A Conditional Allele of the Saccharomyces Cerevisiae Hop1 Gene Is Suppressed by Overexpression of Two Other Meiosis-Specific Genes: Red1 and Rec104

The HOP1 gene of Saccharomyces cerevisiae is believed to encode a protein component of the synaptonemal complex, the structure formed when homologous chromosomes synapse during meiotic prophase. Five new mutant alleles (three conditional, two nonconditional) of HOP1 were identified by screening EMS-mutagenized cells for a failure to complement the spore viability defect of a hop1 null allele. Two high copy plasmids were found that partially suppress the temperature-sensitive spore inviability phenotype of one of these alleles, hop1-628. The suppression is allele-specific; no effect of the plasmids is observed in hop1 null diploids. Mutation of either of the two suppressor genes results in recessive spore lethality, indicating that these genes play important roles during meiosis. The DNA sequence of one high copy suppressor gene matched that of RED1, a previously identified meiosis-specific gene. Our data strongly support the idea that RED1 protein is also a component of the synaptonemal complex and further suggest that the RED1 and HOP1 gene products may interact. The second suppressor maps to the right arm of chromosome VIII distal to CDC12 and is REC104, a meiosis-specific gene believed to act early in meiosis.     

The Hyper-Gene Conversion Hpr5-1 Mutation of Saccharomyces Cerevisiae Is an Allele of the Srs2/Radh Gene

The HPR5 gene has been defined by the mutation hpr5-1 that results in an increased rate of gene conversion. This mutation suppresses the UV sensitive phenotype of rad18 mutations in hpr5-1 rad18 double mutants by channeling the aborted repair events into a recombination repair pathway. The HPR5 gene has been cloned and is shown to be allelic to the SRS2/RADH gene, a putative DNA helicase. The HPR5 gene, which is nonessential, is tightly linked to the ARG3 locus chromosome X. The hpr5-1 allele contains missense mutation in the putative ATP binding domain. A comparison of the recombination properties of the hpr5-1 allele and the null allele suggests that recombination events in hpr5 defective strains can be generated by several mechanisms. We propose that the HPR5 gene functions in the RAD6 repair pathway.     

Uptake and utilization of lyso-phosphatidylethanolamine by Saccharomyces cerevisiae

Phosphatidylethanolamine (PtdEtn) is synthesized by multiple pathways located in different subcellular compartments in yeast. Strains defective in the synthesis of PtdEtn via phosphatidylserine (PtdSer) synthase/decarboxylase are auxotrophic for ethanolamine, which must be transported into the cell and converted to phospholipid by the cytidinediphosphate-ethanolamine-dependent Kennedy pathway. We now demonstrate that yeast strains with psd1Delta psd2Delta mutations, devoid of PtdSer decarboxylases, import and acylate exogenous 1-acyl-2-hydroxyl-sn-glycero-3-phosphoethanolamine (lyso-PtdEtn). Lyso-PtdEtn supports growth and replaces the mitochondrial pool of PtdEtn much more efficiently than and independently of PtdEtn derived from the Kennedy pathway. Deletion of both the PtdSer decarboxylase and Kennedy pathways yields a strain that is a stringent lyso-PtdEtn auxotroph. Evidence for the specific uptake of lyso-PtdEtn by yeast comes from analysis of strains harboring deletions of the aminophospholipid translocating P-type ATPases (APLTs). Elimination of the APLTs, Dnf1p and Dnf2p, or their noncatalytic beta-subunit, Lem3p, blocked the import of radiolabeled lyso-PtdEtn and resulted in growth inhibition of lyso-PtdEtn auxotrophs. In cell extracts, lyso-PtdEtn is rapidly converted to PtdEtn by an acyl-CoA-dependent acyltransferase. These results now provide 1) an assay for APLT function based on an auxotrophic phenotype, 2) direct demonstration of APLT action on a physiologically relevant substrate, and 3) a genetic screen aimed at finding additional components that mediate the internalization, trafficking, and acylation of exogenous lyso-phospholipids.     

Sterol Uptake in Saccharomyces cerevisiae Heme Auxotrophic Mutants Is Affected by Ergosterol and Oleate but Not by Palmitoleate or by Sterol Esterification

The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17α-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs.     

Enhanced Iron Uptake of Saccharomyces cerevisiae by Heterologous Expression of a Tadpole Ferritin Gene

We genetically engineered Saccharomyces cerevisiae to express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene (TFH) into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody against Escherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 μg per gram (dry cell weight) of the recombinant yeast but was 210 μg per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.     

Molecular Basis of Fructose Utilization by the Wine Yeast Saccharomyces cerevisiae: a Mutated HXT3 Allele Enhances Fructose Fermentation

Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Δ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.     

