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

1996 Fiscal Year Final Research Report Summary

• Project/Area Number: 07456047
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 応用微生物学・応用生物化学
• Research Institution: The University of Tokyo
• Principal Investigator: YODA Koji The University of Tokyo, Graduate School of Agriculrural and Biological Sciences, Professor, 大学院・農学生命科学研究科, 教授 (20143406)
• Project Period (FY): 1995 – 1996
• Keywords: yeast / Saccharomyces cerevisiae / cell wall / intracellular transport / localization of proteins / glucan / mannoprotein / glycosyl chains

Research Abstract

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.