Studies on the Intracellular Protein Transport in Yeast

I.生化分析本研究建立了酿酒酵母细胞内蛋白质的体外转运系统。该系统在美国已经建立，但我们也成功地建立了可重复性的实验条件。在液氮上反复冷冻解冻，获得了稳定的细胞(P细胞)。另一方面，在SP6启动子和大肠杆菌RNA聚合酶的控制下，酿酒酵母α因子基因在体外得到了表达。用~(35)&gt；S-蛋氨酸和酵母裂解产物体外翻译α-因子前体基因，凝胶过滤纯化放射性α-因子前体。在P细胞和放射性因子前体孵育后，我们观察到前体通过蛋白酶抗性标准进入粗面内质网内部，并通过…的核心糖基化增加分子量更多的是移位的前驱物质。此外，我们观察到分子量较大的放射性前体，表明高尔基体进一步糖基化。这一事实表明，阿尔法因子前体在体外从RER转运到高尔基体。在此之前，我们获得并鉴定了酿酒酵母uso1突变体，该突变体在有限的温度下阻止了蛋白质从reR到高尔基体的运输。我们通过我们建立的体外蛋白转运系统检测了uso1突变的影响。然而，无论是在限制性温度下还是在常温下生长的P-USO1细胞，我们都没有检测到任何影响。II.用抑制剂分析白色念珠菌是一种酵母，对抗病毒抗生素布雷菲尔丁A敏感。该药物还抑制酸性磷酸酶的分泌，酸性磷酸酶是一种表面结合的酶。在这项研究中，我们发现该药物阻断了RER和高尔基体之间的细胞内蛋白运输。此外，我们还测试了灯盏花素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不能复制。较少

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