Cytokinin Utilization by Adenine-Requiring Mutants of the Yeast Saccharomyces cerevisiae

By monitoring the growth of several adenine auxotrophs of the yeast Saccharomyces cerevisiae on cytokinin-supplemented media, we have demonstrated that this organism can utilize some of these derivatives as a source of adenine. Growth of a mutant lacking adenylosuccinate synthetase suggests that the conversion of cytokinins to adenine does not involve a hypoxanthine intermediate and may be catalyzed by an enzyme analogous to cytokinin oxidase.     

The Saccharomyces cerevisiae SCS2 Gene Product, a Homolog of a Synaptobrevin-Associated Protein, Is an Integral Membrane Protein of the Endoplasmic Reticulum and Is Required for Inositol Metabolism

The Saccharomyces cerevisiae SCS2 gene has been cloned as a suppressor of inositol auxotrophy of CSE1 and hac1/ire15 mutants (J. Nikawa, A. Murakami, E. Esumi, and K. Hosaka, J. Biochem. 118:39–45, 1995) and has homology with a synaptobrevin/VAMP-associated protein, VAP-33, cloned from Aplysia californica (P. A. Skehel, K. C. Martin, E. R. Kandel, and D. Bartsch, Science 269:1580–1583, 1995). In this study we have characterized an SCS2 gene product (Scs2p). The product has a molecular mass of 35 kDa and is C-terminally anchored to the endoplasmic reticulum, with the bulk of the protein located in the cytosol. The disruption of the SCS2 gene causes yeast cells to exhibit inositol auxotrophy at temperatures of above 34°C. Genetic studies reveal that the overexpression of the INO1 gene rescues the inositol auxotrophy of the SCS2 disruption strain. The significant primary structural feature of Scs2p is that the protein contains the 16-amino-acid sequence conserved in yeast and mammalian cells. The sequence is required for normal Scs2p function, because a mutant Scs2p that lacks the sequence does not complement the inositol auxotrophy of the SCS2 disruption strain. Therefore, the Scs2p function might be conserved among eukaryotic cells.     

酵母豆蔻酰辅酶A：蛋白质N端转酰基酶基因的克隆与表达

利用ＰＣＲ技术,从酵母染色体中扩增得到酵母豆蔻酰－ＣｏＡ：蛋白质Ｎ端转酰基酶（ＹＳＣＮＭＴ）基因,并克隆到ｐＢｌｕｅｓｃｒｉｐｔＫＳ＋载体中。由ＤＮＡ全序测定表明,获得了ＹＳＣＮＭＴ编码基因。进一步构建了Ｔ７Ｐｒｏｍｏｔｅｒ控制下的含上述完整ＹＳＣＮＭＴ编码基因的表达质粒ｐＭＦＴ７－５－ＮＭＴ,转化大肠杆菌ＢＬ２１（ＤＥ３）,进行ＩＰＴＧ诱导表达研究。通过ＳＤＳ－ＰＡＧＥ分析,观察到一与理论分子量一致的诱导条带（约５３ｋＤ）,占全菌蛋白的３９％左右,且可溶性部分约占上清液中全部蛋白的３４％。经一步Ｐ１１磷酸纤维素阳离子交换柱层析,将其纯化到纯度达９７％以上．纯化的表达产物经Ｎ端氨基酸序列分析,所测定的Ｎ端５个氨基酸的序列,与从克隆的ＹＳＣＮＭＴ基因推出的氨基酸序列完全一致（不含Ｎ端Ｍｅｔ）。对所得的ＹＳＣＮＭＴ进行酶活力鉴定,观察到了明显的活力。     

Uptake of Selenite by Saccharomyces cerevisiae Involves the High and Low Affinity Orthophosphate Transporters

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P_i concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P_i transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P_i medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P_i transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.     

Uptake of nystatin by protoplasts of sensitive and resistant cells of Saccharomyces cerevisiae

The uptake of nystatin by protoplasts derived from sensitive and resistant cells of Saccharomyces cerevisiae has been studied as a function of nystatin concentration, temperature and pH. The presence or absence of glucose in the uptake experiments was also studied. Activation energies (Ea) for nystatin uptake revealed profound differences between protoplasts derived from sensitive and resistant cells. Those for the latter closely resembled their whole cell counterparts. The values of Ea for the uptake of nystatin under all the conditions studied indicate the importance of the cell wall in the uptake process.     

Uptake of 14C-chlorhexidine gluconate by Saccharomyces cerevisiae, Candida albicans and Candida glabrata

Uptake of radiolabelled chlorhexidine gluconate (¹⁴C-CHG) to Saccharomyces cerevisiae, Candida albicans and C. glabrata was very rapid and near maximal within 30 s. The organism, S. cerevisiae , most sensitive to the lethal action of chlorhexidine, took up significantly more biocide than the other organisms. Cells from cultures of different ages took up different amounts of ¹⁴C-CHG.     

