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タンパク質の膜透過・分泌の機構

Research Project

Project/Area Number 61060001
Research Category

Grant-in-Aid for Specially Promoted Research

Allocation TypeSingle-year Grants
Research InstitutionThe University of Tokyo (1988-1989)
Nagoya University (1986-1987)

Principal Investigator

水島 昭二  東京大学, 応用微生物研究所, 教授 (50013313)

Co-Investigator(Kenkyū-buntansha) 市原 茂幸  名古屋大学, 農学部, 助手 (30092993)
山田 寿美  名古屋大学, 農学部, 教務職員 (30089859)
内田 欣哉  東京大学, 応用微生物研究所, 助手 (60013330)
松山 伸一  東京大学, 応用微生物研究所, 助手 (50183108)
Project Period (FY) 1986 – 1989
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥109,000,000 (Direct Cost: ¥109,000,000)
Fiscal Year 1989: ¥18,000,000 (Direct Cost: ¥18,000,000)
Fiscal Year 1988: ¥35,000,000 (Direct Cost: ¥35,000,000)
Fiscal Year 1987: ¥20,000,000 (Direct Cost: ¥20,000,000)
Fiscal Year 1986: ¥36,000,000 (Direct Cost: ¥36,000,000)
Keywordsタンパク質の分泌 / タンパク質の膜透過 / SecA / ATP / シグナルペプチド / 大腸菌 / ビブリオ菌 / 膜タンパク質 / タンパク質膜透過のin vitro系 / タンパク質膜透過のエネルギー要求性 / バイオエナジエティックス / SecAタンパク質 / シグナルペプチターゼ / シグナルペプチドペプチダーゼ / タンパク質の膜透過のエネルギー要求性 / バイオエナジェティックス / プロテアーゼ【IV】
Research Abstract

1)膜透過に関与する因子の機能解析:精製SecAおよびSecA断片を用いて、ATPとの相互作用、分泌型タンパク質との相互作用におけるSecAの各ドメインの役割を明らかにした。またSecAが二量体を形成していることを明らかにした。
2)膜透過過程の反応速度論的解析:1分子の分泌型タンパク質が膜を通過する過程を解析する目的で、繰返し構造をもつ巨大人工タンパク質をコードする人工遺伝子を作成した。しかし、in vitroで効率よく膜透過させることには成功していない。
3)膜透過反応へのエネルギーの関与:ATPがSecAのN末近くと相互作用することを明らかにした。またΔμ^^ーH^+はATPの分泌装置(恐らくはその中のSecA分子)に対する親和性を高める働きをしていることを明らかにした。
4)分泌型タンパク質の構造と膜透過性:シグナルペプチドのN末端正荷電の重要性を明らかにした。また中央部疎水領域の特性、同領域とN末端領域が機能的に関連しあっていることを、人工シグナルペプチドを用いて明らかにした。
5)タンパク質と膜との相互作用:シグナルペプチドの膜内配向性についての研究成果をもとに、膜タンパク質の膜内配向性を決定する因子として、疎水領域とそれに隣接する正荷電の組合せが重要であることを、in vitro実験によって示した。
6)再構成系を用いての解析:膜透過再構成系の作成に成功した。リン脂質、膜構造、膜タンパク質、SecY、SecA、ATPが必要なことを再構成系で示すことに成功した。
7)ビブリオ菌でのin vitro膜透過系をつくることに成功し、この細菌特有のNa^+ポンプが膜透過に関与することを示した。

Report

(4 results)
  • 1989 Annual Research Report
  • 1988 Annual Research Report
  • 1987 Annual Research Report
  • 1986 Annual Research Report
  • Research Products

    (32 results)

All Other

All Publications (32 results)

