1994 年度実績報告書

原核細胞における蛋白質分泌の分子機構

研究課題

研究課題/領域番号	06248103
研究機関	東京薬科大学
研究代表者	水島昭二東京薬科大学, 生命科学部, 教授 (50013313)
研究分担者	山根國男筑波大学, 生物科学系, 教授 (20013336) 伊藤維昭京都大学, ウイルス研究所, 教授 (90027334) 徳田元東京大学, 分子細胞生物学研究所, 助教授 (40125943)
キーワード	蛋白質の分泌 / 蛋白質の膜透過 / Sec蛋白質 / SecY / SecA / SecG / Ydr / FtsH
研究概要	水島グループ:(1)SecAがシグナルペプチドの疎水領域を認識しうること、この認識には酸性リン脂質が関与することを明らかにした。そしてこの成果をもとに蛋白質分泌におけるSecAの役割に関して新しいモデルを提示した。(2)SecAのATP機能部位を構成していると推定されている133番目のアスパラギン酸をアスパラギンに変えたところ、SecAの多くの機能が失われた。この結果をもとにATP機能部位の膜透過における役割を考察した。(3)変異proOmpAを用いてタンパク質の膜透過が20-30アミノ酸残基毎にstepwiseに進行することを示す結果をさらに蓄積した。徳田グループ:大腸菌の膜透過機構を再構成実験系を用いて解析し以下の成果を得た。(1)再構成活性を上昇させる分子量約12,000の膜内在性因子SecGを見い出した。(2)SecGの遺伝子をクローニングし塩基配列を決定した。SecG遺伝子破壊株を構築し、SecGは低温での蛋白質膜透過に重要であることを明らかにした。(3)SecAの受容体となるもはSecYよよびSecEであり、SecGは受容体活性に影響しないが、膜透過に共役したSecAのATPase活性を顕著に上昇させることを明らかにした。伊藤グループ:単独SecYの速やかな分解に、膜ATPase FtsHの機能が必須であること、その他に少なくとも2種類の遺伝子産物が関与することを明らかにした。FisHがN末端で相互作用しているオリゴマーを形成していることを明らかにし、さらに,ATPaseドメインを変異体の作製により解析した。FtsHは膜蛋白質を認識して、状況に応じてアセンブリーあるいは分解へと導く一種のシャペロンであるとの仮説を提唱した。種々のsecY変異株の膜小胞の解析から、遺伝子学的解析推定されたSceYの「活性部位(C5,C6ドメイン)」の多くの変異体はin vitroでSecAのATPase促進活性が著しく低いことを見いだした。また、Syd(Ydr)蛋白質は、SecY24変異蛋白質の特異的阻害剤としてはたらくことをin vitroで示した。山根グループ:枯草菌における蛋白質分泌装置構成因子の一つと考えられるシグナル認識粒子を精製し、本粒子が8-9Sの大きさであること、scRNAとFfh以外に少なくとも2種類の蛋白質成分を含んでいることを明らかにした。secRNAがFfh蛋白質と結合するためにはFfhのMドメインに存在する2つのα-ヘリックス構造が必要であった。また枯草菌SecAのアミノ末端234残基からなるペプチドは大腸菌secA52変異を非許容温度で相補できるが、相補の際には枯草菌SecA234ペプチド・大腸菌SecA52蛋白質・ATP-dependent Protease Laの3者が大腸菌内で複合体を形成していることを明らかにした。

研究成果
(24件)

すべてその他

すべて文献書誌 (24件)

