2-ts1 mutation was suppressed . Three strains were classified as containing a mitochondrial suppressor: these lost the ability to suppress aep2-ts1 when their mitochondrial genome was replaced with wild-type mitochondrial DNA (mtDNA) . Many other isolates were classified as containing dominant nuclear suppressors . The three mitochondrion-encoded suppressors were localized to the oli1 region of mtDNA using rho- genetic mapping techniques coupled with PCR analysis; DNA sequencing revealed, in each case, a T-to-C nucleotide transition in mtDNA 16 nucleotides upstream of the oli1 reading frame . It is inferred that the suppressing mutation in the 5' untranslated region of oli1 mRNA restores subunit 9 biosynthesis by accommodating the modified structure of Aep2p generated by the aep2-ts1 mutation (shown here to cause the substitution of proline for leucine at residue 413 of Aep2p) . This mode of mitochondrial suppression is contrasted with that mediated by heteroplasmic rearranged rho- mtDNA genomes bypassing the participation of a nuclear gene product in expression of a particular mitochondrial gene . In the present study, direct RNA-protein interactions are likely to form the basis of suppression. Genetics, 1999 Apr, 151(4), 1315 - 25 Suppressor analysis of mutations in the 5'-untranslated region of COB mRNA identifies components of general pathways for mitochondrial mRNA processing and decay in Saccharomyces cerevisiae; Chen W et al.; The cytochrome b gene in Saccharomyces cerevisiae, COB, is encoded by the mitochondrial genome . Nuclear-encoded Cbp1 protein is required specifically for COB mRNA stabilization . Cbp1 interacts with a CCG element in a 64-nucleotide sequence in the 5'-untranslated region of COB mRNA . Mutation of any nucleotide in the CCG causes the same phenotype as cbp1 mutations, i.e., destabilization of both COB precursor and mature message . In this study, eleven nuclear suppressors of single-nucleotide mutations in CCG were isolated and characterized . One dominant suppressor is in CBP1, while the other 10 semidominant suppressors define five distinct linkage groups . One group of four mutations is in PET127, which is required for 5' end processing of several mitochondrial mRNAs . Another mutation is linked to DSS1, which is a subunit of mitochondrial 3' --> 5' exoribonuclease . A mutation linked to the SOC1 gene, previously defined by recessive mutations that suppress cbp1 ts alleles and stabilize many mitochondrial mRNAs, was also isolated . We hypothesize that the products of the two uncharacterized genes also affect mitochondrial RNA turnover. Genetics, 1999 Apr, 151(4), 1261 - 72 Suppressor analysis of the Saccharomyces cerevisiae gene REC104 reveals a genetic interaction with REC102; Salem L et al.; REC104 is a gene required for the initiation of meiotic recombination in Saccharomyces cerevisiae . To better understand the role of REC104 in meiosis, we used an in vitro mutagenesis technique to create a set of temperature-conditional mutations in REC104 and used one ts allele (rec104-8) in a screen for high-copy suppressors . An increased dosage of the early exchange gene REC102 was found to suppress the conditional recombinational reduction in rec104-8 as well as in several other conditional rec104 alleles . However, no suppression was observed for a null allele of REC104, indicating that the suppression by REC102 is not "bypass" suppression . Overexpression of the early meiotic genes REC114, RAD50, HOP1, and RED1 fails to suppress any of the rec104 conditional alleles, indicating that the suppression might be specific to REC102. Genetics, 1999 Apr, 151(4), 1245 - 59 CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae . Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates; Vedel M et al.; We have examined meiotic recombination at the CYS3 locus . Genetic analysis indicates that CYS3 is a hotspot of meiotic gene conversion, with a putative 5'-3' polarity gradient of conversion frequencies . This gradient is relieved in the presence of msh2 and pms1 mutations, indicating an involvement of mismatch repair functions in meiotic recombination . To investigate the role of mismatch repair proteins in meiotic recombination, we performed a physical analysis of meiotic DNA in wild-type and msh2 pms1 strains in the presence or absence of allelic differences at CYS3 . Neither the mutations in CYS3 nor the absence of mismatch repair functions affects the frequency and distribution of nearby recombination-initiating DNA double-strand breaks (DSBs) . Processing of DSBs is also similar in msh2 pms1 and wild-type strains . We conclude that mismatch repair functions do not control the distribution of meiotic gene conversion events at the initiating steps . In the MSH2 PMS1 background, strains heteroallelic for frameshift mutations in CYS3 exhibit a frequency of gene conversion greater than that observed for either marker alone . Physical analysis revealed no modification in the formation of DSBs, suggesting that this marker effect results from subsequent processing events that are not yet understood. Biotechnol Bioeng, 1999 May 20, 63(4), 410 - 7 Oxygen, pH value, and carbon source induced changes of the mode of oscillation in synchronous continuous culture of Saccharomyces cerevisiae; Beuse M et al.; Oscillations of measured process parameters occur in continuous cultures of Saccharomyces cerevisiae owing to a partial synchronization of budding . Intentional changes of the oxygen concentration, pH value, and carbon source cause effects on the period length similar to those known from variations of the dilution rate . The generation times of parent and daughter cells frequently differ in synchronous culture . To analyze the oscillation the term mode IJ of oscillation is used, which is defined as the ratio IJ of the generation times of parent and daughter cells . When the dissolved oxygen concentration was reduced to zero, the mode of oscillation changed within two periods from mode 12 to mode 11, caused by a decrease of the generation time of daughter cells and an increase of that of the parent cells . When the pH value was slowly reduced from 5.0 to 3.9, a change from mode 112 to mode 13 was observed . Mode 13, representing one parent and three daughter cell populations (the start of budding of each of the three being delayed by one period), denotes an elongated generation time of the daughter cells compared to mode 112, marked by one parent and two different daughter cell classes . When the carbon source galactose was replaced by glucose a mode change from mode 12 to mode 11 was observed . This alteration of the mode was found to be dependent on the status of the cell cycle at the time when the carbon source is changed . The population distribution in batch cultures with glucose or galactose as a substrate was analysed by dyeing the DNA and counting the bud scars . Galactose provoked higher growth rates for the older cells . According to the model for stationary synchronous growth parameters like DO, pH value or the type of carbon source can be varied within a certain range without effecting the period length . If the variation imposes a certain stress, the culture switches to a new mode . These kinds of parameters therefore provide selective measures to influence the period lengths and the modes of oscillation . Biotechnol Bioeng, 1999 May 5, 63(3), 285 - 9 Use of an ethanol sensor for feedback control of growth and expression of TBV25H in Saccharomyces cerevisiae; Noronha SB et al.; A process for production of a malaria transmission blocking vaccine candidate under the control of the ADH2 promoter in Saccharomyces cerevisiae was developed . Monitoring and controlling the ethanol concentration during the process is essential for successful expression of the recombinant protein . A simple sensor accomplishing this task has been developed, the principle of its operation is the following: air-flow through silicone tubing submerged in the media picks up ethanol, which is detected by an alcohol sensor that relays a signal to a controller regulating the amount of ethanol added to the culture . The sensor was used successfully in high cell density cultures of various scales . Biotechnol Bioeng, 1998 Nov 20, 60(4), 474 - 82 Growth and energy metabolism in aerobic fed-batch cultures of Saccharomyces cerevisiae: simulation and model verification; Pham HT et al.; A kinetic model of overflow metabolism in Saccharomyces cerevisiae was used for simulation of aerobic fed-batch cultivations . An inhibitory effect of ethanol on the maximum respiration of the yeast was observed in the experiments and included in the model . The model predicts respiration, biomass, and ethanol formation and the subsequent ethanol consumption, and