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.     

Mutations in Fks1p affect the cell wall content of beta-1,3- and beta-1,6-glucan in Saccharomyces cerevisiae

Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. Analysis of fks1 delta mutants showed a partial K1 killer toxin-resistant phenotype and a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1 delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. Overexpression of FKS2 suppressed the killer toxin phenotype of fks1 delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1 delta cells. In addition, eight out of 12 fks1ts fks2 delta mutants had altered beta-glucan levels at the permissive temperature: the partial killer resistant FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive allele FKS1T605I M761T increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. We analysed Fks1p and Fks2p in beta-1,6-glucan deficient mutants and found that they were mislocalized and that the mutants had reduced in vitro glucan synthase activity, possibly contributing to the observed beta-1,6-glucan defects.     

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.     

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.     

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.     

Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β‐Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9‐fold higher than that of the wild‐type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild‐type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild‐type strain. The relative value (mnn2 deletion mutant/wild‐type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β‐glucosidase activity using p‐nitrophenyl‐β‐glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high‐molecular‐weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.     

CDC15, an essential cell cycle gene in Saccharomyces cerevisiae, encodes a protein kinase domain

The cell division cycle gene CDC15 is essential for the late nuclear division in the yeast Saccharomyces cerevisiae. The amino acid sequence of the 974 amino acids/110 kDa CDC15 gene product, as deduced from the nucleotide sequence, includes an aminoterminal protein kinase domain which contains a primary sequence mosaic showing patterns specific for protein serine/threonine kinases besides those for protein tyrosine kinases. Many protein kinases non-essential for growth are known. CDC15 represents an essential protein kinase like CDC7 and CDC28. A carboxyterminal deletion of 32 amino acids renders the protein inactive.     

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。      

β‐1,6‐glucan synthesis‐associated genes are required for proper spore wall formation in Saccharomyces cerevisiae

The yeast spore wall is an excellent model to study the assembly of an extracellular macromolecule structure. In the present study, mutants defective in β ‐1,6‐glucan synthesis, including kre1? , kre6? , kre9? and big1? , were sporulated to analyse the effect of β ‐1,6‐glucan defects on the spore wall. Except for kre6? , these mutant spores were sensitive to treatment with ether, suggesting that the mutations perturb the integrity of the spore wall. Morphologically, the mutant spores were indistinguishable from wild‐type spores. They lacked significant sporulation defects partly because the chitosan layer, which covers the glucan layer, compensated for the damage. The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer. Among the double mutants, the most severe spore wall deficiency was observed in big1? spores. The majority of the big1?chs3? mutants failed to form visible spores at a higher temperature. Given that the big1? mutation caused a failure to attach a GPI‐anchored reporter, Cwp2‐GFP, to the spore wall, β ‐1,6‐glucan is involved in tethering of GPI‐anchored proteins in the spore wall as well as in the vegetative cell wall. Thus, β ‐1,6‐glucan is required for proper organization of the spore wall. Copyright © 2017 John Wiley & Sons, Ltd.     

RCS1, a gene involved in controlling cell size in Saccharomyces cerevisiae

Cloning and sequencing of RCS1, Saccharomyces cerevisiae gene whose product seems to be involved in timing the budding event of the cell cycle, is described. A haploid strain in which the 3'-terminal region of the chromosomal copy of the gene has been disrupted produces cells that are, on average, twice the size of cells of the parental strain. The critical size for budding in the mutant is similarly increased, and the disruption mutation is dominant in a diploid heterozygous for the RCS1 gene. Spores from this diploid have a reduced ability to germinate, the effect being more pronounced in the spores carrying the disrupted copy of RCS1. However, disrupted cells recover from alpha-factor treatment equally as well as wild-type cells.     

