Glucan structure in a fragile mutant of Saccharomyces cerevisiae

The phenotype of VY1160 fragile Saccharomyces cerevisiae mutant is characterized by cell lysis upon transfer to hypotonic solutions and increased permeability of cells growing in osmotically stabilized media. Two mutations, srb1 and ts1, have been identified in VY1160 cells and previous studies have shown that the increased permeability is due to the ts1 mutation which causes a shortening of mannan side-chains. Here we report that the srb1 mutation, which is the genetic determinant of cell lysis, is responsible for quantitative and structural changes of glucans. Experiments with isogenic single mutation strains, genetic studies coupled with quantitative measurements of glucan content per cell, and methylation analysis of glucans provide evidence that srb1 mutation leads to i) formation of mechanically unstable cell wall network made of insoluble glucan fibrils which are shorter and contain beta(1-6) inter-residue linkages and ii) insufficient filling of the space between the fibrils due to a shortage of the alkali-soluble glucan. Although growing exponentially in osmotically stabilized media, the srb1 cells cannot resist an osmotic shock and, hence, burst immediately.     

酿酒酵母温度敏感性突变株的选育

为获得酵母胞内产物的高效分泌,以酿酒酵母二倍体(2n)及单倍体(n)为出发菌,经紫外诱变处理,筛选获得5株可能具有温度敏感性的突变株。为进一步鉴定,以野生菌Y_1为对照菌,测定其胞外核酸、蛋白质及果糖-1,6-二磷酸(FDP)的含量,发现突变菌株Ts_4(2n)自溶程度最高,其核酸、蛋白质渗透率达1.709,1.483,胞外FDP浓度为38μg/mL,较Y_1(2n)提高了24.1%。此外突变株Ts~*_2(n)、Ts~*_3(n)也会发生一定程度的自溶。因此,该试验共获得温度敏感性突变株3株,即Ts_4(2n)、Ts~*_2(n)和Ts~*_3(n)。     

酿酒酵母低渗敏感性突变株的选育

为促进酿酒酵母胞内产物的有效释放,以其野生型二倍体(2n)菌株Y_1为出发菌株,经紫外诱变处理,通过测定其胞外FDP浓度及核酸、蛋白质渗透率,对比各突变株在低渗条件下的自溶程度。结果表明:突变株Hs_5~*(n)低渗培养时,条件性自溶程度最高,其胞外FDP浓度达25.82μg/mL,低渗培养6h时,其核酸、蛋白质渗透率高达1.86,2.07。此外突变株Hs_2~*(n)、Hs_1(2n)也具有较好的条件性自溶能力。试验共筛选获得3株低渗敏感性突变株,与野生型菌株相比,条件性自溶的低渗敏感性突变株能有效促进胞内大分子物质的外泌。     

Membrane transport in an osmotically fragile mutant of Saccharomyces cerevisiae

Transport properties of the osmotically fragile strain VY1160 of Saccharomyces cerevisiae were compared with those of the parent S288c strain. Mediated diffusion of 6-deoxy-D-glucose was practically unaffected; membrane-potential-dependent transport of D-glucosamine was very much depressed in the fragile strain. The H+-driven transport of L-lysine and L-proline, as well as that of the hitherto uninvestigated D-glucose-6-phosphate, were also very depressed. 2-Deoxy-D-glucose transport displayed slightly different kinetic parameters. Primary H+ extrusion by the plasma membrane H-ATPase was not diminished although the ATP-splitting activity was depressed by about 50%. The overall proton-motive force (pmf) of the fragile mutant at pH 5.5 was only 20 mV while in the parent strain it was 108 mV. In parallel with this, spontaneous acidification of the external medium (a CO2-associated event) was only about 2% of that in the parent strain. The defect in this, together with the inability to stimulate transport protein synthesis by glucose, may account for the generally poorer transport performance of the fragile mutant.     

Cell fusion of Saccharomyces cerevisiae fragile mutants

Fragile mutants of Saccharomyces cerevisiae are defective in the structure of the cell wall and plasma membrane. The mutant cells lyse in hypotonic solutions but grow exponentially when osmotic stabilizer is included in the medium. These mutants display a general increase in the permeability of the plasma membrane. We show here that fragile yeast cells of the same mating type can fuse without protoplast formation. The frequency of cell x cell fusion is lower than that observed for protoplast x protoplast fusion and can be significantly increased if the cells of one partner are converted to protoplasts. Microscopic observations and genetic analysis demonstrate that the hybrids obtained are fusion products. The fusion between fragile cells is explained in terms of the existence of local defects on their surface where the cell wall is thinner (or even missing), thus allowing a direct contact of cells by means of their plasma membranes.     

