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Studies on the Intracellular Protein Transport in Yeast

Research Project

Project/Area Number 01470121
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 発酵・醸造
Research InstitutionThe University of Tokyo

Principal Investigator

YAMASAKI Makari  Univ. of Tokyo, Fac. of Agriculture, Professor, 農学部, 教授 (60011889)

Project Period (FY) 1989 – 1990
Project Status Completed (Fiscal Year 1990)
Budget Amount *help
¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1989: ¥4,800,000 (Direct Cost: ¥4,800,000)
Keywordsyeast / intracellular protein transport / brefeldin A / alpha-facter / alkaline phosphatase / acid phosphatase / Candida albicans / ブレフェルデインA / アルカリ性ホスファタ-ゼ / <Candida albicans>___ー / α-ファクタ-
Research Abstract

I. Biochemical analysis
In 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 inhibitors
Candida 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

Report

(3 results)
  • 1990 Annual Research Report   Final Research Report Summary
  • 1989 Annual Research Report
  • Research Products

    (7 results)

All Other

All Publications (7 results)

  • [Publications] Harushi Nakajima et al.: "A cytoskelton related gene,USO1,is required for intracellular protein transport in Saccharomyces cerevisiae" J.Cell Biol.,in press. (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Manabu Arioka et al.: "Brefeldin A blocks protein transport from the ER to the Golgi apparatus in Candida albicans" J.Gen.Microbiol.,in press. (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] H. Nakajima et al.: "A cytoskelton related gene, USO1, is required for intracellular protein transport in Sachharomyces cerevisiae." J. Cell Biol.,. (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] M. Arioka et al.: "Brefeldin A blocks protein transport from the ER to the Golgi apparatus in Candida albicans." J. Gen. Microbiol.(1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Harushi Nakajima <et>___ー <al>___ー.: "A cytoskelton related gene,<USOl>___ー,is required for intracellular protein transport in <Saccharomyces>___ー <cerevisiae>___ー" J.Cell Biol.(1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] Manabu Arioka <et>___ー <al>___ー.: "Brefeldin A blocks protein transport from the ER to the Golgi apparatus in <Candida>___ー <albicans>___ー" J.Gen.Microbiol.(1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] M.Arioka: "Brefeldin A blocks protein transport from the ER to the Golgi apparatus in Candida albicans"

    • Related Report
      1989 Annual Research Report

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Published: 1989-04-01   Modified: 2016-04-21  

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