was experimentally validated in fed-batch cultivations . Oscillating sugar feed with resulting oscillating carbon dioxide production did not influence the maximum respiration rate, which indicates that the pyruvate dehydrogenase complex is not involved as a bottleneck causing aerobic ethanol formation . Biotechnol Bioeng, 1998 Oct 20, 60(2), 180 - 9 Simple generic model for dynamic experiments with Saccharomyces cerevisiae in continuous culture: decoupling between anabolism and catabolism; Duboc P et al.; The dynamic behavior of a continuous culture of Saccharomyces cerevisiae subjected to a sudden increase in the dilution rate has been successfully modelled for anaerobic growth on glucose, and for aerobic growth on acetate, on ethanol, and on glucose . The catabolism responded by an immediate jump whereas biosynthesis did not . Thus catabolism was in excess to anabolism . The model considers the decoupling between biosynthesis and catabolism, both types of reactions being modelled by first-order kinetic expressions evolving towards maximal values . Yield parameters and maximal reaction rates were identified in steady state continuous cultures or during batch experiments . Only the time constant of biosynthesis regeneration, tauX, and the time constant of catabolic capacity regeneration, taucat, had to be identified during transient experiments . In most experiments tauX was around 3 h, and taucat varied between 2 and 2.5 h for the different metabolisms investigated . Biotechnol Bioeng, 1998 Aug 5, 59(3), 286 - 93 Leader peptide efficiency correlates with signal recognition particle dependence in Saccharomyces cerevisiae; Arnold CE et al.; Secretion of bovine pancreatic trypsin inhibitor (BPTI) in Saccharomyces cerevisiae was examined with four different leader peptides: the invertase signal peptide, the mfalpha1 signal peptide, a synthetic signal peptide, and a synthetic pre pro leader . BPTI secretion from a low-copy CEN plasmid varies from 1.8 to 10.4 microgram/mL among these constructs . Secretion titers correlate with dependence on signal recognition particle (SRP), with greatest secretion from the most SRP-dependent construct . Examination of co- vs post-translational translocation pathways and overall translocation efficiency by ubiquitin translocation assay (UTA) does not provide insight into the variation in BPTI secretion efficiency, perhaps due to alteration in translocation kinetics from the additional polypeptide fusion required by the assay . BPTI translocation efficiency (as measured by UTA) is found to drop markedly upon depletion of Srp54p, prior to any observable growth defect . Subsequent to stress response induction and the onset of slow growth (15-h doubling time), BPTI translocation efficiency recovers to the level observed prior to SRP depletion . Biotechnol Bioeng, 1998 Jan 20, 57(2), 245 - 9 Simple approach to reducing proteolysis during secretory production of human parathyroid hormone in Saccharomyces cerevisiae; Chung BH et al.; A gene coding for human parathyroid hormone (hPTH) was synthesized and cloned into a yeast expression and secretion vector containing the mating factor alpha pre-pro leader sequence and the galactose-inducible promoter, GAL10 . The intact hPTH(1-84) was found to be secreted into the culture medium . As observed in the previous reports on the secretory production of hPTH in yeast, however, the proteolytic cleavage occurred as the culture proceeded, resulting in a significant loss of the intact hPTH . Attempts were therefore made to reduce the extent of proteolysis by simply controlling the culture conditions . The proteolytic cleavage was significantly reduced by the addition of an excess amount of l-arginine (>/=0.2M) to the culture medium, which resulted in a marked improvement in the yield of intact hPTH . To examine whether l-arginine affects the activities of intracellular proteases such as KEX2 endoproteinase or extracellular proteases, the proteolysis experiments were performed by incubating the commercial intact hPTH in a yeast host culture supernatant . The results demonstrated that l-arginine at high concentrations reduced the rate of hPTH proteolysis by inhibiting extracellular proteases . FEBS Lett, 1999 Mar 5, 446(1), 9 - 14 Caspase-3 inhibits growth in Saccharomyces cerevisiae without causing cell death; Wright ME et al.; Caspase-3, a member of the caspase family of cell death proteases, cleaves cytoplasmic and nuclear substrates and promotes apoptotic cell death in mammalian cells . Although yeast homologs of apoptotic genes have not been identified, some components of apoptotic pathways retain function in yeast . Here we show that the expression of caspase-3 delays cell growth in Saccharomyces cerevesiae without causing cell death . Mutation of the caspase-3 QACRG active site abolished effects on yeast growth . Co-expression of caspase inhibitors alleviated growth inhibition in yeast as did the tripeptide caspase inhibitor ZVAD-fmk . These results suggest that substrates for caspase-3 are present in S . cerevesiae and may participate in the normal cell growth and division processes. Z Naturforsch {C}, 1999 Jan-Feb, 54(1-2), 84 - 93 Guanosine 3':5'-cyclic monophosphate-dependent particulate protein kinase activity from yeast (Saccharomyces cerevisiae); Eckstein H et al.; Continuing our studies on cGMP in growing yeast we detected a particulate cGMP dependent protein kinase (Pk-G), which was solubilized by detergents and NaCl . It achieves maximum activity at 25 degrees C and pH = 6.8, high concentrations of substrate proteins or cGMP produce saturation . Casein and histones are appropriate substrates, phosphatase-pretreated histone H-2a provokes outstandingly high activity . Pk-G differs from cAMP-dependent protein kinase (Pk-A) with respect to pH optimum, temperature tolerance above 50 degrees C, and stability . Partial purification is achieved by chromatography with DEAE-cellulose, Sepharose, and cGMP-substituted Sepharose . The latter step also markedly removes Pk-A . At least three proteins with Pk-G-activity and high cGMP-affinity are separated by polyacrylamide-gel-electrophoresis . Their apparent molecular masses, as deduced from comigrating marker proteins, differ considerably from those of other Pk-G's, but also of Pk-A's. Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3757 - 62 Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy; Pietrasanta LI et al.; Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA . The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore . We have examined the structure of the CBF3-CEN DNA complex by atomic force microscopy . Assembly of CBF3-CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III . Atomic force microscopy images showed DNA molecules with attached globular bodies . The contour length of the DNA containing the complex is approximately 9% shorter than the DNA alone, suggesting some winding of DNA within the complex . The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data . The CEN DNA is bent approximately 55 degrees at the site of complex formation . A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes . These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis. Eur J Biochem, 1999 Mar, 260(2), 461 - 9 Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae; Jorgensen MU et al.; Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome . The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands . We have studied ligand binding to Vps10p by introducing deletions in the lumenal region . This region contains two domains with homology to each other . Domain 2 binds carboxypeptidase Y (CPY), proteinase A (PrA) and hybrids of these proteases with invertase . Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p . The native proteases compete for binding to domain 2 . Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p . Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p-dependent sorting of CPY-invertase . Apparently, domain 2 contains two different binding sites; one for APY, CPY and PrA, and one for CPY-invertase and PrA-invertase . The latter interaction seems not to be sequence specific, and we suggest that an unfolded structure in these ligands is recognized by Vps10p. Gene, 1999 Mar 18, 229(1-2), 37 - 45 Mass-murdering: deletion of twenty-three ORFs from Saccharomyces cerevisiae chromosome XI reveals five genes essential for growth and three genes conferring detectable mutant phenotype; Wysocki R et al.; In the frame of the European Network for Functional Analysis (EUROFAN), two regions from chromosome XI covering 54kb have been subjected to 'mass-murder' . Ten deletions covering 23 novel open reading frames (ORFs) were constructed in haploid and diploid strains . Six deletions were lethal in haploid strains . One deletion caused slow germination of spores and slow cellular growth, and another one was associated with both cellular growth thermosensitivity and poor growth on glycerol . These two defects were assigned to two different genes . All mutant phenotypes were complemented by a single gene, enabling us to identify five genes essential for vegetative growth, three genes with detectable phenotype and 15 dispensable genes under standard physiological conditions. Mol Microbiol, 1998 Nov, 30(4), 895 - 903 D-Erythroascorbic acid is an important antioxidant molecule in Saccharomyces cerevisiae; Huh WK et al.; D-Arabinono-1,4-lactone oxidase catalysing the final step of D-erythroascorbic acid biosynthesis was purified from the mitochondrial fraction of Saccharomyces cerevisiae . Based on the amino acid sequence analysis of the enzyme, an unknown open reading frame (ORF), YML086C, was identified as the ALO1 gene encoding the enzyme . The ORF of ALO1 encoded a polypeptide consisting of 526 amino acids with a calculated molecular mass of 59493Da . The deduced amino acid sequence of the enzyme shared 32% and 21% identity with that of L-gulono-1,4-lactone oxidase from rat and L-galactono-1,4-lactone dehydrogenase from cauliflower, respectively, and contained a putative transmembrane segment and a covalent FAD binding site . Blot hybridization analyses showed that a single copy of the gene was present in the yeast genome and that mRNA of the ALO1 gene was 1.8kb in size . In the alo1 mutants, D-erythroascorbic acid and the activity of D-arabinono-1,4-lactone oxidase could not be detected . The intracellular concentration of D-erythroascorbic acid and the enzyme activity increased up to 6.9-fold and 7.3-fold, respectively, in the transformant cells carrying ALO1 in multicopy plasmid . The alo1 mutants showed increased sensitivity towards oxidative stress, but overexpression of ALO1 made the cells more resistant to oxidative stress. Mol Microbiol, 1998 Nov, 30(4), 855 - 64 Ssa1p chaperone interacts with the guanine nucleotide exchange factor of ras Cdc25p and controls the cAMP pathway in Saccharomyces cerevisiae; Geymonat M et al.; We have found that the guanine nucleotide exchange factor for ras, Cdc25p, interacts with Ssa1p in Saccharomyces cerevisiae . This interaction was observed with GST-fused Cdc25p polypeptides and confirmed by coimmunoprecipitation with the endogenous Cdc25p . Hsp82 appeared also to be co-immunoprecipitated with Cdc25p, albeit to a lower level than Hsp70 . In a strain deleted for SSA1 and SSA2, we observed a reduced cellular content of Cdc25p . Consistent with a reduced activity of the cAMP-dependent PKA pathway, the rate of accumulation of both trehalose and glycogen was stimulated in the ssa-deleted strain . Expression of SSA1 reversed these effects, whereas co-expression of SSA1 and PDE2 restored high accumulation . The expression of genes repressed by cAMP, GAC1 and TPS1, fused to beta-galactosidase, was also stimulated by deletion of SSA genes . The effect of ssa deletion on glycogen accumulation was lost in a strain deleted for CDC25 rescued by the RAS2ile152 allele . Altogether, these results lead to the conclusion that Ssa1p positively controls the cAMP pathway through Cdc25p . We propose that this connection plays a critical role in the adaptation of cells to stress conditions. RNA, 1999 Mar, 5(3), 360 - 8 The identification and characterization of a novel splicing protein, Isy1p, of Saccharomyces cerevisiae; Dix I et al.; We have identified a novel splicing factor, Isy1p, through two-hybrid screens for interacting proteins involved in nuclear pre-mRNA splicing . Isy1p was tagged and demonstrated to be part of the splicing machinery, associated with spliceosomes throughout the splicing reactions . At least a portion of the Isy1 protein population is associated with snRNAs; low levels of U5 and U6 snRNAs are coimmunoprecipitated specifically with Isy1p . When the ISY1 gene was knocked out, no defect in vegetative growth was observed . Using a sensitive in vivo splicing assay, however, we observed lower splicing efficiency in the isy1 null mutant compared to wild-type, indicating that Isy1 p is important in the optimization of splicing. J Mol Evol, 1999 Mar, 48(3), 313 - 6 Evolution of microsatellites in the yeast Saccharomyces cerevisiae: role of length and number of repeated units; Pupko T et al.; The observed and expected frequencies of occurrence of microsatellites in the yeast Saccharomyces cerevisiae were investigated . In all cases, the observed frequencies exceeded the expected ones . In contrast to predictions by Messier et al . (1996), there is no critical number of repeats beyond which the observed frequencies of microsatellites significantly exceed the frequencies expected in a random DNA sequence of the same size . Rather, the degree of deviation from expectation was found to be dependent on the length of the microsatellite . That is, a fourfold concatemeric repeat of 3 bp was found to deviate from expectation as much as three-fold concatemeric repeat of 4 bp . These findings suggest that microsatellites evolve through strand-slippage events, rather than recombination events . This, in turn, suggests that the chances of erroneous hybridizations leading to strand-slippage are length dependent. J Biol Chem, 1999 Apr 2, 274(14), 9564 - 72 Motile properties of the kinesin-related Cin8p spindle motor extracted from Saccharomyces cerevisiae cells; Gheber L et al.; We have developed microtubule binding and motility assays for Cin8p, a kinesin-related mitotic spindle motor protein from Saccharomyces cerevisiae . The methods examine Cin8p rapidly purified from crude yeast cell extracts . We created a recombinant form of CIN8 that fused the biotin carrying polypeptide from yeast pyruvate carboxylase to the carboxyl terminus of Cin8p . This form was biotinated in yeast cells and provided Cin8p activity in vivo . Avidin-coated glass surfaces were used to specifically bind biotinated Cin8p from crude extracts . Microtubules bound to the Cin8p-coated surfaces and moved at 3.4 +/- 0.5 micrometer/min in the presence of ATP . Force production by Cin8p was directed toward the plus ends of microtubules . A mutation affecting the microtubule-binding site within the motor domain (cin8-F467A) decreased Cin8p's ability to bind microtubules to the glass surface by >10-fold, but reduced gliding velocity by only 35% . The cin8-3 mutant form, affecting the alpha2 helix of the motor domain, caused a moderate defect in microtubule binding, but motility was severely affected . cin8-F467A cells, but not cin8-3 cells, were greatly impaired in bipolar spindle forming ability . We conclude that microtubule binding by Cin8p is more important than motility for proper spindle formation. J Biol Chem, 1999 Apr 2, 274(14), 9531 - 8 Phosphorylation and regulation of choline kinase from Saccharomyces cerevisiae by protein kinase A; Kim KH et al.; The CKI1-encoded choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) from Saccharomyces cerevisiae was phosphorylated in vivo on multiple serine residues . Activation of protein kinase A activity in vivo resulted in a transient increase in the phosphorylation of choline kinase . This phosphorylation was accompanied by a stimulation in choline kinase activity . In vitro, protein kinase A phosphorylated choline kinase on a serine residue with a stoichiometry (0.44 mol of phosphate/mol of choline kinase) consistent with one phosphorylation site/choline kinase subunit . The major phosphopeptide derived from the enzyme phosphorylated in vitro by protein kinase A was common to one of the major phosphopeptides derived from the enzyme phosphorylated in vivo . Protein kinase A activity was dose- and time-dependent and dependent on the concentrations of ATP (Km 2.1 microM) and choline kinase (Km 0.12 microM) . Phosphorylation of choline kinase with protein kinase A resulted in a stimulation (1.9-fold) in choline kinase activity whereas alkaline phosphatase treatment of choline kinase resulted in a 60% decrease in choline kinase activity . The mechanism of the protein kinase A-mediated stimulation in choline kinase activity involved an increase in the apparent Vmax values with respect to ATP (2.6-fold) and choline (2.7-fold) . Overall, the results reported here were consistent with the conclusion that choline kinase was regulated by protein kinase A phosphorylation. J Biol Chem, 1999 Apr 2, 274(14), 9253 - 7 Intermediate length Rieske iron-sulfur protein is present and functionally active in the cytochrome bc1 complex of Saccharomyces cerevisiae; Nett JH et al.; To investigate the relationship between post-translational processing of the Rieske iron-sulfur protein of Saccharomyces