Molecular mechanism of protein translocation in Escherichia coli

• Project/Area Number: 07408015
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Tokyo University of Pharmacy and Life Science
• Principal Investigator: SHISHIDO Katsuko (1996-1997) Tokyo University of Pharmacy and Life Science, School of Life Sci. Assistant prof., 生命科学部, 講師 (40266896); 水島 昭二 (1995) 東京薬科大学, 生命科学部, 教授 (50013313)
• Co-Investigator(Kenkyū-buntansha): MORI Hiroyuki Tokyo University of Pharmacy and Life Science, School of Life Sci. Research asso, 生命科学部, 助手 (10243271) ; HATSUZAWA Kiyotaka Tokyo University of Pharmacy and Life Science, School of Life Sci. Research asso, 生命科学部, 助手 (20256655) ; TAGAYA Mitsuo Tokyo University of Pharmacy and Life Science, School of Life Sci. Professor, 生命科学部, 教授 (30179569) ; 谷 佳津子 東京薬科大学, 生命科学部, 講師 (40266896)
• Project Period (FY): 1995 – 1997
• Project Status: Completed (Fiscal Year 1997)
• Budget Amount: ¥29,600,000 (Direct Cost: ¥29,600,000); Fiscal Year 1997: ¥4,800,000 (Direct Cost: ¥4,800,000); Fiscal Year 1996: ¥8,800,000 (Direct Cost: ¥8,800,000); Fiscal Year 1995: ¥16,000,000 (Direct Cost: ¥16,000,000)
• Keywords: translocation / secretory protein / Escherichia coli / signal sequence / 膜蛋白質 / 小胞体 / Stepwise Translocation / SecA / 前駆体タンパク質 / 結晶解析 / シグナル認識粒子

Research Abstract

The protein translocation machinery in Escherichia coli consists of SecA,translocation ATPase, and the membrane-embedded complex comprising SecY,SecE and SecG.Secretory proteins are passed through a hydrophilic tunnel formed by the membrane-embedded complex, and then released into the periplasm via a mechanism involving SecD and SecF.Secretory proteins contain a signal sequence at the amino-terminus. Signal sequences consist of positively charged amino-terminal region and a hydrophobic core region. We analyzed the mechanism of protein translocation in E.coli at the molecular level and obtained the following results.
1. SecA interacts with the hydrophobic core region of the signal peptide in the presence of acidic phospholipids and can mediate the translocation of secretory proteins that do not contain positively charged amino acid residues in the signal sequence.
2. Short hydrophobic segments consisting of 4-5 amino acid residues in the mature region of a secretory protein, proOmpA,are a determinant for the rate of translocation across the membrane. A duplicated short hydrophobic segment can act as a stop-transfer sequence.
3. The amino-terminal region and Asp-133 of SecA are important for the interaction with SecG and ATP hydrolysis, respectively.
4. The hydrophobic core of the signal sequence is also important for translocation across the endoplasmic reticulum membrane in animal cells.

