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Reconstitution of the E.coli protein translocation machinery

Research Project

Project/Area Number 05454620
Research Category

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

Allocation TypeSingle-year Grants
Research Field Structural biochemistry
Research InstitutionUniversity of Tokyo, Institute of Molecular and Cellular Biosciences

Principal Investigator

TOKUDA Hajime  University of Tokyo, Institute of Molecular and Cellular Biosciences, Associate Professor., 分子細胞生物学研究所, 助教授 (40125943)

Project Period (FY) 1993 – 1994
Project Status Completed (Fiscal Year 1994)
Budget Amount *help
¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 1994: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1993: ¥3,300,000 (Direct Cost: ¥3,300,000)
KeywordsProtein translocation machinery / Reconstitution system / Proteoliposome / SecG / Membrane protein / 蛋白質膜透過 / 再構成系 / 遺伝子破壊 / p12
Research Abstract

Several Sec factors are involved in the protein translocation across the E.coli cytoplasmic membrane. To elucidate the mechanism of protein translocation, we have established conditions for the reconstitution of the translocation machinery with purified Sec factors. Using this reconstitution system, we have revealed followings.
1.Discovery of a novel factor, SecG.
SecG was found to stimulate the reconstituted activity of proteoliposomes. Furthermore, SecG was found to be a membrane protein having a molecular mass of 12kDa.
2.Genetic analysis of SecG
A gene encoding SecG was cloned and sequenced. Furthermore, E.coli DELTASecG strain was constructed and examined for protein secretion in vivo. The DELTASecG strain was defective in secretion at 20゚C but not at 37゚C,indicating that the SecG function is important for protein translocation at low temparature.
3.Roles of SecG.
Roles of SecG was examined in detail using the reconstitution system. It was found that both SecY and SecE are required for SecA receptor activity. On the the other hand, SesG was found to stimulate the translocation-coupled ATPase of SecA,indicating that SecG function after SecA targets to the membrane.

Report

(3 results)
  • 1994 Annual Research Report   Final Research Report Summary
  • 1993 Annual Research Report
  • Research Products

    (23 results)

All Other

All Publications (23 results)

  • [Publications] Kawasaki,S.: "Membrane vesicles containing overproduced SecY and SecE exhibit high translocation ATPase activity and counter movement of protons in a SecA-and presecretory protein-dependent manner." J.Biol.Chem.268. 8193-8198 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nishiyama,K.: "A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli." EMBO J.12. 3409-3415 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokuda,H.: "Histidine residues are involred in translocation-coupled ATP hydrolysis of SecA protein." Biochem.Biophys.Res.Commun.195. 1415-1421 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokuda,H.: "Biochemical characterization of the presecretory protein translocation machinery of Escherichia coli." FEBS Lett.346. 65-68 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nishiyama,K.: "Disruption of the gene encoding p12(SecG)reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli.at low temperature." EMBO J.13. 3272-3277 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Hanada,M.: "Reconstitution of an efficient protein translocation machinery comprising SecA and the three membrane protein,SecY,SecE,and SecG(p12)." J.Biol.Chem.269. 23625-23631 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Kawasaki, S.: "Membrane vesicles containing overproduced SecY and SecE exhibit high translocation ATPase activity and counter movement of protons in a SecA-and presecretory protein-dependent manner." J.Biol.Chem.268. 8193-8198 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nishiyama, K.: "A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli." EMBO J.12. 3409-3415 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokuda, H.: "Histidine residues are involved in translocation-coupled ATP hydrolysis of SecA protein." Biochem.Biophys.Res.Commun.195. 1415-1421 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokuda, H.: "Biochemical characterization of the presecretory protein translocation machinery of Escherichia cali." FEBS Lett.346. 65-68 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nishiyama, K.: "Disruption of the gene encoding p12 (SecG) reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli at low temperature." EMBO J.13. 3272-3277 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Hanada, M.: "Reconstitution of an efficient protein translocation machinery comprising SecA and the three membrane proteins, SecY,SecE,and SecG (p12)." J.Biol.Chem.269. 23625-23631 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokuda,Hajime: "Biochemical characterization of the presecretory protein translocation machinery of Escherichia coli." FEBS Lett.346. 65-68 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Nishiyama,Ken-ichi: "Disruption of the gene encoding p12(SecG)reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli at low temperature." EMBO J.13. 3272-3277 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Douville,K.: "Band 1 subunit of Escherichia coli preprotein translocase and integral membrane export factor p12 are the same protein.(Communication)" J.Biol.Chem.269. 18705-18707 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Hanada,Mitsuharu: "Reconstitution of an efficient protein translocation machinery comprising SecA and the three membrane proteins,SecY,SecE,and SecG(p12)." J.Biol.Chem.269. 23625-23631 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 徳田 元: "大腸菌の蛋白質膜透過系とリポ蛋白質外膜局在化のメカニズム" 日本農芸化学会誌. 69. 39-42 (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] Kawasaki,S.: "Membrane vesicles containing overproduced SecY and SecE exhibit high translocation ATPase activity and counter movement of protons in a SecA- and presecretory protein-dependent manner." J.Biol.Chem.268. 8193-8198 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Nishiyama,K.: "A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli." EMBO J.12. 3409-3415 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Tokuda,H.: "Histidine residues are involved in translocation-coupled ATP hydrolysis by the SecA Protein." Biochem.Biophys.Res.Commun.192. 1415-1421 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 徳田 元: "大腸菌蛋白質分泌の分子機構" 蛋白質・核酸,酵素. 38. 947-956 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 徳田 元: "“Annual Review細胞生物学1993(矢原,御子柴,月田編)"中外医学社" 大腸菌における分泌型蛋白質の細胞質膜透過系, 37-48 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 徳田 元: "“最新生体膜システム(水島,宇井編)"講談社" タンパク質の細胞内輸送, 61-69 (1993)

    • Related Report
      1993 Annual Research Report

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Published: 1993-04-01   Modified: 2016-04-21  

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