cerevisiae and its assembly into the mitochondrial cytochrome bc1 complex we used iron-sulfur proteins in which the presequences had been changed by site-directed mutagenesis of the cloned iron-sulfur protein gene, so that the recognition sites for the matrix processing peptidase or the mitochondrial intermediate peptidase (MIP) had been destroyed . When yeast strain JPJ1, in which the gene for the iron-sulfur protein is deleted, was transformed with these constructs on a single copy expression vector, mitochondrial membranes and bc1 complexes isolated from these strains accumulated intermediate length iron-sulfur proteins in vivo . The cytochrome bc1 complex activities of these membranes and bc1 complexes indicate that intermediate iron-sulfur protein (i-ISP) has full activity when compared with that of mature sized iron-sulfur protein (m-ISP) . Therefore the iron-sulfur cluster must have been inserted before processing of i-ISP to m-ISP by MIP . When iron-sulfur protein is imported into mitochondria in vitro, i-ISP interacts with components of the bc1 complex before it is processed to m-ISP . These results establish that the iron-sulfur cluster is inserted into the apoprotein before MIP cleaves off the second part of the presequence and that this second processing step takes place after i-ISP has been assembled into the bc1 complex. Protein Expr Purif, 1999 Apr, 15(3), 327 - 35 High-level expression of Rhizopus niveus lipase in the yeast Saccharomyces cerevisiae and structural properties of the expressed enzyme; Kohno M et al.; Rhizopus niveus lipase (RNL) has a unique structure consisting of two noncovalently bound polypeptides (A-chain and B-chain) . To improve this enzyme's properties by protein engineering, we have developed a new expression system for the production of recombinant lipase in the yeast Saccharomyces cerevisiae . For the present study, we developed a more efficient expression system using the strain ND-12B and the multicopy-type plasmid pJDB219 . We purified two types of recombinant lipases, each to a single peak by gel-filtration HPLC, although they were found to be heterogeneous by SDS-PAGE . Analysis of reversed-phase HPLC, N-terminal amino acid sequence, and sugar content showed that the difference between the two types of lipases was due mainly to their sugar content (high or low mannose type) . Moreover, there were two species within each type of lipase . One kind was processed to the A-chain and B-chain as in the native lipase, while the other remained unprocessed . Although these yeast-purified lipases contained several posttranslational modifications and different glycosylations, their secondary structures were the same as those of the native lipase as measured by circular dichroism spectra and determination of disulfide bonding . This suggests that protein folding of the recombinant lipase occurred correctly in yeast . Eur J Biochem, 1999 Feb, 260(1), 22 - 30 Screening of an intragenic second-site suppressor of purine-cytosine permease from Saccharomyces cerevisiae . Possible role of Ser272 in the base translocation process; Ferreira T et al.; The purine-cytosine permease from Saccharomyces cerevisiae mediates the active transport through the plasma membrane of adenine, hypoxanthine, guanine and cytosine using the proton electrochemical potential difference as an energy source . Analysis of the activity of strains mutated in a hydrophilic segment (371-377) of the polypeptidic chain has shown the involvement of this segment in the maintenance of the active three-dimensional structure of the carrier . In an attempt to identify permease domains that could interact functionally and/or physically with this segment, we looked for second-site mutations that could suppress the effects of amino acid changes in this region . This paper describes a positive screen that has allowed the isolation of one suppressor from a permease mutant displaying the N374I change (fcy2-20 allele), a substitution that induces a dramatic decrease in the affinity of the carrier for adenine, cytosine and hypoxanthine . The second-site mutation corresponds to the replacement of the Ser272 residue by Leu . Its suppressive effect is shown to be a partial restoration of the binding of cytosine and hypoxanthine to the permease . To test whether this second-site mutation is specific for the fcy2-20 allele, two double mutants were constructed (Fcy2pT213I, S272L and Fcy2pS272L, N377G) . Results obtained with these two double mutants showed that the suppressive effect of S272 L replacement was not specific for the original N374I change . To understand the general effect of this amino acid replacement for the three distinct double mutants, a strain overexpressing Fcy2pS272I, was constructed . Kinetic analysis of this strain showed that, by itself, the S272 L change induced an improvement in the base-binding step that could account for its global suppressive effect . Moreover, S272 L induced a decrease in the turnover of the permease, thus showing the involvement of S272 in the translocation process . Taking into account the topological model of the permease proposed here, this Ser residue is probably located in a transmembrane amphipathic alpha-helix (TM5) . The location and the observed decrease in the turnover of the carrier observed with the S272 L change lead us to propose that S272 could be part of a hydrophilic pore involved in the translocation of the base and/or the proton. Appl Microbiol Biotechnol, 1999 Feb, 51(2), 185 - 92 A variant of Saccharomyces cerevisiae pep4 strain with improved oligotrophic proliferation, cell survival and heterologous secretion of alpha-amylase; Chen DC et al.; A variant of Saccharomyces cerevisiae pep4 strain 20B12, with improved oligotrophic proliferation, cell survival and secretion of heterologous mouse alpha-amylase, is described . Previously we reported a procedure to enrich NI transformants that are not inhibited by cytotoxic expression of hepatitis B virus surface antigen in the secretion pathway of the protease-A-deficient (pep4) strain . To use the NI cells as a host for heterologous expression, we tried to amend the introduced pYAS/12S vector and obtain a host strain, NI-C, with stable NI phenotype and trp1 marker restored . Southern analysis of genomic DNA of NI-C suggested that the original pYAS/12S was abnormally rearranged and not completely corrected . Further assay showed that the viability and mitotic ability of the NI-C strain were increased . While using the NI-C strain as host for plasmid transformation and heterologous expression of mouse alpha-amylase, we observed that transformed colonies grew more quickly and secreted more alpha-amylase than general yeast strains . A further test showed that the NI-C strain was able to use mouse alpha-amylase as a positive selection marker to form transformed colonies on nitrogen-starved plates that contain starch as the sole carbon source . The results imply that the NI-C variant is an improved pep4 strain that can be used for heterologous expression and for the development of new selective markers in the yeast transformation system. Appl Microbiol Biotechnol, 1999 Feb, 51(2), 176 - 84 N-glycosylation is involved in the sensitivity of Saccharomyces cerevisiae to HM-1 killer toxin secreted from Hansenula mrakii IFO 0895; Kimura T et al.; Saccharomyces cerevisiae rhk mutants were previously shown to have a phenotype that is resistant to HM-1 killer toxin secreted from Hansenula mrakii IFO 0895 . The RHK1/ALG3 gene encodes a mannosyl-transferase that is involved in the synthesis of an oligosaccharide in protein N-glycosylation . Previously, this gene was cloned and shown to complement the rhk1 mutation . In this study, the RHK2 gene, which complements the rhk2 mutation, was cloned . The RHK2 gene was found to be identical to the essential gene STT3, which encodes a subunit of the oligosaccharyl-transferase complex . This complex transfers the core oligosaccharide to proteins . The rhk2 mutants showed supersensitivity to several drugs (Calcofluor White, caffeine and FK506), suggesting that these strains have cell-wall defects . Activity staining of invertase in an acrylamide gel indicated that it was underglycosylated . These results suggest that one or more mannoproteins are involved in the cytocidal process of HM-1. Genes Dev, 1999 Mar 15, 13(6), 698 - 708 The boundaries of the silenced HMR domain in Saccharomyces cerevisiae; Donze D et al.; The chromosomes of eukaryotes are organized into structurally and functionally discrete domains that provide a mechanism to compact the DNA as well as delineate independent units of gene activity . It is believed that insulator/boundary elements separate these domains . Here we report the identification