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Studies on the Intracellular Protein Transport in Yeast

酵母细胞内蛋白质转运的研究

基本信息

批准号：
01470121
负责人：
YAMASAKI Makari
金额：
$ 3.9万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

I. Biochemical analysisIn this research, we established in vitro intracellular protein transport system of Saccharomyces cerevisiae. This system using the permeabilized yeast cell has already been established in U. S. A., but we also succeeded in the setting up reproducible experimental conditions. The permiabilized cell (P-cell) was obtained by the repeated freezing and thawing above liquid nitrogen. On the other hand, alpha-facter gene of S. cerevisiae was expressed in vitro under the control of SP6 promoter and E. coli RNA polymerase. The alpha-facter precursor mRNA was translated in in vitro with ^<35>S-Met and the yeast lysates and the resulted radioactive alpha-facter precursor was purified by gel filtration. Upon the incubation of the P-cell and the radioactive aloha-facter precursor, we observed the translocation of the precursor into the inside of the rough endoplasmic reticulum by the criteria of protease resistance and the increase of molecular weight by core-glycosylation o … More f the translocated precursor. Furthermore, we observed the radioactive precursors with larger molecular weight suggesting the further glycosylation at Golgi apparatus. This fact suggests the in vitro transport of the alpha-facter precursor from rER to Golgi. Previously we obtained and characterized uso1 mutatant of S. cerevisiae which was blocked the protein transport from rER to Golgi at restrictive temperature. We examined the effect of uso1 mutation by our established in vitro protein transport system. We coulldnot detect any effect, however, at either the restrictive temperature or the normal temperature with P-uso1 cell grown at the restrictive temperature.II. Analysis with inhibitorsCandida albicans, an yeast, is sensitive to brefeldin A, an antiviral antibiotic. The drug also inhibited the secretion of acid phosphatase, a surface-bound enzyme. In this study, we revealed that the drug blocked the intracellular protein transport between rER and Golgi apparatus. Furthermore, we tested the effect of brefeldin A in the in vitro protein transport system mentioned above. The transport of alpha-facter precurcor, however, was not reproduced in the in vitro system. Less

I.生化分析本研究建立了酿酒酵母细胞内蛋白质转运系统。这种利用透性化酵母细胞的系统已经在美国建立。S.一、但我们也成功地建立了可重复的实验条件。通过在液氮上反复冻融获得透性细胞（P细胞）。另一方面，S.酿酒酵母在SP 6启动子和E. coli RNA聚合酶。α-因子前体mRNA在体外用β-<35>S-Met和酵母裂解物翻译，所得放射性α-因子前体通过凝胶过滤纯化。在P细胞和放射性α因子前体孵育后，我们观察到前体移位到粗面内质网内部，通过蛋白酶抗性和通过核心糖基化增加分子量， ...更多信息 f易位的前体。此外，我们观察到具有较大分子量的放射性前体，表明高尔基体进一步糖基化。这一事实表明，在体外运输的α-因子前体从rER到高尔基体。以前我们获得并鉴定了S. cerevisiae在限制性温度下阻断了rER向高尔基体的蛋白转运。我们通过我们建立的体外蛋白转运系统检测uso 1突变的影响。然而，在限制性温度下生长的P-uso 1细胞在限制性温度和正常温度下均未检测到任何影响。抑制剂分析白色念珠菌是一种酵母菌，对布雷菲德菌素A（一种抗病毒抗生素）敏感。这种药物还能抑制酸性磷酸酶（一种表面结合酶）的分泌。在这项研究中，我们发现，药物阻断rER和高尔基体之间的细胞内蛋白质转运。此外，我们测试了布雷菲德菌素A在上述体外蛋白转运系统中的作用。然而，在体外系统中没有再现α-因子β-肾上腺皮质激素的转运。少