不同菌龄酿酒酵母细胞壁蛋白差异性分析

以酿酒酵母为研究对象,比较了完整细胞提取法、稀碱缓冲液提取法及溶菌酶和β-葡聚糖酶复合酶法等三种酵母菌细胞壁蛋白提取方法,分析了不同菌龄酵母细胞壁差异性蛋白。结果表明:溶菌酶和β-葡聚糖酶复合酶液水解纯化好细胞壁提取蛋白的方法具有所得胞壁蛋白条带较多,且纯度较高的优点,确定了此方法为提取酵母细胞壁蛋白的最佳提取方法。同时,通过SDS-PAGE电泳分析发现,不同菌龄酵母细胞壁蛋白存在着较大的差异性,并确定了分子质量在36 ku、17 ku和12 ku为不同酵母代数细胞壁的3个主要差异性蛋白,其中36 ku、17 ku处条带蛋白随着菌龄的增加酵母细胞壁蛋白表达量逐渐减少,而12 ku处条带蛋白随着菌龄的增加酵母细胞壁蛋白表达量逐渐增加。     

青蛤凝集素CSL对酿酒酵母细胞形态的影响

通过研究青蛤凝集素CSL与酵母细胞壁肽聚糖的相互作用及CSL与酿酒酵母细胞结合后其表面积的变化,分析CSL对酿酒酵母细胞作用的影响。结果表明,固相吸附测试中,CSL可与酵母细胞壁肽聚糖结合,两者的结合具有浓度依赖关系,该结合位点受甘露糖（D-Mannose）及N-乙酰-D-半乳糖胺（N-acetyl-D-galactosamine）的抑制。CSL与酿酒酵母细胞壁相互作用,导致了它的表面积发生了改变,这表明其细胞壁发生了变化。对照组与CSL添加组两组酵母菌内的Ca²⁺含量未发生明显变化。扫描电镜观察表明,对照组酵母细胞表面较圆润光滑,CSL添加组酵母细胞表面有较多赘附凸起。研究结果为CSL促进酵母发酵能力的机理提供基础数据。      

Importance of cell wall mannoproteins for septum formation in Saccharomyces cerevisiae

The mannosyltransferase mutants mnn9 and mnn10 were isolated in a genetic screen for septation defects in Saccharomyces cerevisiae. Ultrastructural examination of mutant cell walls revealed markedly thin septal structures and occasional failure to construct trilaminar septa, which then led to the formation of bulky default septa at the bud neck. In the absence of a functional septation apparatus, mnn10 mutants are unable to complete cytokinesis and die as cell chains with incompletely separated cytoplasms, indicating that mannosylation defects impair the ability to form remedial septa. We could not detect N-linked glycosylation of the beta(1,3)glucan synthase Fks1p and mnn10 defects do not change the molecular weight or abundance of the protein. We discuss a model explaining the pleiotropic effects of impaired N-linked protein glycosylation on septation in S. cerevisiae.     

Functional,comparative and cell biological analysis of Saccharomyces cerevisiae Kre5p

Saccharomyces cerevisiae kre5delta mutants lack beta-1,6-glucan, a polymer required for proper cell wall assembly and architecture. A functional and cell biological analysis of Kre5p was conducted to further elucidate the role of this diverged protein glucosyltransferase-like protein in beta-1,6-glucan synthesis. Kre5p was found to be a primarily soluble N-glycoprotein of approximately 200 kDa, that localizes to the endoplasmic reticulum. The terminal phenotype of Kre5p-deficient cells was observed, and revealed a severe cell wall morphological defect. KRE6, encoding a glucanase-like protein, was identified as a multicopy suppressor of a temperature-sensitive kre5 allele, suggesting that these proteins may participate in a common beta-1,6-biosynthetic pathway. An analysis of truncated versions of Kre5p indicated that all major regions of the protein are required for viability. Finally, Candida albicans KRE5 was shown to partially restore growth to S. cerevisiae kre5delta cells, suggesting that these proteins are functionally related.     

Inhibitory effect of arginine on proline utilization in Saccharomyces cerevisiae

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. Although the presence of other nitrogen sources under fermentation conditions is likely to interfere with proline utilization, the inhibitory mechanisms of proline utilization remain unclear. In this study, we examined the effect of arginine on proline utilization in S. cerevisiae. We first constructed a proline auxotrophic yeast strain and identified an inhibitory factor by observing the growth of cells when proline was present as a sole nitrogen source. Intriguingly, we found that arginine, and not ammonium ion, clearly inhibited the growth of proline auxotrophic cells. In addition, arginine prevented the proline consumption of wild-type and proline auxotrophic cells, indicating that arginine is an inhibitory factor of proline utilization in yeast. Next, quantitative polymerase chain reaction (PCR) analysis showed that arginine partially repressed the expression of genes involved in proline degradation and uptake. We then observed that arginine induced the endocytosis of the proline transporters Put4 and Gap1, whereas ammonium induced the endocytosis of only Gap1. Hence, our results may involve an important mechanism for arginine-mediated inhibition of proline utilization in yeast. The breeding of yeast that utilizes proline efficiently could be promising for the improvement of wine quality.     