  • [Publications] Kawasaki,H.;Matsuyama,S.;Akita,M.;Mizushima,S: "SecA protein is directly involved in protein secretion in Escherichia coli." FEBS Letters. 242. 431-434 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Yamada,H.;Tokuda,H.;Mizushima,S.: "Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli." J.Biol.Chem.264. 1723-1728 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Matsuyama,S.;Mizushima,S.: "Energy-dependent in vitro translocation of a model protein into Escherichia coli inverted membrane vesicles can take place efficiently in the complete absence of the cytosol fraction." J.Biol.Chem.264. 3583-3587 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Ichige,A.;Matsutani,S.;Oishi,K.;Mizushima,S.: "Establishment of gene transfer systems for and construction of the genetic map of a marine Vibrio strain." J.Bacteriol.171. 1825-1834 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Shinkai,A.;Yamada,H.;Mizuno,T.;Mizushima,S.: "Insertion of signal peptide-derived hydrophobic segment into the mature domain of OmpC,an outer membrane protein,does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E.coli." J.Biochem.106. 323-330 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Yamada,H.;Matsuyama,S.;Tokuda,H.;Mizushima,S.: "A high concentration of SecA allows proton motive force-independent translocation of a model secretory protein into E.coli membrane vesicles." J.Biol.Chem.264. 18577-18581 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Tani,K.;Shiozuka,K.;Tokuda,H.;Mizushima,S.: "In vitro analysis of the process of translocation of OmpA across the Escherichia coli cytoplasmic membrane." J.Biol.Chem.264. 18582-18588 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Sasaki,S.;Matsuyama,S.;Mizushima,S.: "In vitro kinetic analysis of the role of the positive charge at the amino-terminal region of signal peptides in translocation of secretory protein across the cytoplasmic membrane in Escherichia coli." J.Biol.Chem.

    • Related Report
      1989 Annual Research Report
  • [Publications] Mizushima,S.;Tokuda,H.: "In vitro translocation of bacterial secretory proteins and energy requirements" J.Bioenerg.Biomemb.

    • Related Report
      1989 Annual Research Report
  • [Publications] Akita,M.;Sasaki,S.;Matsuyama,S.;Mizushima,S.: "SecA interacts with secretory proteins by recognizing the positive charge at the amino-terminus of the signal peptide in Escherichia coli." J.Biol.Chem.

    • Related Report
      1989 Annual Research Report
  • [Publications] Yamane.K.;Matsuyama.S.;Mizushima.S.: J.Biol.Chem.263. 5368-5372 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Ichige.A.;Oishi.K.;Mizushima.S.: J.Bacteriol.170. 3537-3542 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Yamane.K.;Mizushima.S.: J.Biol.Chem.263. 19690-19696 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Kawasaki.H.;Matsuyama.S.;Sasaki.S.;Akita.M.;Mizushima.S.: FEBS Letters. 242. 431-434 (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] Yamada.H.;Tokuda.H.;Mizushima.S.: J.Biol.Chem.264. 1723-1728 (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] Matsuyama.S.;Mizushima.S.: J.Biol.Chem.264. (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] 松山伸一,水島昭二: "BIOmedica" 北隆館, 47-51 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Yamane, K., Ichihara, S., and Mizushima, S.: J. Biol. Chem.262. 2358-2362 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] Suzuki, T., Itoh, A., Ichihara, S., and Mizushima, S.: J. Bacteriol.169. 2523-2528 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] Matsuyama, S., and Mizushima, S.: J. Mol. Biol.195. 847-853 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] Aiba, H., Matsuyama, S., Mizuno, T., and Mizushima, S.,: J. Bacteriol.169. 3007-3012 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] Choi, D.-S., Yamada, H., Mizuno, T., and Mizushima, S.: J. Biochem.102. 975-983 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] Mizuno, T., Kato, M., Jo, Y.-L., and Mizushima, S.: J. Biol. Chem.263. (1988)

    • Related Report
      1987 Annual Research Report
  • [Publications] Shoji Mizushima: "細胞工学 別冊2" 東京化学同人, 12 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] Ichihara shigeyuki and Shoji Mizushima: "現代化学 増刊10" 東京化学同人, 17 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] Masao Itoh: Agr.Biol.Chem.50. 1295-1302 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] Dong-Seong Choi: J.Biol.Chem.261. 8953-8957 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] Shigeyuki Ichihara: J.Biol.Chem.261. 9405-9411 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] Kazuhiko Yamane: J.Biol.Chem.

    • Related Report
      1986 Annual Research Report
  • [Publications] Hisami Yamada: J.Biol.Chem.(1987)

    • Related Report
      1986 Annual Research Report
  • [Publications] Shoji Mizushima: "Nippon Nougeikagaku Kaishi" Nippon Nogeikagaku Kai, 3 (1987)

    • Related Report
      1986 Annual Research Report
  • [Publications] Shoji Mizushima: "Bacterial Outer Membranes as Model Systems" John Wiley & Sons,Inc., 23 (1986)

    • Related Report
      1986 Annual Research Report

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Published: 1987-03-31   Modified: 2016-04-21  

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