1995 年度実績報告書

大腸菌細胞を中心とした蛋白質膜透過機構の先端的研究

研究課題

研究課題/領域番号	07308069
研究種目	総合研究(A)
研究機関	東京大学
研究代表者	徳田元東京大学, 分子細胞生物学研究所, 教授 (40125943)
研究分担者	山根国男筑波大学, 生物科学系, 教授 (20013336) 平田肇姫路工業大学, 理学部, 教授 (40049052) 渡辺誠大分医科大学, 医学部, 教授 (20142408) 遠藤斗志也名古屋大学, 理学部, 教授 (70152014) 伊藤維昭京都大学, ウィルス研究所, 教授 (90027334)
キーワード	蛋白質膜透過 / 原核細胞 / 再構成 / SecY / SecE / SecG / SecA
研究概要	蛋白質膜透過反応中に、SecGの膜内配向性は反転・復帰を繰り返していることを見出した。この構造変化によって、SecAの膜内への挿入と離脱が促進され、膜透過反応の効率が上昇することを示している(徳田)。変異蛋白質の膜透過をin vitroで解析し、膜透過は約30アミノ酸残基単位で段階的に進行することを明らかにした(森)。膜透過反応初期過程を詳細に解析する系を開発した。SecEとSecGは直接結合していること、この結合にはsecYのそれぞれ異なる領域との相互作用が重要であることを明らかにした。SecAの膜挿入に影響するSecY変異を見出した(伊藤)。エンドウ葉緑体SecAと、シロイヌナズナ葉緑体SecYの遺伝子のクローニングに成功した。エンドウSecAはシアノバクテリアSecAと一次構造上、高い相同性を示し、両者の進化的近縁性が示唆された。エンドウSecAがタンパク質のチラコイド膜透過に直接関与していることを証明した。(遠藤)。リン脂質平面膜法を用い以下を明らかにした。精製SecYおよびSecEはイオンチャンネルを形成し、SecAはこのチャンネルを閉鎖する。SecGはチャンネルのコンダクタンスを上昇させる(平田)。枯草菌のSRP様粒子の構成成分、Ffhが前駆体蛋白質と複合体を形成すること、またFfhは枯草菌のscRNAとも結合することを明らかにした。Ffh変異株を用い、蛋白質前駆体とscRNAの結合部位は異なることを示した(山根)。

研究成果

(18件)

すべてその他

すべて文献書誌 (18件)

[文献書誌] Kontinen, V. P.: "Overexpression of phosphatidylglycerophosphate synthase restores protein translocation in a secG deletion mutant of Escherichia coli at low temperature." FEBS Lett.364. 157-160 (1995)
[文献書誌] Matsuyama, S.: "A novel periplasmic carrier protein involved in the sorting and transport of E. coli lipoproteins destined for the outer membrane." EMBO J.14. 3365-3372 (1995)
[文献書誌] Nishiyama, K.: "Preferential interaction of SecG with SecE stabilizes an unstable SecE derivative in the Escherichia coli cytoplasmic membrane." Biochem. Biophys. Res. Commun.217. 217-223 (1995)
[文献書誌] Uchida, K.: "Stepwise movement of preproteins in the process of translocation across the cytoplasmic membrane of Escherichia coli." J. Biol. Chem.270. 30862-30868 (1995)
[文献書誌] Breukink, E.: "The C terminus of SecA is involved in both Iipid binding and SecB binding." J. Biol. Chem.270. 7902-7907 (1995)
[文献書誌] Tagaya, M.: "Syntaxin 1 (HPC-1) is associated with chromaffin granules." J. Biol. Chem.270. 15930-15933 (1995)
[文献書誌] Shimoike, T.: "Product of a new gene, syd, functionally interacts with SecY when overproduced in Escherichia coli." J. Biol. Chem.270. 5519-5526 (1995)
[文献書誌] Kihara, A.: "FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase subunit." Proc. Natl. Acad. Sci. USA. 92. 4532-4536 (1995)
[文献書誌] Akiyama, Y.: "FtsH, a membrane-bound ATPase, forms a complex in the cytoplasmic membrane of Escherichia coli." J. Biol. Chem.270. 23485-23490 (1995)
[文献書誌] Endo, T.: "Roles of Sec proteins in protein transport within chloroplasts" Photosynthesis: from Light to Biosphere. 3. 811-814 (1995)
[文献書誌] Nakai, M.: "Cloning and characterization of sec and ffh genes from the cyanobacterium Synechococcus PCC7942" Photosynthesis: from Light to Biosphere. 3. 505-508 (1995)
[文献書誌] Nohara, M.: "Isolation and characterization of the cDNA for pea chloroplast SecA. Evolutionary conservation of the bacterial-type SecA-dependent protein transport within chloroplasts" FEBS Lett.364. 305-308 (1995)
[文献書誌] Shinomiya, H.: "Complete Primary Structure and Phosphorylation Site of the 65 kDa Macrophage Protein Phosphorylated by Stimulation with Bacterial Lipopolysaccharide." J. Immunol.154. 3471-3478 (1995)
[文献書誌] Murai, N.: "A novel insertion sequence (IS) -like element of the thermophilic bacterium PS3 promotes expression of the alanine carrier protein-encoding gene." Gene. 163. 103-107 (1995)
[文献書誌] Oguro, A.: "The effect of expression of srb, encoding a homologue of mammalian SRP receptor a-subunits, on cell growth and protein translocation in Bacillus subtilis and srb operon structure." Gene. (in press). (1996)
[文献書誌] Nakamura, K.: "Small cytoplasmic RNA (scRNA) gene from Clostridium perfringens can replaced the gene for the Bacillus subtilis scRNA in both growth and sporulation." Microbiology. 141. 2956-2975 (1995)
[文献書誌] Shibata, T.: "Depletion of small cytoplasmic RNA confers fusidic-acid resistance on Bacillus subtilis." Biochemical and Biophysical Research Communications.210. 317-323 (1995)
[文献書誌] Watanabe, M.: "High Affinity Binding of Escherichia coli SecB to the Signal Sequence Region of a Presecretory Protein." Proc. Natl. Acad. Sci. USA. (in press). (1996)

