1993 Fiscal Year Annual Research Report

大腸菌蛋白質分泌の分子機構

Research Project

Project/Area Number	04259102
Research Institution	Tokyo University of Pharmacy and Life Science
Principal Investigator	水島昭二東京薬科大学, 生命科学部, 教授 (50013313)
Co-Investigator(Kenkyū-buntansha)	伊藤維昭京都大学, ウィルス研究所, 教授 (90027334) 徳田元東京大学, 分子細胞生物学研究所, 助教授 (40125943)
Keywords	蛋白質の分泌 / 蛋白質の膜透過 / Sec蛋白質 / SecY / SecA / SecG / Ydr / FtsH
Research Abstract	1.水島グループの研究成果:(1)膜透過反応の初期過程で中心的役割を果すSecAと前駆体蛋白質の相互作用を著しく促進する蛋白質を発見し、蛋白質、遺伝子両レベルで詳細な解析を行った。(2)前駆体蛋白質がシグナルペプチドの疎水領域を外側に突出するような形でSecA分子と相互作用すること、またこの相互作用によってSecF自身も疎水領域を外部に露出させることをNMR解析によって示した。(3)SecAがSecD、SecYの膜内アッセンブリーに関与することを示した。(4)以上の成果を組み込んだ分泌型蛋白質の膜透過モデルを提示した。 2.徳田グループの研究成果:大腸菌の蛋白質膜透過系は、SecA、SecY、SecEを中心として構成されており、これら3種の因子のみを再構成したプロテオリポゾームの活性を顕著に促進させる新因子p12を細胞質膜中に見いだし、これを均一に精製した。p12の部分アミノ酸配列を基に構造遺伝子を同定しその全塩基配列を決定した。p12遺伝子の破壊株を構築し、細胞での蛋白質分泌が阻害されていることを示した。以上の結果から、p12が大腸菌蛋白質膜透過系を構成する新因子であることを明らかにし、SecGと命名した。 3.伊藤グループの研究成果:SecY‐SecE複合形成にはSecYの細胞質ドメイン4を介した細胞質領域どうしの相互作用が重要であること、SecY‐SecEの結合は合成後直ちに起こり、一旦形成されたSecY‐SecE複合体は細胞内で安定に保持され、Biekerらの解離会合モデルは考え難いことを示した。In vitroでSecY複合体形成や種々の変異体の活性変化を調べる系の構築を進めた。Ydr遺伝子産物には野性型SecY分子の選択的安定化および、SecY‐SecE相互作用への阻害的介入の機能があることを示した。FtsH機能とチャペロン機能のオーバーラップおよびFtsHの単独SecYの分解への関与を見出した。

Research Products

(19 results)

All Other

All Publications (19 results)

[Publications] S.Matsuyama et al.: "SecD is involved in the release of translocated secretory proteins from the cytoplasmic membrane of Escherichia coli." EMBO J.12. 265-270 (1993)
[Publications] S.Ichihara et al.: "Molecular cloning,sequencing,and mapping of the gene encoding protease I and characterization of proteinase and proteinase‐defective Escherichia coli mutants." J.Bacteriol.175. 1032-1037 (1993)
[Publications] M.Kato et al.: "Translocation of conjugated presecretory proteins possessing an intemal non‐peptide domain into everted membrane vesicles in Escherichia coli." J.Biol.268. 3586-3593 (1993)
[Publications] D.Fourel et al.: "Specific regions of Escherichia coli OmpF protein involved in antigenic/colicin receptor sites and in stable trimerization." J.Bacteriol.175. 2754-2757 (1993)
[Publications] S.Kawasaki et al.: "Membrane vesicles containing overproduced SecY and SecE exhibit high translocation ATPase activity and countermovement of proteins in a SecA‐and presecretory protein‐dependent manner." J.Biol.Chem.268. 8193-8198 (1993)
[Publications] D.A.Phoenix et al.: "OmpF‐Lpp signal sequence mutants with varying charge hydrophobicity ratios provide evidence for a phosphatidylglycerol‐signal sequence interaction during protein translocation across the Escherichia coli inner membrane." J.Biol.Chem.268. 17069-17073 (1993)
[Publications] D.A.Phoenix et al.: "Phosphatidylglycerol dependent protein translocation across the Escherichia coli inner membrane is inhibited by the anticancer drug doxorubicin." FEBS Letters. 324. 113-116 (1993)
[Publications] K.Nishiyama et al.: "A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli." EMBO.J.12. 3409-3415 (1993)
[Publications] Hong-mei Lu et al.: "A periplasmic intermediate in the extracellular secretion pathway of Pseudomonas aeruginosa exotoxin A." J.Bacteriol.175. 7463-7467 (1993)
[Publications] H.Tokuda et al.: "Histidine residucs are invloved in translocation‐coupled ATP hydrolysis by the SecA" Biochem.Biophys.Res.Commun.192. 1415-1421 (1993)
[Publications] 徳田元: "大腸菌蛋白質分泌の分子機構" 蛋白質、核酸、酸素. 38. 947-956 (1993)
[Publications] Y.Akiyama.et al.: "Folding and assembly of bacterial alkaline phosphatase in vitro and in vivo." J.Biol.Chem.268. 8146-8150 (1993)
[Publications] T.Taura et al.: "Determinants of quantity of the stable SecY complex in the Escherichia coli cell." J.Bacteriol.175. 7771-7775 (1993)
[Publications] C.Herman et al.: "Cell growth and lambda phage development controlled by the same essential Escherichia coli gene,ftsH/hflB." Proc.NatI.Acad.Sci.USA. 90. 10861-10865 (1993)
[Publications] Y.Akiyama et al.: "Involvement of FtsH in protein assembly into and throuhgh the membrane.I.Mutations that reduce retention efficiency of a cytoplasmic reporter." J.Biol.Chem.269(in press). (1994)
[Publications] Y.Akiyama et al.: "Involvement of FtsH in protein assembly into and through the membrane.II.Dominant mutations affecting FtsH functions." J.Biol.Chem.269(in press). (1994)
[Publications] T.Tauyra et al. 20GB17:Genetic analysis of SecY:additional export defective mutations and factors affecting their phenotypes.: Mol.Gen.Genet.,. (in press). (1994)
[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)
[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)

