Genetic and Biochemical Study on the Mechanism of Biosynthesis of Yeast Cell Wall

酵母细胞壁生物合成机制的遗传学和生化研究

• 批准号: 07456047
• 负责人: YODA Koji
• 金额: $ 4.86万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07456047/
• 关键词: yeast; Saccharomyces cerevisiae; cell wall; intracellular transport; localization of proteins; glucan; mannoprotein; glycosyl chains; Saccharomyces cerevisial

We have analyzed mutants and cloned genes to elucidate the mechanism of biosynthesis and localization of the components of yeast cell wall. (A) As for the gene products of VIG genes which concern mannosylation of secretory and cell wall proteins, we demonstrated that VIG9 encodes the enzyme which synthesizes GDP-mannose from mannose-1-phosphate and GTP.By using polyclonal antibodies or epitope tagging, we showed Vig1/Van1, Vig3/Anp1, Vig4/Van2, and Vig6/Mnn9 localize on the Golgi membrane. As Vig1, Vig6, and the mannosyltransferase Vig7/Ochi were co-purified with GST-Vig1 by glutathione-Sepharose from the detergent-solubilized membrane, they should be in a same complex. (B) As for osmotic stabilizer-dependent mutants, the mutation in JS23 was in PKC1 and the mutation in JS30 was in VIG9. (C) By overproduction of the novel acid phosphatase PhoKM of Kluyveromyces marxianus with the aid of GAP promoter, we could detect PhoKM by Western blots and proved that it is covalently linked to the cell wall glucan. Thus, PhoKM is useful as a marker of wall protein which has easily detectable enzyme activity.

我们通过分析突变体和克隆基因来阐明酵母细胞壁成分的生物合成和定位机制。(A)对于与分泌蛋白和细胞壁蛋白甘露糖基化有关的VIG基因的基因产物，我们证明了VIG9编码由甘露糖-1-磷酸和GTP合成gdp -甘露糖的酶。通过多克隆抗体或表位标记，我们发现Vig1/Van1、Vig3/Anp1、Vig4/Van2和Vig6/Mnn9定位在高尔基膜上。由于Vig1、Vig6和甘露糖基转移酶Vig7/Ochi是用谷胱甘肽- sepharose从洗涤剂溶解膜中与GST-Vig1共纯化的，因此它们应该在同一个络合物中。(B)对于依赖渗透稳定剂的突变体，JS23突变在PKC1， JS30突变在VIG9。(C)利用GAP启动子过量生产马氏克卢维菌的新型酸性磷酸酶PhoKM， Western blots检测PhoKM，证实其与细胞壁葡聚糖共价连接。因此，PhoKM作为壁蛋白的标记物是有用的，它具有易于检测的酶活性。

项目成果

Hisashi Yamakawa: "Usol protein is a dimer with two globular heads and a long coiled-coil tail" J.Structural Biology. (印刷中). (1996)

Hisashi Yamakawa：“Usol 蛋白是一种具有两个球状头部和一个长卷曲尾部的二聚体”J.Structural Biology（出版中）。

Masahiro Kito: "Calcium and SLY genes suppress the tesnperature-sensitive secretion defect of Sacclaromyces cesevisiae usol matant" Biochem. Biophys. Res.Commum.(印刷中). (1996)

Masahiro Kito：“钙和 SLY 基因抑制酿酒酵母的温度敏感性分泌缺陷”Biochem。Res.Commum（出版中）。