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

我们分析了突变体和克隆的基因，以阐明酵母细胞壁组分的生物合成和定位机制。(A)VIG基因的基因产物涉及分泌蛋白和细胞壁蛋白的甘露糖基化，我们证明VIG 9编码由1-磷酸甘露糖和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VIG 1/Van 1、Vig 3/Anp 1、Vig 4/Van 2和Vig 6/Mnn 9定位于高尔基体膜上。由于Vig 1、Vig 6和甘露糖基转移酶Vig 7/Ochi与GST-Vig 1通过谷胱甘肽-琼脂糖凝胶从洗涤剂溶解的膜中共纯化，因此它们应该在同一复合物中。(B)对于渗透稳定剂依赖性突变体，JS 23中的突变在PKC 1中，JS 30中的突变在VIG 9中。(C)利用GAP启动子过量表达马克思克鲁维酵母新的酸性磷酸酶PhoKM，通过Western印迹检测PhoKM，证明PhoKM与细胞壁葡聚糖共价连接。因此，PhoKM可用作具有容易检测的酶活性的壁蛋白的标记物。

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.