Studies on the Intracellular Protein Transport in Yeast

在本研究中，我们建立了酿酒酵母菌体外细胞内蛋白转运系统。该系统已在美国建立，但我们也成功地建立了可重复的实验条件。在液氮上反复冻融，得到p细胞（permabilized cell）。另一方面，在SP6启动子和大肠杆菌RNA聚合酶的控制下，体外表达酿酒酵母α因子基因。用^<35>S-Met和酵母裂解物在体外翻译α -因子前体mRNA，所得放射性α -因子前体经凝胶过滤纯化。在p细胞和放射性aloha因子前体的孵育过程中，我们观察到前体以蛋白酶抗性为标准向粗内质网内部易位，并通过核糖基化观察到易位前体的分子量增加。此外，我们还观察到具有较大分子量的放射性前体，表明在高尔基体中进一步糖基化。这一事实表明α因子前体在体外从内质网转运到高尔基体。在此之前，我们获得并鉴定了酿酒酵母的uso1突变体，该突变体在限制性温度下阻断了蛋白质从内质网到高尔基体的转运。我们通过建立的体外蛋白质转运系统检测了uso1突变的影响。然而，在限制温度和正常温度下，P-uso1细胞在限制温度下生长，我们没有发现任何影响。白色念珠菌是一种酵母菌，对一种抗病毒抗生素brefeldin A敏感。该药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，我们发现药物阻断了内质网和高尔基体之间的细胞内蛋白质运输。此外，我们还测试了brefeldin A在上述体外蛋白质转运系统中的作用。然而，α因子前体的转运在体外系统中没有复制。少

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