[文献書誌] Sagara,K.: "SecF Stabilizes SecD and SecY,Components of the Protein Translocation Machinery of the Escherichia coli Cytoplasmic Membrane." J.Bacteriol.176. 4111-4116 (1994)
[文献書誌] Hanada,M.: "Reconstitution of aneffidient ptoein translocation machinery comprising SecA and the three membrane proteins,SecY,SecE and SecG(p12)." J.Biol.Chem.269. 23625-23632 (1994)
[文献書誌] Dauville,K.: "The band 1 subunit of Escherichia coli prepreotein translocase and the integral membrane export factor P12 are the same protein." J.Biol.Chem.269. 18705-18707 (1994)
[文献書誌] Kawasaki,S.: "Membrane vesicles containing overproduced SecY and SecE exbibit high translocation ATPase activity and counter movement of protons in a SecA-and presecretory protein-dependent manner." J.Biol.Chem.268. 8193-8198 (1993)
[文献書誌] Nishiyama,K.: "A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli." EMBO J.12. 3409-3415 (1993)
[文献書誌] Tokuda,H.: "Histidine residues are involved in translocation-coupled ATP hydrolysis of SecA protein." Biochem.Biophys.Res.Commun.195. 1415-1421 (1993)
[文献書誌] Tokuda,H.: "Biochemical characterization of the presecretory protein translocation machinery of Escherichia coli." FEBA Lett.(Minireview). 346. 65-68 (1994)
[文献書誌] 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)
[文献書誌] 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)
[文献書誌] Shimoike,T.,Taura,T.,Kihara,A.,Yoshihisa,T.,Akiyama,Y.,Cannon,K.and Ito,K.: "Product of a new gene,syd,functionally interacts with SecY when overproduced in Escherichia coli." J.Biol.Chem.(in press). (1995)
[文献書誌] Kihara,A.,Akiyama,Y.and Ito,K.: "FtsH is required for proteolytic elimination of uncomplexed forms of SecY,an essential protein translocase subunit." Proc.Natl.Acad.Sci.USA. (in press). (1995)
[文献書誌] Akiyama,Y.,Ogura,T.and Ito,K.: "Involvement of FtsH in protein assembly into and through the membrane.I.Mutations that reduce retention efficiency of a cytoplasmic reporter." J.Biol.chem.269. 5218-5224 (1994)
[文献書誌] Akiyama,Y.,Shirai,Y.and Ito,K.: "Involvement of FtsH in protein assembly into and through the membrane.II.Dominant mutations affecting FtsH functions." J.Biol.chem.269. 5225-5229 (1994)
[文献書誌] Baba,T.,Taura,T.,Shimoike,T.,Akiyama,Y.,Yoshihisa,T.and Ito,K.: "A cytoplasmic domain is important for the formation of a SecY-SecE translocator complex." Proc.Natl.Acad.Sci.USA. 91. 4539-4543 (1994)
[文献書誌] Taura,T.,Akiyama,Y.and Ito,K.: "Genetic analysis of SecY:additional export defective mutations and factors affecting their phenotypes." Mol.Gen.Genet.243. 261-269 (1994)
[文献書誌] M.Nishiguchi: "structural requirements of Bacillus subtilis small cytoplasmic RNA for cell growth,sporulation,and extracellular enzyme prooduction." Journal of Bacteriology. 176. 157-165 (1994)
[文献書誌] K.Nakamura: "The Bacillus subtilis SRP54 homologue,Ffh,has an intrinsic GTPase activity and formes a ribonucleoprotein complex with small cytoplasmic RNA in vivo." Biochemical and Biophisical Research Communication. 199. 1394-1399 (1994)
[文献書誌] H.Takamatsu: "A truncated Bacillus subtilis SecA protein consisting of N-terminal 234 amino acid residues forms a complex with Escherichia coli SecA51(ts) protein and complements the protein translocation defect of the sacA51 mutant." J.Biochem.116. 1287-1294 (1994)
[文献書誌] A.Nakane: "Acquisition of azide-resistance by elevated ATPase activity confers azide-resistance upon cell growth and protein translocation in Bacillus subtilis." Microbiology. 141. 113-121 (1994)
[文献書誌] K.Haga.: "Crystallization and preliminary x-ray studies of cyclodexterin glucano-transferase from alklophilic Bacillus sp.1011." J.Mol.Biol.237. 163-164 (1994)
[文献書誌] A.Nakamura: "Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp.1011:effects of replacements on substrate binding and cyclization characteristics." Biochemistry. 33. 9929-9936 (1994)
[文献書誌] H.Takamatsu: "The rapid degradation of mutant SecA protein in the Bacillus subtilis secA341(ts)mutant causes a protein translocation defect in the cell." Biosci.Biotech.Biochemistry. 58. 1845-1850 (1994)
[文献書誌] 中村幸治: "枯草菌における蛋白質分泌系と分泌装置" 日本農芸化学会誌. 69. 43-46 (1995)
[文献書誌] A.Nakamura: "Enzyme Chemistry and Molecular Biology of Amylases and Related" CRC Press, 29 (1994)

1994 年度 実績報告書

原核細胞における蛋白質分泌の分子機構

研究代表者

水島 昭二 東京薬科大学, 生命科学部, 教授 (50013313)

研究成果

[文献書誌] Sagara,K.: "SecF Stabilizes SecD and SecY,Components of the Protein Translocation Machinery of the Escherichia coli Cytoplasmic Membrane." J.Bacteriol.176. 4111-4116 (1994)