and characterization of boundary elements that flank the transcriptionally repressed HMR locus in the yeast Saccharomyces cerevisiae . Deletion of these boundary elements led to the spread of silenced chromatin, whereas the ectopic insertion of these elements between a silencer and a promoter blocked the repressive effects of the silencer on that promoter at HMR and at telomeres . Sequence analysis indicated that the boundary element contained a TY1 LTR, and a tRNA gene and mutational analysis has implicated the Smc proteins, which encode structural components of chromosomes, in boundary element function. Genes Dev, 1999 Mar 15, 13(6), 686 - 97 Characterization of the imitation switch subfamily of ATP-dependent chromatin-remodeling factors in Saccharomyces cerevisiae; Tsukiyama T et al.; We have identified and characterized two Imitation Switch genes in Saccharomyces cerevisiae, ISW1 and ISW2, which are highly related to Drosophila ISWI, encoding the putative ATPase subunit of three ATP-dependent chromatin remodeling factors . Purification of ISW1p reveals a four-subunit complex with nucleosome-stimulated ATPase activity, as well as ATP-dependent nucleosome disruption and spacing activities . Purification of ISW2p reveals a two-subunit complex also with nucleosome-stimulated ATPase and ATP-dependent nucleosome spacing activities but no detectable nucleosome disruption activity . Null mutations of ISW1, ISW2, and CHD1 genes cause synthetic lethality in various stress conditions in yeast cells, revealing the first in vivo functions of the ISWI subfamily of chromatin-remodeling complexes and demonstrating their genetic interactions . A single point mutation within the ATPase domain of both ISW1p and ISW2p inactivated all ATP-dependent biochemical activities of the complexes, as well as the ability of the genes to rescue the mutant phenotypes . This demonstrates that the ATP-dependent chromatin-remodeling activities are essential for the in vivo functions of both ISW1 and ISW2 complexes. J Cell Biol, 1999 Mar 8, 144(5), 977 - 87 Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae; Maddox P et al.; We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae . Mating begins when two cells of opposite mating type come into proximity . The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection . Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism . Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts . This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min) . Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB . This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus . Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy) . Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion . During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud . There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints . Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid cell fusion in budding yeast may forgo cell cycle checkpoints present in vegetative mitosis. J Cell Biol, 1999 Mar 8, 144(5), 947 - 61 Control of mitotic spindle position by the Saccharomyces cerevisiae formin Bni1p; Lee L et al.; Alignment of the mitotic spindle with the axis of cell division is an essential process in Saccharomyces cerevisiae that is mediated by interactions between cytoplasmic microtubules and the cell cortex . We found that a cortical protein, the yeast formin Bni1p, was required for spindle orientation . Two striking abnormalities were observed in bni1Delta cells . First, the initial movement of the spindle pole body (SPB) toward the emerging bud was defective . This phenotype is similar to that previously observed in cells lacking the kinesin Kip3p and, in fact, BNI1 and KIP3 were found to be in the same genetic pathway . Second, abnormal pulling interactions between microtubules and the cortex appeared to cause preanaphase spindles in bni1Delta cells to transit back and forth between the mother and the bud . We therefore propose that Bni1p may localize or alter the function of cortical microtubule-binding sites in the bud . Additionally, we present evidence that other bipolar bud site determinants together with cortical actin are also required for spindle orientation. Biochem J, 1999 Apr 1, 339 ( Pt 1), 37 - 42 Only one of the charged amino acids located in membrane-spanning regions is important for the function of the Saccharomyces cerevisiae uracil permease; Pinson B et al.; The transport of uracil into the yeast Saccharomyces cerevisiae is mediated by uracil permease, a specific co-transporter encoded by the FUR4 gene . Uracil permease is a multispan membrane protein that is delivered to the plasma membrane via the secretory pathway . Experimental results led to the proposal of a two-dimensional model of the protein's topology . According to this model, the membrane domain of Fur4p contains three charged amino acid residues (Glu-243, Lys-272 and Glu-539) that are conserved in the members of the FUR family of yeast transporters . We have previously shown that a mis-sense mutation leading to the replacement of Lys-272 by Glu severely impairs the function of uracil permease . In the present paper, the role of the three charged residues present in the membrane-spanning regions of Fur4p was further investigated by using site-directed mutagenesis . The variant permeases were correctly targeted to the plasma membrane and their stabilities were similar to that of the wild-type permease . The effect of the mutations was studied by measuring the uptake constants for uracil on whole cells and equilibrium binding parameters on plasma membrane-enriched fractions . We found no evidence for ionic interaction between either of the glutamic residues in transmembrane segments 3 and 9 and the lysine residue in transmembrane segment 4 . Of the three charged residues, only Lys-272 was important for the transport activity of the transporter . Its replacement by Ala, Glu or even Arg strongly impaired both the binding and the translocation of uracil. Biochem J, 1999 Apr 1, 339 ( Pt 1), 21 - 32 Functional production and reconstitution of the human equilibrative nucleoside transporter (hENT1) in Saccharomyces cerevisiae . Interaction of inhibitors of nucleoside transport with recombinant hENT1 and a glycosylation-defective derivative (hENT1/N48Q); Vickers MF et al.; We have produced recombinant human equilibrative nucleoside transporter (hENT1) in the yeast Saccharomyces cerevisiae and have compared the binding of inhibitors of equilibrative nucleoside transport with the wild-type transporter and a N-glycosylation-defective mutant transporter . Equilibrium binding of 3H-labelled nitrobenzylmercaptopurine ribonucleoside inverted question mark6-{(4-nitrobenzyl)thio}-9-beta-d-ribofuranosyl purine; NBMPR inverted question mark to hENT1-producing yeast revealed a single class of high-affinity sites that were shown to be in membrane fractions by (1) equilibrium binding (means+/-S.D.) of {3H}NBMPR to intact yeast (Kd 1.2+/-0.2 nM; Bmax 5.0+/-0.5 pmol/mg of protein) and membranes (Kd 0.7+/-0.2 nM; Bmax 6.5+/-1 pmol/mg of protein), and (2) reconstitution of hENT1-mediated {3H}thymidine transport into proteoliposomes that was potently inhibited by NBMPR . Dilazep and dipyridamole inhibited NBMPR binding to hENT1 with IC50 values of 130+/-10 and 380+/-20 nM respectively . The role of N-linked glycosylation in the interaction of NBMPR with hENT1 was examined by the quantification of binding of {3H}NBMPR to yeast producing either wild-type hENT1 or a glycosylation-defective mutant (hENT1/N48Q) in which Asn-48 was converted into Gln . The Kd for binding of NBMPR to hENT1/N48Q was 10 . 5+/-1.6 nM, indicating that the replacement of an Asn residue with Gln decreased the affinity of hENT1 for NBMPR . The decreased affinity of hENT1/N48Q for NBMPR was due to an increased rate of dissociation (koff) and a decreased rate of association (kon) of specifically bound {3H}NBMPR because the values for hENT1-producing and hENT1/N48Q-producing yeast were respectively 0.14+/-0.02 and 0 . 36+/-0.05 min-1 for koff, and (1.2+/-0.1)x10(8) and (0.40+/-0 . 