Effects of a novel DNA-damaging agent on the budding yeast Saccharomyces cerevisiae cell cycle

We have investigated the effects on Saccharomyces cerevisiae of a novel antitumour agent (FCE24517 or Tallimustine) which causes selective alkylations to adenines in the minor groove of DNA. Tallimustine, added to wild-type cells for short periods, reduced the growth rate and increased the percentage of budded cells and delayed the cell cycle in the late S+G₂+M phases. In the rad9Δ null mutant cells, Tallimustine treatment did not affect growth rate and the percentage of budded cells but greatly reduced cell viability compared to isogenic cells. Consistent with a role of RAD9 in inducing a transient delay in G₂ phase which preserves cell viability, the potent cytotoxic effect of the drug on rad9Δ cells was alleviated by treatment with nocodazole. Tallimustine was also found to delay the resumption from G₁ arrest of wild-type but not of rad9Δ cells. These data indicate that the effects of Tallimustine on cell cycle progression in yeast are mediated by the RAD9 gene product. From our data it appears that yeast could be a valuable model system to study the mode of action of this alkylating drug and of minor groove alkylators in general.     

耐盐酵母的选育及其遗传特性的初步研究

以酿酒酵母单倍体T-27(α，trp^-，ura^-)为出发菌株，通过紫外线和氯化锂复合诱变，选育出耐盐酵母突变株T-27-12(α，trp^-，ura^-)，其耐盐度由10％提高到16％；分析了耐盐突变株产生的原因及初步的遗传特性，同时确定了其耐盐遗传决定子，并对决定耐盐基因的显隐性进行了初步测定。     

Genetic control of chromosome stability in the yeast Saccharomyces cerevisiae

We have identified four new genetic loci: CHL2 (on chromosome XII), CHL3 (on chromosome XII); CHL4 (on chromosome IV), and CHL5 (on chromosome IX), controlling mitotic transmission of yeast chromosomes. The frequency of loss of chromosomes is 10-100-fold higher in chl5, chl2, chl3 and chl4 mutants than observed in wild-type strains. The mutants also show unstable maintenance of artificial circular minichromosomes with various chromosomal replicators (ARS) and one of the centromeric loci (CEN3, CEN4, CEN5 or CEN6). The instability of minichromosomes in the chl5, chl2, and chl4 mutants is due to the loss of minichromosomes in mitosis (1:0 segregation). In the chl3 mutant the instability of artificial minichromosomes is due to nondisjunction (2:0 segregation). The CHL3 gene therefore appears to affect the segregation of chromosomes during cell division.     

The linkage of (1–3)-β-glucan to chitin during cell wall assembly in Saccharomyces cerevisiae

Pulse-chase experiments with [¹⁴C]glucose demonstrated that in the cell wall of wild-type Saccharomyces cerevisiae alkali-soluble (1–3)-β-glucan serves as a precursor for alkali-insoluble (1–3)-β-glucan. The following observations support the notion that the insolubilization of the glucan is caused by linkage to chitin: (i) degradation of chitin by chitinase completely dissolved the glucan, and (ii) disruption of the gene for chitin synthase 3 prevented the formation of alkali-insoluble glucan. These cells, unable to form a glucan–chitin complex, were highly vulnerable to hypo-osmotic shock indicating that the linkage of the two polymers significantly contributes to the mechanical strength of the cell wall. Conversion of alkali-soluble glucan into alkali-insoluble glucan occurred both early and late during budding and also in the ts-mutant cdc24-1 in the absence of bud formation.     

Study of Two Pools of Glycogen in Saccharomyces cerevisiae and their Role in Fermentation Performance

Glycogen in Saccharomyces cerevisiae is present in two pools as cell wall bound and intracellular glycogen. The content of the cell wall bound glycogen was found to be almost three times higher than the intracellular glycogen. The content varied with the sugar concentration in the medium and an optimum value of 22.11 mg glycogen/g yeast was observed for the cell wall bound glycogen, while it was 7 mg/g yeast for the intracellular glycogen at a 12% medium sugar content. The two pools also varied with fermentation time reaching an optimal value at 36 h of fermentation. The cell wall bound glycogen was reduced by 85% during the first three hours after pitching, when sugar uptake was minimal and started to accumulate when almost 50% of the medium sugar was utilized. It was the cell wall bound glycogen that correlated with fermentation performance. Cells grown in 8% sugar content and rich in cell wall bound glycogen, when pitched into a 1% sugar medium showed an enhancement in ethanol content by 21%. The depletion of glycogen also affected fermentation performance.     

