Studies on the Intracellular Protein Transport in Yeast

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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