1995 年度 実績報告書

大腸菌細胞を中心とした蛋白質膜透過機構の先端的研究

研究代表者

徳田 元 東京大学, 分子細胞生物学研究所, 教授 (40125943)

研究成果

[文献書誌] Kontinen, V. P.: "Overexpression of phosphatidylglycerophosphate synthase restores protein translocation in a secG deletion mutant of Escherichia coli at low temperature." FEBS Lett.364. 157-160 (1995)

[文献書誌] Matsuyama, S.: "A novel periplasmic carrier protein involved in the sorting and transport of E. coli lipoproteins destined for the outer membrane." EMBO J.14. 3365-3372 (1995)

[文献書誌] Nishiyama, K.: "Preferential interaction of SecG with SecE stabilizes an unstable SecE derivative in the Escherichia coli cytoplasmic membrane." Biochem. Biophys. Res. Commun.217. 217-223 (1995)

[文献書誌] Uchida, K.: "Stepwise movement of preproteins in the process of translocation across the cytoplasmic membrane of Escherichia coli." J. Biol. Chem.270. 30862-30868 (1995)

[文献書誌] Breukink, E.: "The C terminus of SecA is involved in both Iipid binding and SecB binding." J. Biol. Chem.270. 7902-7907 (1995)

[文献書誌] Tagaya, M.: "Syntaxin 1 (HPC-1) is associated with chromaffin granules." J. Biol. Chem.270. 15930-15933 (1995)

[文献書誌] Shimoike, T.: "Product of a new gene, syd, functionally interacts with SecY when overproduced in Escherichia coli." J. Biol. Chem.270. 5519-5526 (1995)

[文献書誌] Kihara, A.: "FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase subunit." Proc. Natl. Acad. Sci. USA. 92. 4532-4536 (1995)

[文献書誌] Akiyama, Y.: "FtsH, a membrane-bound ATPase, forms a complex in the cytoplasmic membrane of Escherichia coli." J. Biol. Chem.270. 23485-23490 (1995)

[文献書誌] Endo, T.: "Roles of Sec proteins in protein transport within chloroplasts" Photosynthesis: from Light to Biosphere. 3. 811-814 (1995)

[文献書誌] Nakai, M.: "Cloning and characterization of sec and ffh genes from the cyanobacterium Synechococcus PCC7942" Photosynthesis: from Light to Biosphere. 3. 505-508 (1995)

[文献書誌] Nohara, M.: "Isolation and characterization of the cDNA for pea chloroplast SecA. Evolutionary conservation of the bacterial-type SecA-dependent protein transport within chloroplasts" FEBS Lett.364. 305-308 (1995)

[文献書誌] Shinomiya, H.: "Complete Primary Structure and Phosphorylation Site of the 65 kDa Macrophage Protein Phosphorylated by Stimulation with Bacterial Lipopolysaccharide." J. Immunol.154. 3471-3478 (1995)

[文献書誌] Murai, N.: "A novel insertion sequence (IS) -like element of the thermophilic bacterium PS3 promotes expression of the alanine carrier protein-encoding gene." Gene. 163. 103-107 (1995)

[文献書誌] Oguro, A.: "The effect of expression of srb, encoding a homologue of mammalian SRP receptor a-subunits, on cell growth and protein translocation in Bacillus subtilis and srb operon structure." Gene. (in press). (1996)

[文献書誌] Nakamura, K.: "Small cytoplasmic RNA (scRNA) gene from Clostridium perfringens can replaced the gene for the Bacillus subtilis scRNA in both growth and sporulation." Microbiology. 141. 2956-2975 (1995)

[文献書誌] Shibata, T.: "Depletion of small cytoplasmic RNA confers fusidic-acid resistance on Bacillus subtilis." Biochemical and Biophysical Research Communications.210. 317-323 (1995)

[文献書誌] Watanabe, M.: "High Affinity Binding of Escherichia coli SecB to the Signal Sequence Region of a Presecretory Protein." Proc. Natl. Acad. Sci. USA. (in press). (1996)

1995 年度実績報告書

徳田元東京大学, 分子細胞生物学研究所, 教授 (40125943)