1993 Fiscal Year Annual Research Report

大腸菌蛋白質分泌の分子機構

Principal Investigator

水島 昭二 東京薬科大学, 生命科学部, 教授 (50013313)

Research Products

[Publications] S.Matsuyama et al.: "SecD is involved in the release of translocated secretory proteins from the cytoplasmic membrane of Escherichia coli." EMBO J.12. 265-270 (1993)

[Publications] S.Ichihara et al.: "Molecular cloning,sequencing,and mapping of the gene encoding protease I and characterization of proteinase and proteinase‐defective Escherichia coli mutants." J.Bacteriol.175. 1032-1037 (1993)

[Publications] M.Kato et al.: "Translocation of conjugated presecretory proteins possessing an intemal non‐peptide domain into everted membrane vesicles in Escherichia coli." J.Biol.268. 3586-3593 (1993)

[Publications] D.Fourel et al.: "Specific regions of Escherichia coli OmpF protein involved in antigenic/colicin receptor sites and in stable trimerization." J.Bacteriol.175. 2754-2757 (1993)

[Publications] S.Kawasaki et al.: "Membrane vesicles containing overproduced SecY and SecE exhibit high translocation ATPase activity and countermovement of proteins in a SecA‐and presecretory protein‐dependent manner." J.Biol.Chem.268. 8193-8198 (1993)

[Publications] D.A.Phoenix et al.: "OmpF‐Lpp signal sequence mutants with varying charge hydrophobicity ratios provide evidence for a phosphatidylglycerol‐signal sequence interaction during protein translocation across the Escherichia coli inner membrane." J.Biol.Chem.268. 17069-17073 (1993)

[Publications] D.A.Phoenix et al.: "Phosphatidylglycerol dependent protein translocation across the Escherichia coli inner membrane is inhibited by the anticancer drug doxorubicin." FEBS Letters. 324. 113-116 (1993)

[Publications] K.Nishiyama et al.: "A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli." EMBO.J.12. 3409-3415 (1993)

[Publications] Hong-mei Lu et al.: "A periplasmic intermediate in the extracellular secretion pathway of Pseudomonas aeruginosa exotoxin A." J.Bacteriol.175. 7463-7467 (1993)

[Publications] H.Tokuda et al.: "Histidine residucs are invloved in translocation‐coupled ATP hydrolysis by the SecA" Biochem.Biophys.Res.Commun.192. 1415-1421 (1993)

[Publications] 徳田元: "大腸菌蛋白質分泌の分子機構" 蛋白質、核酸、酸素. 38. 947-956 (1993)

[Publications] Y.Akiyama.et al.: "Folding and assembly of bacterial alkaline phosphatase in vitro and in vivo." J.Biol.Chem.268. 8146-8150 (1993)

[Publications] T.Taura et al.: "Determinants of quantity of the stable SecY complex in the Escherichia coli cell." J.Bacteriol.175. 7771-7775 (1993)

[Publications] C.Herman et al.: "Cell growth and lambda phage development controlled by the same essential Escherichia coli gene,ftsH/hflB." Proc.NatI.Acad.Sci.USA. 90. 10861-10865 (1993)

[Publications] Y.Akiyama et al.: "Involvement of FtsH in protein assembly into and throuhgh the membrane.I.Mutations that reduce retention efficiency of a cytoplasmic reporter." J.Biol.Chem.269(in press). (1994)

[Publications] Y.Akiyama et al.: "Involvement of FtsH in protein assembly into and through the membrane.II.Dominant mutations affecting FtsH functions." J.Biol.Chem.269(in press). (1994)

[Publications] T.Tauyra et al. 20GB17:Genetic analysis of SecY:additional export defective mutations and factors affecting their phenotypes.: Mol.Gen.Genet.,. (in press). (1994)

[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)

[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)

水島昭二東京薬科大学, 生命科学部, 教授 (50013313)