酿酒酵母丝氨酸/苏氨酸蛋白磷酸酯酶的功能与调控

从酿酒酵母蛋白磷酸酯酶的分类和结构特征入手,阐述了该蛋白家族中的亚家族成员丝氨酸/苏氨酸蛋白磷酸酯酶的功能和表达调控.深入研究酿酒酵母丝氨酸/苏氨酸蛋白磷酸酯酶,特别是PP2C蛋白磷酸酯酶的细胞功能及其调控,将对新药研发和疾病干预治疗提供重要基础.     

Uptake and phosphorylation of 2-deoxy-D-glucose by wild-type and single-kinase strains of Saccharomyces cerevisiae

The role of phosphorylation in sugar transport in baker's yeast was studied using 2-deoxy-D-glucose. In wild-type baker's yeast, 2-deoxy-D-glucose is accumulated as a mixture of the free sugar and several derivatives. Pool labeling experiments, designed to determine the temporal order of appearance of labeled 2-deoxy-D-glucose in the intracellular pools, have confirmed previous reports that 2-deoxy-D-glucose first appears in the sugar phosphate pool. Such results are consistent with a transport associated phosphorylation mechanism. Since wild-type yeasts contain three enzymes which could participate in such a process, hexokinase isozymes PI and PII and glucokinase, pool labeling experiments were carried out with single-kinase mutant strains containing only one of these enzymes. Results similar to those for wild-type strains were obtained for all three single-kinase strains, suggesting that if transport associated phosphorylation does occur in baker's yeast, it is not a function of the specific kinase present in the cell. While the results of the pool labeling experiments are consistent with a transport associated phosphorylation mechanism for 2-deoxy-D-glucose, caution is urged in interpreting the results of experiments with whole cells where problems of compartmentation and multiple pools are difficult to assess.     

Expression of 135-kDa Insecticidal Protein Gene from Bacillus thuringiensis in the Yeast Saccharomyces cerevisiae

Bacillus thuringiensis subsp. aizawai produces 130-kDa and 135-kDa (CrylA(a)) insecticidal proteins. When Saccharomyces cerevisiae was transformed by the vector carrying a cryIA(a) gene, the gene expression could not be observed. When the 5′-upstream region from the initiation codon was removed using a synthetic oligonucleotide, the CryIA(a) protein was successfully synthesized in yeast. The yeast extract containing CryIA(a) protein had insecticidal activity against Plutella xylostella larvae.     

Uptake of GABA and putrescine by UGA4 on the vacuolar membrane in Saccharomyces cerevisiae

The product of the UGA4 gene in Saccharomyces cerevisiae, which catalyzes the transport of 4-aminobutyric acid (GABA), also catalyzed the transport of putrescine. The Km values for GABA and putrescine were 0.11 and 0.69 mM, respectively. The UGA4 protein was located on the vacuolar membrane as determined by the effects of bafilomycin A1 and by indirect immunofluorescence microscopy. Uptake of both GABA and putrescine was inhibited by spermidine and spermine, although these polyamines are not substrates of UGA4. The UGA4 mRNA was induced by exposure to GABA, but not putrescine over 12h. The growth of an ornithine decarboxylase-deficient strain was enhanced by putrescine, and both putrescine and spermidine contents increased, when the cells were expressing UGA4. The results suggest that a substantial conversion of putrescine to spermidine occurs in the cytoplasm even though UGA4 transporter exists on vacuolar membranes.     

Pex17p of Saccharomyces cerevisiae Is a Novel Peroxin and Component of the Peroxisomal Protein Translocation Machinery

The Saccharomyces cerevisiae pex17-1 mutant was isolated from a screen to identify mutants defective in peroxisome biogenesis. pex17-1 and pex17 null mutants fail to import matrix proteins into peroxisomes via both PTS1- and PTS2-dependent pathways. The PEX17 gene (formerly PAS9; Albertini, M., P. Rehling, R. Erdmann, W. Girzalsky, J.A.K.W. Kiel, M. Veenhuis, and W.-H Kunau. 1997. Cell. 89:83–92) encodes a polypeptide of 199 amino acids with one predicted membrane spanning region and two putative coiled-coil structures. However, localization studies demonstrate that Pex17p is a peripheral membrane protein located at the surface of peroxisomes. Particulate structures containing the peroxisomal integral membrane proteins Pex3p and Pex11p are evident in pex17 mutant cells, indicating the existence of peroxisomal remnants (“ghosts”). This finding suggests that pex17 null mutant cells are not impaired in peroxisomal membrane biogenesis. Two-hybrid studies showed that Pex17p directly binds to Pex14p, the recently proposed point of convergence for the two peroxisomal targeting signal (PTS)-dependent import pathways, and indirectly to Pex5p, the PTS1 receptor. The latter interaction requires Pex14p, indicating the potential of these three peroxins to form a trimeric complex. This conclusion is supported by immunoprecipitation experiments showing that Pex14p and Pex17p coprecipitate with both PTS receptors in the absence of Pex13p. From these and other studies we conclude that Pex17p, in addition to Pex13p and Pex14p, is the third identified component of the peroxisomal translocation machinery.