[文献書誌] Hanada,M.: "Reconstitution of aneffidient ptoein translocation machinery comprising SecA and the three membrane proteins,SecY,SecE and SecG(p12)." J.Biol.Chem.269. 23625-23632 (1994)

[文献書誌] Dauville,K.: "The band 1 subunit of Escherichia coli prepreotein translocase and the integral membrane export factor P12 are the same protein." J.Biol.Chem.269. 18705-18707 (1994)

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

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

[文献書誌] Tokuda,H.: "Histidine residues are involved in translocation-coupled ATP hydrolysis of SecA protein." Biochem.Biophys.Res.Commun.195. 1415-1421 (1993)

[文献書誌] Tokuda,H.: "Biochemical characterization of the presecretory protein translocation machinery of Escherichia coli." FEBA Lett.(Minireview). 346. 65-68 (1994)

[文献書誌] 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)

[文献書誌] 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)

[文献書誌] Shimoike,T.,Taura,T.,Kihara,A.,Yoshihisa,T.,Akiyama,Y.,Cannon,K.and Ito,K.: "Product of a new gene,syd,functionally interacts with SecY when overproduced in Escherichia coli." J.Biol.Chem.(in press). (1995)

[文献書誌] Kihara,A.,Akiyama,Y.and Ito,K.: "FtsH is required for proteolytic elimination of uncomplexed forms of SecY,an essential protein translocase subunit." Proc.Natl.Acad.Sci.USA. (in press). (1995)

[文献書誌] Akiyama,Y.,Ogura,T.and Ito,K.: "Involvement of FtsH in protein assembly into and through the membrane.I.Mutations that reduce retention efficiency of a cytoplasmic reporter." J.Biol.chem.269. 5218-5224 (1994)

[文献書誌] Akiyama,Y.,Shirai,Y.and Ito,K.: "Involvement of FtsH in protein assembly into and through the membrane.II.Dominant mutations affecting FtsH functions." J.Biol.chem.269. 5225-5229 (1994)

[文献書誌] Baba,T.,Taura,T.,Shimoike,T.,Akiyama,Y.,Yoshihisa,T.and Ito,K.: "A cytoplasmic domain is important for the formation of a SecY-SecE translocator complex." Proc.Natl.Acad.Sci.USA. 91. 4539-4543 (1994)

[文献書誌] Taura,T.,Akiyama,Y.and Ito,K.: "Genetic analysis of SecY:additional export defective mutations and factors affecting their phenotypes." Mol.Gen.Genet.243. 261-269 (1994)

[文献書誌] M.Nishiguchi: "structural requirements of Bacillus subtilis small cytoplasmic RNA for cell growth,sporulation,and extracellular enzyme prooduction." Journal of Bacteriology. 176. 157-165 (1994)

[文献書誌] K.Nakamura: "The Bacillus subtilis SRP54 homologue,Ffh,has an intrinsic GTPase activity and formes a ribonucleoprotein complex with small cytoplasmic RNA in vivo." Biochemical and Biophisical Research Communication. 199. 1394-1399 (1994)

[文献書誌] H.Takamatsu: "A truncated Bacillus subtilis SecA protein consisting of N-terminal 234 amino acid residues forms a complex with Escherichia coli SecA51(ts) protein and complements the protein translocation defect of the sacA51 mutant." J.Biochem.116. 1287-1294 (1994)

[文献書誌] A.Nakane: "Acquisition of azide-resistance by elevated ATPase activity confers azide-resistance upon cell growth and protein translocation in Bacillus subtilis." Microbiology. 141. 113-121 (1994)

[文献書誌] K.Haga.: "Crystallization and preliminary x-ray studies of cyclodexterin glucano-transferase from alklophilic Bacillus sp.1011." J.Mol.Biol.237. 163-164 (1994)

[文献書誌] A.Nakamura: "Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp.1011:effects of replacements on substrate binding and cyclization characteristics." Biochemistry. 33. 9929-9936 (1994)

[文献書誌] H.Takamatsu: "The rapid degradation of mutant SecA protein in the Bacillus subtilis secA341(ts)mutant causes a protein translocation defect in the cell." Biosci.Biotech.Biochemistry. 58. 1845-1850 (1994)

[文献書誌] 中村幸治: "枯草菌における蛋白質分泌系と分泌装置" 日本農芸化学会誌. 69. 43-46 (1995)

[文献書誌] A.Nakamura: "Enzyme Chemistry and Molecular Biology of Amylases and Related" CRC Press, 29 (1994)

1994 年度実績報告書

水島昭二東京薬科大学, 生命科学部, 教授 (50013313)