04)x10(8) M-1.min-1 for kon . These results indicated that the conservative conversion of an Asn residue into Gln at position 48 of hENT1 and/or the loss of N-linked glycosylation capability altered the binding characteristics of the transporter for NBMPR, dilazep and dipyridamole. J Biol Chem, 1999 Mar 26, 274(13), 9092 - 7 The Cdc6 protein is ubiquitinated in vivo for proteolysis in Saccharomyces cerevisiae; Sanchez M et al.; The Saccharomyces cerevisiae Cdc6 protein is necessary for the formation of pre-replicative complexes that are required for firing DNA replication at origins at the beginning of S phase . Cdc6p protein levels oscillate during the cell cycle . In a normal cell cycle the presence of this protein is restricted to G1, partly because the CDC6 gene is transcribed only during G1 and partly because the Cdc6p protein is rapidly degraded at late G1/early S phase . We report here that the Cdc6p protein is degraded in a Cdc4-dependent manner, suggesting that phosphorylated Cdc6 is specifically recognized by the ubiquitin-mediated proteolysis machinery . Indeed, we have found that Cdc6 is ubiquitinated in vivo and degraded by a Cdc4-dependent mechanism . Our data, together with previous observations regarding Cdc6 stability, suggest that under physiological conditions budding yeast cells degrade ubiquitinated Cdc6 every cell cycle at the beginning of S phase. J Biol Chem, 1999 Mar 26, 274(13), 8546 - 53 Characterization of the p33 subunit of eukaryotic translation initiation factor-3 from Saccharomyces cerevisiae; Hanachi P et al.; Eukaryotic translation initiation factor-3 (eIF3) is a large multisubunit complex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNAi and mRNA . The molecular mechanism by which eIF3 exerts these functions is incompletely understood . We report here the cloning and characterization of TIF35, the Saccharomyces cerevisiae gene encoding the p33 subunit of eIF3 . p33 is an essential protein of 30,501 Da that is required in vivo for initiation of protein synthesis . Glucose repression of TIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p39 subunits . Expression of histidine-tagged p33 in yeast in combination with Ni2+ affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3 . The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus . Deletion of the C-terminal 71 amino acid residues causes loss of RNA binding, but expression of the truncated form as the sole source of p33 nevertheless supports the slow growth of yeast . These results indicate that the p33 subunit of eIF3 plays an important role in the initiation phase of protein synthesis and that its RNA-binding domain is required for optimal activity. Biochem Biophys Res Commun, 1999 Feb 5, 255(1), 157 - 63 Transcription initiation mediated by initiator binding protein in Saccharomyces cerevisiae; Ohishi-Shofuda T et al.; Many instances of the initiator element in the core promoter of protein-coding genes have been reported in mammalian cells and their viruses, but only one has been reported in the yeast Saccharomyces cerevisiae at the GAL80 gene . The initiator element of GAL80 directs transcription by itself and interacts with a nuclear protein designated yeast initiator binding factor (yIF) . Here we show that yIF in a partially purified sample binds the sequence from -18 to +10 of GAL80 . By employing a selected and amplified binding procedure, we have determined the preferred sequence for yIF binding to be -2 CACTN +3 (N indicates any nucleotide) . Binding affinity of selected sequences to yIF correlated with their initiator-directed transcription in vivo, suggesting that the yIF-initiator interaction mediates transcription from the initiator in yeast . We also suggest that sequences flanking the preferred sequence affect both yIF binding and initiator activity . Antonie Van Leeuwenhoek, 1998 Nov, 74(4), 253 - 63 Physiological characterisation of a pyruvate-carboxylase-negative Saccharomyces cerevisiae mutant in batch and chemostat cultures; de Jong-Gubbels P et al.; A prototrophic pyruvate-carboxylase-negative (Pyc-) mutant was constructed by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces cerevisiae . Its maximum specific growth rate on ethanol was identical to that of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media . Consistent with earlier reports, growth on glucose could be restored by supplying aspartate as a sole nitrogen source . Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures . To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc- strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h-1 on mixtures of glucose and ethanol . In such mixed-substrate chemostat cultures of the Pyc- strain, steady-state growth could only be obtained when ethanol contributed 30% or more of the substrate carbon in the feed . Attempts to further decrease the ethanol content of the feed invariably resulted in washout . In Pyc- as well as in wild-type cultures, levels of isocitrate lyase, malate synthase and phospho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed . Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc- mutant, activities of the glyoxylate cycle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis . This suggests that factors other than glucose repression of alternative routes for oxaloacetate synthesis prevent growth of Pyc- mutants on glucose. Plant Mol Biol, 1999 Jan, 39(1), 117 - 28 The wheat LEA protein Em functions as an osmoprotective molecule in Saccharomyces cerevisiae; Swire-Clark GA et al.; The biased amino acid composition and aperiodic (random coil) configuration of Group 1 late embryogenesis-abundant (LEA) proteins imply that these proteins are capable of binding large amounts of water . While Group 1 LEAs have been predicted to contribute to osmotic stress protection in both embryonic and vegetative tissues, biochemical support has been lacking . We have used Saccharomyces cerevisiae as a model system to test the putative osmoprotective function of a wheat Group 1 LEA protein, Em . We demonstrate that expression of Em protein in yeast cells is not deleterious to growth in media of normal osmolarity and attenuates the growth inhibition normally observed in media of high osmolarity . Enhanced growth is observed in the presence of a variety of osmotically active compounds indicating that Em protein is capable of mitigating the detrimental effect of low water potential in a relatively non-specific manner . These results are the first biochemical demonstration of an osmoprotective function for a Group 1 LEA and suggest that the yeast expression system will be useful in dissecting the mechanism of protection through structure-function studies. Curr Genet, 1999 Mar, 35(2), 68 - 76 RPD3 (REC3) mutations affect mitotic recombination in Saccharomyces cerevisiae; Dora EG et al.; Prior research identified the recessive rec3-1ts mutation in Saccharomyces cerevisiae which, in homozygous diploid cells, confers a conditional phenotype resulting in reduced levels of spontaneous mitotic recombination and loss of sporulation at the restrictive temperature of 36 degrees C . We found that a 3.4-kb genomic fragment that complements the rec3-1ts/rec3-1ts mutation and which maps to chromosome XIV, is identical to RPD3, a gene encoding a histone de-acetylase . Sporulation is reduced in homozygous diploid strains containing the rec3-1ts allele at 24 degrees C, suggesting that this allele of RPD3 encodes a gene product with a reduced function . Sporulation is abolished in diploid strains homozygous for the rpd3Delta or rec3-1ts alleles, as well as in rpd3Delta/rec3-1ts heteroallelic diploids, at the non-permissive temperature . Acid-phosphatase expression has been shown to be RPD3 dependent . We found that acid-phosphatase activity is greater in diploid strains homozygous for the temperature-sensitive rec3-1ts allele than in RPD3/RPD3 strains and increased further when mutant strains are grown at 36 degrees C . We also tested the rpd3Delta/rpd3Delta strains for their effects on spontaneous mitotic recombination . By assaying a variety of intra- and inter-genic recombination events distributed over three chromosomes, we found that in the majority of cases spontaneous mitotic recombination was reduced in diploid rpd3Delta/rpd3Delta cells (relative to a RPD3/RPD3 control) . Finally, although 90% of mitotic recombinant events are initiated in the G1 phase of the growth cycle (i.e., before DNA synthesis) we show that RPD3 is not regulated in a cell-cycle-dependent manner . These data suggest that mitotic recombination, in addition to gene expression, is affected by changes in chromatin architecture mediated by RPD3. Yeast, 1999 Feb, 15(3), 219 - 53 Systematic identification, classification, and characterization of the open reading frames which encode novel helicase-related proteins in Saccharomyces cerevisiae by gene disruption and Northern analysis; Shiratori A et al.; Helicase-related proteins play important roles in various cellular processes incuding DNA replication, DNA repair, RNA processing and so on . It has been well known that the amino acid sequences of these proteins contain several conserved motifs, and that the open reading frames (ORFs) which encode helicase-related proteins make up several gene families . In this study, we have identified 134 ORFs that encode helicase-like proteins in the Saccharomyces genome, based on