Selectable marker replacement in Saccharomyces cerevisiae

Selectable markers integrated by the ‘gamma’ deletion method (Sikorski and Hieter, 1989) can be efficiently replaced in vivo with other markers by transformation with homologous plasmids. Transformation frequencies in experiments designed to replace original selectable markers with an alternate marker were high and molecular analysis confirmed that all transformants that exhibited the expected phenotypes (loss of the original prototrophy and gain of the alternate prototrophy) resulted from homologous recombination between plasmid sequences at the target locus. This technique involves no plasmid construction and greatly facilitates the generation of yeast cells containing multiple gene disruptions.     

Genetic/genomic evidence for a key role of polarized endocytosis in filamentous differentiation of S. cerevisiae

Unicellular S. cerevisiae cells switch from the yeast form to pseudohyphal or filamentous form in response to environmental cues. We report that wild-type BY diploids (in which yeast ORFs have been systematically deleted) undergo normal HU-induced filamentous growth and discernable nitrogen starvation-induced filamentous growth, despite their perceived filamentation-deficient S288C genetic background. This finding allowed us to perform a genome-wide survey for non-essential genes that are required for filamentous growth with the homozygous deletion strains. We report that genes involved in endocytosis are required for both HU-induced and nitrogen starvation-induced filamentous growth. Surprisingly, no known genes involved in exocytosis are required. Despite the fact that polarized growth involves transport of vesicles to the site of growth, we failed to obtain genetic/genomic evidence that exocytosis plays an essential role in filamentous growth. A possible key role of polarized endocytosis (from the growth tip) is consistent with the proposed biological function of filamentous growth as a foraging behaviour. In addition, BUD8 that encodes the distal landmark in yeast-form bipolar budding is required for nitrogen starvation-induced but not HU-induced filamentous growth. Moreover, BUD5, SPA2, PEA2 and BUD6 that regulate bipolar bud site selection do not regulate the unipolar distal budding pattern in HU-induced filamentous growth.     

Cooperative functions of the mannoprotein-encoding genes in the biogenesis and maintenance of the cell wall in Saccharomyces cerevisiae.

To elucidate the roles of genes involved in the cell wall biogenesis and function in Saccharomyces cerevisiae, we isolated and characterized mutants that were lethal in a strain in which the SED1 gene encoding a cell wall mannoprotein was disrupted. Thus, double mutants of SED1 and either MNN9 or MNN10 were unable to grow and YOL155c on a multicopy plasmid could suppress their synthetic lethality. A Yol155cp-GFP fusion protein was found to localize to the cell wall, suggesting that it might also be a cell wall mannoprotein. Subsequently, we analysed the effects of the shut-off of SED1 in a sed1 and mnn9 double mutant: cells after the shut-off showed anomalous cellular morphology and died in the mitotic M phase. From these and other results, we postulate that these genes function cooperatively with each other and in a cell cycle-dependent manner in the biogenesis and maintenance of cell wall in S. cerevisiae.     

Alterations of the glucose metabolism in a triose phosphate isomerase-negative Saccharomyces cerevisiae mutant

The absence of triose phosphate isomerase activity causes an accumulation of only one of the two trioses, dihydroxyacetone phosphate, and this produces a shift in the final product of glucose catabolism from ethanol to glycerol (Compagno et al., 1996). Alterations of glucose metabolism imposed by the deletion of the TPI1 gene in Saccharomyces cerevisiae were studied in batch and continuous cultures. The Deltatpi1 null mutant was unable to grow on glucose as the sole carbon source. The addition of ethanol or acetate in media containing glucose, but also raffinose or galactose, relieved this effect in batch cultivation, suggesting that the Crabtree effect is not the primary cause for the mutant's impaired growth on glucose. The addition of an energy source like formic acid restored glucose utilization, suggesting that a NADH/energy shortage in the Deltatpi1 mutant could be a cause of the impaired growth on glucose. The amount of glycerol production in the Deltatpi1 mutant could represent a good indicator of the fraction of carbon source channelled through glycolysis. Data obtained in continuous cultures on mixed substrates indicated that different contributions of glycolysis and gluconeogenesis, as well as of the HMP pathway, to glucose utilization by the Deltatpi1 mutant may occur in relation to the fraction of ethanol present in the media.