Research Products

• [Publications] Hatsuzawa et al.: "The hydrophobic region of signal peptides is a determinant for SRP recognition and protein translocation across the ER membrane." J.Biochem. 121. 270-277 (1997)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Sato et al.: "Short hydrophobic segments in the mature domain of proOmpA determine its stepwise movement during translocation across the cytoplasmic membrane of Escherichia coli." J.Biol.Chem.271. 880-5886 (1997)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Mori et al.: "The hydrophobic region of signal peptides is involved in the intereaction with membrane-bound SecA." Biochem.Biophys.Acta. 1326. 23-36 (1997)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Sato et al.: "In vitro analysis of the stop-transfer process during translocation across the cytoplamic membrane of Escherichis coli." J.Biol.Chem.272. 20082-20087 (1997)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Uchida et al.: "Stepwise movement of preproteins in the process of translocation across the cytoplasmic membrane of Escherichis coli." J.Biol.Chem.270. 30862-30868 (1995)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nishiyama et al.: "Preferential interaction of Sec-G with Sec-E stabilizes an unstable Sec-E derivative in the Escherichis coli cytoplasmic membrane." Biochem.Biophys.Res.Commun.217. 217-223 (1995)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Hatsuzawa et al.: "The hydrophobic region of signal peptides is a determinant for SRP recognition and protein translocation across the ER membrane." J.Biochem.121. 270-277 (1997)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Sato et al.: "Short hydrophobic segments in the mature domain of proOmpA determine its stepwise movement during translocation across the cytoplasmic membrane of Escherichia coli." J.Biol.Chem.272. 5880-5886 (1997)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Mori et al.: "The hydrophobic region of signal peptides is involved in the interaction with membrane-bound SecA." Biochim.Biophys.Acta. 1326. 23-36 (1997)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Sato et al.: "In vitro analysis of the stop-transfer process during trans location across the cytoplasmic membrane of Escherichia coli." J.Biol.Chem.272. 20082-20087 (1997)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Uchida et al.: "Stepwise movement of preproteins in the process of translocation across the cytoplasmic membrane of Escherichia coli." J.Biol.Chem.270. 30862-30868 (1995)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nishiyama et al.: "Preferential interaction of Sec-G with Sec-E stabilizes an unstable Sec-E derivative in the Escherichia coli cytoplasmic membrane." Biochem.Biophys.Res.Commun.217. 217-223 (1995)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Hatsuzawa, et al.: "The hydrophobic region of signal peptides is a determinant for SRP recognition and protein translocation across the ER membrane." J.Biochem. 121. 270-277 (1997)
• [Publications] Sato, et al.: "Short hydrophobic segments in the mature domain of proOmpA determine its stepwise movement during translocation across the cytoplasmic membrane of Escherichia coli." J.Biol.Chem.271. 880-5886 (1997)
• [Publications] Mori, et al.: "The hydrophobic region of signal peptides is involved in the intereaction with membrane-bound SecA." Biochem.Biophys.Acta. 1326. 23-36 (1997)
• [Publications] Sato, et al.: "In vitro analysis of the stop-transfer process during translocation across thecytoplasmic membrane of Escherichia coli." J.Biol.Chem.272. 20082-20087 (1997)
• [Publications] Sato,K.et al.: "Characterization of a potential catalytic reside,Asp-133 in the high affinity ATP-binding site of Escherichia coli SecA,translocation ATPase." J.Biol.Chem.271. 17439-17444 (1996)
• [Publications] Hatsuzawa,K.et al.: "The hydrophobic region of signal peptides is a determinant for SRP recognition and protein translocation across the ER membrane." J.Biochem.121. 111-118 (1997)
• [Publications] Sato,K.et al.: "Short hydrophobic segments in the mature domain of proOmpA determine its stepwise movement during translocation across the cytoplasmic membrane of Escherichia coli." J.Biol.Chem.272. 5880-5886 (1997)
• [Publications] Mori,H.et al.: "The hydrophobic region of signal peptides is involved in the interaction with membrane bound SecA." Biochim.Biophys.Acta. (in press). (1997)
• [Publications] S. Matsuyama et al.: "Biochemical Analyses of components comprising the protein translocation machinery of Escherichia coli." Adv. in Cell. Mol. Biol.4. 61-84 (1995)
• [Publications] E. Breukink et al.: "The C Terminus of SecA Is Involved in Both Lipid Binding and SecB Binding." J. Biol. Chem.270. 7902-7907 (1995)
• [Publications] M. Tagay et al.: "Systaxin 1 (HPC-1)Is Associated with Chromaffin Granules." J. B. C.270. 15930-15933 (1995)
• [Publications] K. Uchida et al.: "Stepwise movement of preproteins in the process of translocation across the cytoplasmic membrane of Escherichia coli." J. Biol.Chem.217. 30862-30868 (1995)
• [Publications] K. Nishiyama et al.: "Preferential interaction of Sec-G with Sec-E S tabilizes and unstable SecE derivative in the E. coli. membrane." Biochem. Biocphys. Res. Commun.217. (1995)