A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae

A reliable acid hydrolysis method for quantitative determination of the proportion of β-glucan, mannan and chitin in Saccharomyces cerevisiae cell wall is reported together with a simple extraction procedure to quantify within a standard error of less than 2% the proportion of the wall per gram of cell dry mass. This method is an optimized version of Saeman's procedure based on sulfuric acid hydrolysis of complex polysaccharides. It resulted in an almost complete release of glucose, mannose and glucosamine residues from cell wall polysaccharides. After complete removal of sulfate ions by precipitation with barium hydroxide, the liberated monosaccharides were separated and quantified by high performance anion-exchange chromatography with pulsed amperometric detection. The superiority of this method over the hydrolysis in either trifluoroacetic or hydrochloric acid resides in its higher efficiency regarding the release of glucose from β1,6-glucan and of glucosamine from chitin. The sulfuric acid method was successfully applied to determine the β-glucan, mannan and chitin contents in cell walls of genetically well-characterized yeast mutants defective in cell wall biosynthesis, and in Schizosaccharomyces pombe cell walls. The simplicity and reliability of this procedure make it the method of choice for the characterization of cell walls from S. cerevisiae mutants generated in the EUROFAN programme, as well as for other pharmacological and biotechnological applications. © 1998 John Wiley & Sons, Ltd.     

Absence of cell wall chitin in Saccharomyces cerevisiae leads to resistance to Kluyveromyces lactis killer toxin

Kluyveromyces lactis killer toxin causes sensitive strains of a variety of yeasts to arrest at the G1 stage of the cell cycle, and to lose viability. We describe here the isolation and characterization of a class of recessive mutations in Saccharomyces cerevisiae that leads to toxin resistance and a temperature-sensitive phenotype. These mutant cells arrest growth at 37°C with a characteristic phenotype of elongated buds. Cloning of the gene complementing these defects revealed it to be CAL1, coding for chitin synthase 3 activity. Calcofluor staining of the mutant cells indicated that chitin is absent both at 23°C and 37°C. Given that the CAL1 activity is responsible for the synthesis of most of chitin in yeast cells, and that in its absence the cells are viable but resistant to the killer toxin, our results strongly suggest that chitin might represent the receptor for this killer toxin.     

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。     

Localization of a protein a-tagged kex2 protein to the vacuole of Saccharomyces cerevisiae allows rapid purification of vacuolar membranes

We have previously reported an immunoisolation procedure which allows purification of Kex2p-containing Golgi membranes from lysed yeast cells. In order to evaluate the use of tagging procedures in organelle isolation we set out to isolate the same Golgi membrane fraction using a version of the Kex2 protease that had been affinity-tagged at its C-terminus. This protein is found to be localized in the vacuole, providing the basis of a method for the affinity-purification of vacuolar membranes.     

细胞膜对酿酒酵母乙醇耐受性影响的研究进展

酿酒酵母因其发酵工艺成熟主要被用于燃料乙醇生产及酿造行业。然而发酵过程中乙醇积累对酵母细胞的毒害是限制乙醇产量的主要因素之一,乙醇积累引起的细胞膜变化是研究酵母细胞乙醇耐受性的重要方面。该文介绍了乙醇对酵母细胞膜的作用机理,以及膜脂质,膜蛋白,膜特性与乙醇耐受性之间的关系,提出了细胞膜在酵母乙醇耐受方面所起的重要作用。     

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.     

Analysis of β-glucans and chitin in a Saccharomyces cerevisiae cell wall mutant using high-performance liquid chromatography

We have previously shown that mutations in the yeast KNR4 gene resulted in pleiotropic cell wall defects, including resistance to killer 9 toxin, elevated osmotic sensitivity to SDS and increased resistance to zymolyase, a (1→3)-β-glucanase. In this report, we further demonstrated that knr4 mutant cells were more permeable to a chromogenic substrate, X-GAL, suggesting that the mutant cell walls were leakier to certain non-permeable molecules. To determine if these defects resulted from structural changes in the cell walls, we analysed the alkali-insoluble cell wall components using HPLC assays developed for this purpose. Comparative analysis using four isogenic strains from a ‘knr4 disrupted’ tetrad demonstrated that mutant cell walls contained much less (1→3)-β-glucan and (1→6)-β-glucan; however, the level of chitin, a minor cell wall component, was found to be five times higher in the mutant strains compared to the wild-type strains. The data suggested that the knr4 mutant cell walls were dramatically weakened, which may explain the pleiotropic cell wall defects.