Molecular mechanism of protein translocation stimulation by SecDF using proton motive force

• Project/Area Number: 17K07334
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Functional biochemistry
• Research Institution: Kyoto University
• Principal Investigator: Mori Hiroyuki 京都大学, ウイルス・再生医科学研究所, 准教授 (10243271)
• Project Period (FY): 2017-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2017: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: SecDF / プロトン駆動力 / タンパク質膜透過 / PMF / 細菌 / 分泌 / PpiD / in vivo光架橋実験 / 膜タンパク質 / エネルギー変換

Outline of Final Research Achievements

The membrane protein complex SecDF promotes protein translocation in bacteria using proton-driven force, but the details of its molecular mechanism remain unclear. In this study, by identifying factors that are in close proximity to SecDF in living cells, we found that the periplasmic chaperone PpiD is involved in the protein translocation of a substrate by working together with SecDF. In addition, we determined high-resolution crystal structures of another conformational state of SecDF. These results provide important insights into the mechanism of SecDF-mediated stimulation of protein translocation.

Academic Significance and Societal Importance of the Research Achievements

タンパク質の膜透過反応は全ての生物に必須の現象であり、その分子機構の理解は細胞生物学の重要な研究課題の１つである。加えて、近年病原性細菌の多剤耐性化による院内感染の増加は大きな社会問題となっており、これまでとは作用機序の異なる薬の開発が待たれている。SecDFは細菌に特有の因子であるのに加え、大きな機能ドメインを細胞質外に持つことから、創薬の有用なターゲット候補として期待されている。効率良い創薬を進めるには、本研究による分子機構の理解は非常に意義が大きい。