similarity with the ORFs of authentic helicase and helicase-related proteins . Multiple alignment of the ORF sequences resulted in the 134 ORFs being classified to 11 clusters . Seven out of 21 previously uncharacterized ORFs (YDL031w, YDL070w, YDL084w, YGL150c, YKL078w, YLR276c, and YMR128w) were identified by systematic gene disruption, to be essential for vegetative growth . Three (YDR332w, YGL064c, and YOL095c) out of the remaining 14 dispensable ORFs exhibited the slow-growth phenotype at 30 degrees C and 37 degrees C . Furthermore, the expression profiles of transcripts from 43 ORFs were examined under seven different growth conditions by Northern analysis and reverse transcription-polymerase chain reaction, indicating that all of the 43 tested ORFs were transcribed . Interestingly, we found that the level of transcript from 34 helicase-like genes was markedly increased by heat shock . This suggests that helicase-like genes may be involved in the biosynthesis of nucleic acids and proteins, and that the genes can be transcriptionally activated by heat shock to compensate for the repressed synthesis of mRNA and protein. Yeast, 1999 Feb, 15(3), 205 - 18 Spontaneous mutation, oxidative DNA damage, and the roles of base and nucleotide excision repair in the yeast Saccharomyces cerevisiae; Scott AD et al.; The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) . When compared to wild-type, ogg1 mutants show an increase in the frequency of GC to TA transversions, indicating a role for Ogg1 in the repair of 8-OxoG . Here we report an increased frequency of forward mutation to canavanine resistance in mutants defective in the nucleotide excision repair (NER) gene RAD14 . This was not increased further in strains additionally defective in OGG1 . However, when compared to strains solely defective in OGG1, ogg1radl4 mutants displayed an increase in spontaneous GC to TA transversions . Intriguingly, reversion of the lys1-1 ochre allele was not increased in rad14 mutants, suggesting that oxidative base damage may only represent a substrate for NER in certain regions of the genome . We also examined repair of oxidative DNA damage by transforming mutant strains with plasmid DNA treated with methylene blue plus visible light . Mutants defective in OGG1 showed no significant reduction in transformation efficiency compared with wild-type strains . In contrast, disruption of RAD14 reduced the efficiency of transformation, yet there was no further decrease in an ogg1rad14 mutant . This strongly supports a role for NER in the repair of oxidative base damage in yeast, and differs from similar experiments carried out in E . coli, where transformation efficiency is only reduced in mutants defective in both fpg and uvrA . Finally, the repair of Fpg-sensitive sites was examined at the MATalpha and HMLalpha mating type loci, and NER was found to play a role in their removal. FEMS Microbiol Lett, 1999 Feb 15, 171(2), 167 - 72 Involvement of Nha1 antiporter in regulation of intracellular pH in Saccharomyces cerevisiae; Sychrova H et al.; The Nha1 antiporter is involved in regulation of intracellular pH in Saccharomyces cerevisiae . We report that deletion of the NHA1 gene resulted in an increase of cytoplasmic pH in cells suspended in water or acidic buffers . Addition of KCl or NaCl to exponentially growing cells lowered the internal pH but the difference between cells with or without NHA1 was maintained . Addition of KCl to starved cells resulted in much higher alkalinization of cytoplasmic pH in a strain lacking Nha1p compared to the wild-type or Nha1p-overexpressing strains . The H+/K+(Na+) exchange mechanism of Nha1p was confirmed in reconstituted plasma membrane vesicles. FEMS Microbiol Lett, 1999 Feb 15, 171(2), 81 - 7 Acquisition of aluminum tolerance in Saccharomyces cerevisiae by expression of the BCB or NtGDI1 gene derived from plants; Ezaki B et al.; Eleven aluminum stress-induced genes derived from plants (wheat, Arabidopsis and tobacco) were introduced into Saccharomyces cerevisiae to test if expression of these genes confers Al tolerance . Al sensitivity tests showed that expression of two genes, either an Arabidopsis gene for blue copper binding protein (BCB), or a tobacco gene for the GDP dissociation inhibitor (NtGDI1), conferred Al tolerance . Determinations of total content and localization of Al ions in these transformants suggested that the BCB gene product functions in restricting Al uptake, while expression of the NtGDI1 gene promotes release of Al ions after uptake. Int J Biol Macromol, 1999 Jan, 24(1), 27 - 35 Phosphofructokinase-1 from Saccharomyces cerevisiae: analysis of molecular structure and function by electron microscopy and self-catalysed affinity labelling; Kuicke J et al.; Conventional and cryoelectron microscopy portray native octameric yeast phosphofructokinase-1 (PFK) as consisting of two identical heterotetrameric tetrahedron-like moieties being rotated relative to each other . Immunoelectron microscopy employing subunit-specific IgG identifies alpha-type subunits in the contact zone of the two tetrahedrons, while beta-chains are recognized exclusively at the tips of the octamer . The chemical reaction of phosphofructokinase with analogues of fructose 6-phosphate followed by autocatalytic phosphoryl transfer from {gamma-32P}-ATP results in a specific labelling of the alpha-subunit . AMP and fructose 2,6-bisphosphate affect labelling by stimulating the binding of substrate analogue; AMP additionally promotes phosphoryl transfer . No stimulation of labelling is observed with proteolytically modified tetrameric 12-S phosphofructokinase. Methods, 1999 Jan, 17(1), 3 - 10 Analysis of mRNA decay pathways in Saccharomyces cerevisiae; He W et al.; The analysis of mRNA turnover often requires a knowledge of the pathway by which a particular mRNA is being degraded . In this article we describe experimental procedures that can be used to determine the mechanism of degradation for yeast transcripts . These approaches include the insertion of strong secondary structures to block exonuclease cleavage and thereby trap decay intermediates . In addition, mRNA decay pathways can be analyzed by using regulatable promoters to perform transcriptional pulse-chase experiments, thereby allowing the determination of precursor-product relationships during the mRNA decay process . Finally, the mechanism of mRNA degradation can also now be determined by using trans-acting mutations specific for distinct mRNA turnover pathways . Most importantly, the combination of these three approaches can often clearly define the mechanism(s) by which a given transcript is degraded . J Biol Chem, 1999 Mar 19, 274(12), 8291 - 8 The essential role of Saccharomyces cerevisiae CDC6 nucleotide-binding site in cell growth, DNA synthesis, and Orc1 association; Wang B et al.; Saccharomyces cerevisiae Cdc6 is a protein required for the initiation of DNA replication . The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotide-binding sites . To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues . We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6 temperature-sensitive mutant cells . Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation . To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis . Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis . In order to test its effect on the G1/S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex . To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins . We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins . To further investigate the molecular mechanism of nucleotide-binding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo . Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used . Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP-binding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G1/S transition. Microbiology, 1999 Feb, 145 ( Pt 2), 309 - 16 Down-regulation of the expression of PKC1 and SRB1/PSA1/VIG9, two genes involved in cell wall integrity in Saccharomyces cerevisiae, causes flocculation; Zhang N et al.; The cell wall integrity determinants PKC1 and SRB1/PSA1/VIG9 of Saccharomyces cerevisiae were expressed under the control of the tightly regulated promoter pMET3 . Substitution of the cell-cycle-regulated SRB1/PSA1 native promoter with pMET3 led to faster cell growth, larger cell volumes, and a twofold reduction of the steady-state SRB1/PSA1 mRNA level . In addition, the new pattern of expression of SRB1/PSA1 resulted in a dominant flocculation phenotype at all phases of batch growth . By contrast, expression of PKC1 from pMET3 increased the flocculation capacity of cells only at stationary phase . Methionine-mediated repression of either PSA1/SRB1 or PKC1 resulted in enhanced cell clumping . Cells in which both these genes had been replaced with their respective pMET3-regulated cassettes were highly flocculent under both expression and repression conditions . These results suggest that greater exposure of flocculin on the cell surface, caused by either cell wall distortion (through depletion of Pkc1p) or aberrant regulation of mannosylation (through constitutive production of Srb1p), results in an increased flocculation ability. Microbiology, 1999 Feb, 145 ( Pt 2), 293 - 9 Farnesol-induced growth inhibition in Saccharomyces cerevisiae by a cell cycle mechanism; Machida K et al.; The growth of budding yeast, Saccharomyces cerevisiae, was inhibited in medium containing 25 microM farnesol (FOH) . The FOH-treated cells were still viable, and were characterized by a transition from budded to unbudded phase as well as a significant loss of intracellular diacylglycerol (DAG) . FOH-induced growth inhibition could be effectively prevented by the coaddition of a membrane-permeable DAG analogue which can activate yeast protein kinase C (PKC) . However, yeast cell growth was not initiated upon addition of the PKC activator when the cells had been pretreated with FOH for 20 min . The failure in cell growth recovery was believed to be due to a signalling-mediated cell cycle arrest in FOH-pretreated cells . Differential display analysis demonstrated that the expression of cell cycle genes encoding DNA ligase (CDC9) and histone acetyltransferase (HAT2) was strongly repressed in FOH-treated cells . Repression of the expression of these genes was effectively cancelled when cells were grown in medium supplemented with DAG . The authors propose an interference with a phosphatidylinositol-type signalling which is involved in cell cycle progression as a cause of FOH-induced growth inhibition in yeast cells. J Virol, 1999 Apr, 73(4), 2893 - 900 The ski7 antiviral protein is an EF1-alpha homolog that blocks expression of non-Poly(A) mRNA in Saccharomyces cerevisiae; Benard L et al.; We mapped and cloned SKI7, a gene that negatively controls the copy number of L-A and M double-stranded RNA viruses in Saccharomyces cerevisiae . We found that it encodes a nonessential 747-residue protein with similarities to two translation factors, Hbs1p and EF1-alpha . The ski7 mutant was hypersensitive to hygromycin B, a result also suggesting a role in translation . The SKI7 product repressed the expression of nonpolyadenylated {non-poly(A)} mRNAs, whether capped or uncapped, thus explaining why Ski7p inhibits the propagation of the yeast viruses, whose mRNAs lack poly(A) . The dependence of the Ski7p effect on 3' RNA structures motivated a study of the expression of capped non-poly(A) luciferase mRNAs containing 3' untranslated regions (3'UTRs) differing in length . In a wild-type strain, increasing the length of the 3'UTR increased luciferase expression due to both increased rates and duration of translation . Overexpression of Ski7p efficiently cured the satellite virus M2 due to a twofold-increased repression of non-poly(A) mRNA expression . Our experiments showed that Ski7p is part of the Ski2p-Ski3p-Ski8p antiviral system because a single ski7 mutation derepresses the expression of non-poly(A) mRNA as much as a quadruple ski2 ski3 ski7 ski8 mutation, and the effect of the overexpression of Ski7p is not obtained unless other SKI genes are functional . ski1/xrn1Delta ski2Delta and ski1/xrn1Delta ski7Delta mutants were viable but temperature sensitive for growth. J Basic Microbiol, 1999, 39(1), 37 - 41 Transformation of a flocculating Saccharomyces cerevisiae using lithium acetate and pYAC4; Venancio A et al.; A flocculating yeast Saccharomyces cerevisiae ura3 was transformed by the method based on treatment of intact cells with lithium acetate plus single-stranded carrier DNA using the shuttle vector pYAC4 . The transformation efficiency was above 10(3) transformants per microgram of plasmid DNA which is similar to other described yeast transformation systems . Under selective pressure, the transformed cells were stable and maintained the flocculation ability . Thus, this simple transformation system can be used for gene expression studies in flocculating yeasts, overcoming disadvantages of conventional methods such as the spheroplast one. Genes Dev, 1999 Mar 1, 13(5), 545 - 55 Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p; Sassoon I et al.; We have investigated the role of protein phosphorylation in regulation of Saccharomyces cerevisiae kinetochores . By use of phosphatase inhibitors and a type 1 protein phosphatase mutant (glc7-10), we show that the microtubule binding activity, but not the centromeric DNA-binding activity, of the kinetochore complex is regulated by a balance between a protein kinase and the type 1 protein phosphatase (PP1) encoded by the GLC7 gene . glc7-10 mutant cells exhibit low kinetochore-microtubule binding activity in vitro and a high frequency of chromosome loss in vivo . Specifically, the Ndc10p component of the centromere DNA-binding CBF3 complex is altered by the glc7-10 mutation; Ndc10p is hyperphosphorylated in glc7-10 extracts . Furthermore, addition of recombinant Ndc10p reconstitutes the microtubule-binding activity of a glc7-10 extract to wild-type levels . Finally, the glc7-10-induced mitotic arrest is abolished in spindle checkpoint mutants, suggesting that defects in kinetochore-microtubule interactions caused by hyperphosphorylation of kinetochore proteins activate the spindle checkpoint. Genes Dev, 1999 Mar 1, 13(5), 517 - 22 Defects in Saccharomyces cerevisiae protein phosphatase type I activate the spindle/kinetochore checkpoint; Bloecher A et al.; A conditional allele of type 1 protein phosphatase (glc7-129) in Saccharomyces cerevisiae causes first cycle arrest in G2/M, characterized by cells with a short spindle and high H1 kinase activity . Point-of-execution experiments indicate Glc7p function is required in G2/M just before anaphase for the completion of mitosis . Loss of the spindle/kinetochore checkpoint in glc7-129 cells abolishes the G2/M cell cycle arrest with a concomitant increase in chromosome loss and reduced viability . These results support a role for Glc7p in regulating kinetochore attachment to the spindle, an event monitored by the spindle/kinetochore checkpoint. Mol Gen Genet, 1999 Feb, 261(1), 152 - 60 Trifluoroleucine resistance and regulation of alpha-isopropyl malate synthase in Saccharomyces cerevisiae; Cavalieri D et al.; Seven spontaneous Saccharomyces cerevisiae mutants that express dominant resistance to 5,5,5-trifluoro-DL-leucine have been characterised at the molecular level . The gene responsible for the resistance was cloned from one of the mutants (FSC2.4) . Determination of its nucleotide sequence showed that it was an allele of LEU4 (LEU4-1), the gene that encodes alpha-isopropyl malate synthase I (alpha-IPM synthase I), and that the mutation involved a codon deletion localised close to the 3' end of the LEU4 ORF . Six different point mutations--four transitions and two transversions--were found in the remaining mutants . Alpha-IPM synthase activity was found to be insensitive to feedback inhibition by leucine in five of the strains . In the other two the enzyme was resistant to Zn2+-mediated inactivation by Coenzyme A, a previously postulated control mechanism in energy metabolism; as far as we know, this represents the first direct in vivo evidence for this mechanism . The seven mutations define a region, the R-region, involved in both leucine feedback inhibition and in Zn2+-mediated inactivation by CoA . Deletion experiments involving the R-region showed that it is also necessary for enzyme activity. Mol Biol Cell, 1999 Mar, 10(3), 713 - 26 A modulatory role for clathrin light chain phosphorylation in Golgi membrane protein localization during vegetative growth and during the mating response of Saccharomyces cerevisiae; Chu DS et al.; The role of clathrin light chain phosphorylation in regulating clathrin function has been examined in Saccharomyces cerevisiae . The phosphorylation state of yeast clathrin light chain (Clc1p) in vivo was monitored by {32P}phosphate labeling and immunoprecipitation . Clc1p was phosphorylated in growing cells and also hyperphosphorylated upon activation of the mating response signal transduction pathway . Mating pheromone-stimulated hyperphosphorylation of Clc1p was dependent on the mating response signal transduction pathway MAP kinase Fus3p . Both basal and stimulated phosphorylation occurred exclusively on serines . Mutagenesis of Clc1p was used to map major phosphorylation sites to serines 52 and 112, but conversion of all 14 serines in Clc1p to alanines {S(all)A} was necessary to eliminate phosph