Study on the regulatory region of the chloroplast ATP synthase by using peptide antibody

• Project/Area Number: 11640643
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 植物生理
• Research Institution: Chemical Resources Laboratory, Tokyo Institute of Technology
• Principal Investigator: HISABORI Toru Chemical Resources Laboratory, Tokyo Institute of Technology Associate Professor, 資源化学研究所, 助教授 (40181094)
• Co-Investigator(Kenkyū-buntansha): TAGUCHI Hideki Chemical Resources Laboratory, Tokyo Institute of Technology Research Associate, 資源化学研究所, 助手 (40272710)
• Project Period (FY): 1999 – 2000
• Project Status: Completed (Fiscal Year 2000)
• Budget Amount: ¥3,600,000 (Direct Cost: ¥3,600,000); Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000); Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
• Keywords: ATP synthase / chloroplast / gamma subunit / thioredoxin / regulation / CF_1 / ジスルフィド結合 / 調節

Research Abstract

To clarify the regulatory mechanism of the chloroplast ATP synthase, we carried out three different approach as follows ; (1) the mutation analysis of the regulatory region of the γ subunit, (2) the investigation of the interaction between thioredoxin and the regulatory region of the γ subunit, (3) the investigation of the interaction between thioredoxin and the chloroplast ATP synthase F_1 part.
We first investigated the change of the regulation by the introduction of the mutation at around the regulatory region including two cysteines on the γ subunit. From this study, we concluded that the conformational change of the regulatory region itself is the most critical for the regulation of the enzyme activity. Furthermore, we found that the deletion of the negative charged cluster in the regulatory region results the inversion of the regulation.
Secondary, we prepared the mutant thioredoxin to investigate the interaction with ATP synthase. From this study, we could clarify that the chloroplast thioredoxin can induce the important conformational change of the γ subunit to facilitate the reduction of the regulatory disulfide bond only by the protein-protein interaction. Although we prepared the antibody against the active region of the chloroplast thiredoxin to investigate the interaction between thioredoxin and 2ATP synthase, the study on this direction is not successful yet.

Research Products

• [Publications] Konno,H.,Yodogawa,M.,Stumpp,M.T.,Kroth,P.,Strotmann,H.,Motohashi,K.,Amano,T.,Hisabori,T.: "Inverse regulation of F_1-ATPase activity by a mutation at the regulatory region on the γ subunit of chloroplast ATP synthase"Biochem.J.. 352. 783-788 (2000)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Bald,D.,Noji,H.,Stumpp,M.T.,Yoshida,M.,Hisabori,T: "ATPsae activity of a highly stable α_3β_3γ subcomplex of thermophilic F_1 can be regulated by the introduced regulatory region of γ subunit of chloroplast F_1"J.Biol.Chem.. 275. 12757-12762 (2000)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Hisabori T,Kondoh A,Yoshida M: "The γ subunit in chloroplast F_1-ATPase can rotate in a unidirectional and counter-clockwise manner"FEBS Lett.. 463. 35-38 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Stumpp,M.T.,Motohashi,K.,Hisabori,T.: "Chloroplast thioredoxin mutants without active site cysteines facilitate the reduction of the regulatory disulfide bridge on the γ subunit of chloroplast ATP synthase"Biochem.J.. 341. 157-163 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Konno, H., Yodogawa, M., Stumpp, M.T., Kroth, P., Strotmann, H., Motohashi, K., Amano, T., Hisabori, T.: "Inverse regulation of F_1-ATPase activity by a mutation at the regulatory region on the γsubunit of chloroplast ATP synthase"Biochem.J.. 352. 783-788 (2000)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Bald, D., Noji, H., Stumpp, M.T., Yoshida, M., Hisabori, T.: "ATPase activity of a highly stable α_3β_3γ subcomplex of thermophilic F_1 can be regulated by the introduced regulatory region of γ subunit of chloroplast F_1"J.Biol.Chem.. 275. 12757-12762 (2000)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Hisabori T, Kondoh A, Yoshida M: "The γ subunit in chloroplast F_1-ATPase can rotate in a unidirectional and counter-clockwise manner"FEBS Lett.. 463. 35-38 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Stumpp, M.T., Motohashi, K., Hisabori, T.: "Chloroplast thioredoxin mutants without active site cysteines facilitate the reduction of the regulatory disulfide bridge on the γ subunit of chloroplast ATP synthase"Biochem.J.. 341. 157-163 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Bald,D.: "ATPase activity of a highly stable α_3β_3γ subcomplex of thermophilic F_1 can be regulated by the introduced regulatory region of γ subunit of chloroplast F_1"Journal of Biological Chemistry. 275. 12757-12762 (2000)
• [Publications] Konno,H.: "Inverse regulation of F_1-ATPase activity by a mutation at the regulatory region of the γ Subunit of chloroplast ATP synthase"Biochemical Journal. 352. 783-788 (2000)
• [Publications] Stumpp M.T.: "Chloroplast thioredoxin mutants without active-site cysteines facilitate the reduction of the regulatory disulphide bridge on the γ-subunit of chloroplast ATP synthase"Biochmical Journal. 341. 157-163 (1999)
• [Publications] Hiroshi T.: "The γ-subunit in chloroplast F-1-ATPase can rotate in a unidirectional and counter-clockwise manner"FEBS Letters. 463. 35-38 (1999)
• [Publications] Bald D.: "ATPase activity of a highly stable α_3β_3γ Subcomplex of thermophilic F1 can be regulated by the introduced regulatory region of γ-subunit of chloroplast F1"J.Biol.Chem.. (in press). (2000)