Research Products

• [Journal Article] Fine interaction profiling of VemP and mechanisms responsible for its translocation-coupled arrest-cancelation (2020)
  • Author(s): Miyazaki Ryoji、Akiyama Yoshinori、Mori Hiroyuki
  • Journal Title: eLife Volume: 9
  • DOI: 10.7554/elife.62623
  • Peer Reviewed / Open Access
• [Journal Article] Reversible auto-inhibitory regulation of Escherichia coli metallopeptidase BepA for selective β-barrel protein degradation. (2020)
  • Author(s): Daimon 他7名
  • Journal Title: Proc. Natl. Acad. Sci. USA. Volume: 117 Issue: 45 Pages: 27989-27996
  • DOI: 10.1073/pnas.2010301117
  • Peer Reviewed / Open Access
• [Journal Article] Tree of motility : A proposed history of motility systems in the tree of life (2020)
  • Author(s): M Miyata, R C Robinson, T QP Uyeda, Y Fukumori, S Fukushima, S Haruta, M Homma, K Inaba, M Ito, C Kaito, K Kato, TKenri, Y Kinosita, S Kojima, T Minamino, H Mori, S Nakamura, D Nakane, K Nakayama, M Nishiyama, S Shibata, K Shimabukuro, M Tamakoshi, A Taoka, Y Tashiro, ITulum, HiWada, K Wakabayashi
  • Journal Title: Genes to Cells Volume: 25 Issue: 1 Pages: 6-21
  • DOI: 10.1111/gtc.12737
  • NAID: 130007881664
  • URL: https://ocu-omu.repo.nii.ac.jp/records/2019864
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A photo-cross-linking approach to monitor protein dynamics in living cells (2019)
  • Author(s): Miyazaki Ryoji、Akiyama Yoshinori、Mori Hiroyuki
  • Journal Title: Biochimica et Biophysica Acta (BBA) - General Subjects Volume: 印刷中 Issue: 2 Pages: 129317-129317
  • DOI: 10.1016/j.bbagen.2019.03.003
  • Peer Reviewed
• [Journal Article] Heat shock transcription factor σ³² defective in membrane transport can be suppressed by transposon insertion into genes encoding a restriction enzyme subunit or a putative autotransporter in <i>Escherichia coli</i> (2018)
  • Author(s): Yura Takashi、Miyazaki Ryoji、Fujiwara Keigo、Ito Koreaki、Chiba Shinobu、Mori Hiroyuki、Akiyama Yoshinori
  • Journal Title: Genes & Genetic Systems Volume: 93 Issue: 6 Pages: 229-235
  • DOI: 10.1266/ggs.18-00040
  • NAID: 130007582071
  • ISSN: 1341-7568, 1880-5779
  • Year and Date: 2018-12-01
  • Peer Reviewed
• [Journal Article] Monitoring substrate enables real-time regulation of a protein localization pathway (2018)
  • Author(s): Ito Koreaki、Mori Hiroyuki、Chiba Shinobu
  • Journal Title: FEMS Microbiology Letters Volume: 365 Issue: 11 Pages: 1-11
  • DOI: 10.1093/femsle/fny109
  • Peer Reviewed
• [Journal Article] A new photo-cross-linking approach for analysis of protein dynamics in vivo. (2018)
  • Author(s): Miyazaki, R., Myogo, N., Mori, H. and Akiyama, Y.
  • Journal Title: J. Biol. Chem. Volume: 293 Issue: 2 Pages: 677-686
  • DOI: 10.1074/jbc.m117.817270
  • Peer Reviewed
• [Journal Article] Identification and characterization of arrest motif in VemP by systematic mutational analysis. (2018)
  • Author(s): Mori, H., Sakashita, S., Ito, J., Ishii, E. and Akiyama, Y.
  • Journal Title: J. Biol. Chem. Volume: 293 Issue: 8 Pages: 2915-2926
  • DOI: 10.1074/jbc.m117.816561
  • Peer Reviewed
• [Journal Article] Tunnel Formation Inferred from the I-Form Structures of the Proton-Driven Protein Secretion Motor SecDF (2017)
  • Author(s): Arata Furukawa, Kunihito Yoshikaie, Takaharu Mori, Hiroyuki Mori, Yusuke V. Morimoto, Yasunori Sugano, Shigehiro Iwaki, Tohru Minamino, Yuji Sugita, Yoshiki Tanaka, Tomoya Tsukazaki
  • Journal Title: Cell Reports Volume: 19 Issue: 5 Pages: 1-7
  • DOI: 10.1016/j.celrep.2017.04.030
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] BepA mediates productive transfer of substrate proteins to the β-barrel assembly machinery (BAM) complex. (2017)
  • Author(s): Daimon, Y., Masui, C., Tanaka, Y., Shiota, T., Suzuki, T., Miyazaki, R., Sakurada, H., Lithgow, T., Dohmae, N., Mori, H., Tsukazaki, T., Narita, S. and Akiyama, Y.
  • Journal Title: Mol. Microbiol. Volume: 106 Issue: 5 Pages: 760-776
  • DOI: 10.1111/mmi.13844
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] 細菌の外膜生合成と品質管理に関わる2機能性プロテアーゼBepAの基質認識機構 (2020)
  • Author(s): 宮﨑亮次、吉谷亘平、森 博幸、秋山芳展
  • Organizer: 第93回日本生化学会大会、web開催
• [Presentation] ビブリオ菌VemPの翻訳途上鎖を介したmRNA分解機構に関する研究 (2019)
  • Author(s): 石井英治、秋山芳展、森 博幸
  • Organizer: 第16回21世紀大腸菌研究会
• [Presentation] 部位特異的in vivo光架橋法によるVemPの翻訳停止解除機構の解析 (2019)
  • Author(s): 宮﨑亮次、秋山芳展、森 博幸
  • Organizer: 第16回21世紀大腸菌研究会
• [Presentation] 部位特異的in vivo光架橋法による大腸菌LptDの生合成過程の解析 (2019)
  • Author(s): 渡邊哲朗、宮﨑亮次、森 博幸、秋山芳展
  • Organizer: 第16回21世紀大腸菌研究会
• [Presentation] 改良型部位特異的in vivo光架橋法による生細胞内での翻訳途上ポリペプチド鎖の相互作用動態解析 (2019)
  • Author(s): 宮﨑亮次、秋山芳展、森 博幸
  • Organizer: 第92回日本生化学会大会
• [Presentation] Vibrio alginolyticusにおける新生ポリペプチド鎖依存的なmRNA分解機構 (2019)
  • Author(s): 石井英治、秋山芳展、森 博幸
  • Organizer: 第53回ビブリオシンポジウム
• [Presentation] ビブリオ属細菌における新生ポリペプチド鎖を介した遺伝子発現制御機構について (2019)
  • Author(s): 石井 英治、秋山芳展、森 博幸
  • Organizer: 発酵研究所寄付講座開設記念シンポジウム「IFOがつなぐ京大微生物学のフロントライン」
• [Presentation] 部位特異的in vivo光架橋法による大腸菌LptDの生合成過程の解析 (2019)
  • Author(s): 渡邊哲朗、宮﨑亮次、森 博幸、秋山芳展
  • Organizer: 発酵研究所寄付講座開設記念シンポジウム「IFOがつなぐ京大微生物学のフロントライン」
• [Presentation] 細菌の翻訳途上鎖による翻訳アレストを介したmRNA分解機構の解明 (2019)
  • Author(s): 石井英治、秋山芳展、森 博幸
  • Organizer: 第42回日本分子生物学会年会
• [Presentation] 部位特異的in vivo光架橋法による大腸菌外膜タンパク質LptDの成熟過程の解析 (2019)
  • Author(s): 宮﨑亮次、渡邊哲朗、森 博幸、秋山芳展
  • Organizer: 第42回日本分子生物学会年会
• [Presentation] Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis. (2018)
  • Author(s): Mori, H., Sakashita, S., Ito, J., Ishii, E. and Akiyama Y.
  • Organizer: International symposium Proteins: From the Cradle to the Grave
• [Presentation] Analysis of cis and trans factors involved in export and translation arrest of VemP by an in vivo photo cross-linking approach. (2018)
  • Author(s): Miyazaki, R., Akiyama Y. and Mori, H.
  • Organizer: International symposium Proteins: From the Cradle to the Grave
• [Presentation] Roles of mRNA secondary structure of vemP-secD2 intergenic region in VemP translation arrest coupled SecD2 expression (2018)
  • Author(s): Ishii, E., Sakashita, S., Akiyama, Y. and Mori, H.
  • Organizer: International symposium Proteins: From the Cradle to the Grave
• [Presentation] SRP経路を介したVemPの膜透過装置への輸送と翻訳アレスト解除. (2018)
  • Author(s): 宮崎亮次、秋山芳展、森 博幸
  • Organizer: 第5回ribosome meeting
• [Presentation] Structure and Physiological Function of SecDF That Enhances Protein Export. (2017)
  • Author(s): Mori, H.
  • Organizer: International symposium 'Harmonized supermolecular motility machinery and its diversity'
  • Int'l Joint Research / Invited
• [Presentation] Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis. (2017)
  • Author(s): Mori, H., Sakashita, S., Ito, J., Ishii, E. and Akiyama, Y.
  • Organizer: 日本生物物理学会第55回年会 シンポジウム「実験と理論計算で明らかになってきた細胞環境での蛋白質間相互作用」
  • Invited
• [Presentation] Protein translocation motor SecDF. (2017)
  • Author(s): Tsukazaki, T., Furukawa, A., Yoshikaie, K., Mori, T., Mori, H., Morimoto, V. Y., Sugano, T., Iwaki, S., Minamino, T., Sugita, Y. and Tanaka, T
  • Organizer: 日本生物物理学会第55回年会 シンポジウム「構造生命科学の新しい潮流」
  • Invited
• [Presentation] Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of a marine bacterium, Vibrio alginolyticus. (2017)
  • Author(s): Mori, H.
  • Organizer: Commemorative symposium for 33rd international prize for biology 'Marine biology opens a frontier for the future'.
  • Int'l Joint Research / Invited