新生鎖VemPの翻訳伸長停止／解除機構の解明

• Project Area: Nascent-chain Biology
• Project/Area Number: 17H05666
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kyoto University
• Principal Investigator: 森 博幸 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 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥12,610,000 (Direct Cost: ¥9,700,000、Indirect Cost: ¥2,910,000); Fiscal Year 2018: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2017: ¥6,110,000 (Direct Cost: ¥4,700,000、Indirect Cost: ¥1,410,000)
• Keywords: VemP / SecDF / タンパク質膜透過 / ビブリオ菌 / 翻訳伸長停止 / 翻訳停止 / 発現制御 / アレストモチーフ / 蛋白質 / 細菌

Outline of Annual Research Achievements

Vibrio菌は、2種類のSecDFパラログを保持しており、高Na+環境下ではNa+駆動型SecDF1のみを利用するが、低Na+条件下ではプロトン駆動型のSecDF2を特異的に発現誘導し、タンパク質の膜透過能を維持する機構を持つ。VemP (Vibrio protein export monitoring polypeptide)は、159アミノ酸残基からなる分泌タンパク質であり、上記の発現制御機構に必須の役割を持つ。VemPは、菌の膜透過活性をモニターし、活性が低下した際には自身の翻訳伸長をC末端近傍で安定に停止させ、同一オペロン下流に位置するsecDF2の発現量を増加させる。この翻訳伸長の停止は、膜透過が正常な時は一過的であり速やかに解除される。本研究では、１）VemPの翻訳停止能を簡便に評価できるレポーターシステムを構築し、VemPのC末端高度保存領域の徹底的な変異解析を通してVemP翻訳アレストモチーフを同定し、翻訳停止の分子機構について考察した。２）VemPの翻訳停止／解除の分子機構を明らかにすることを目指して、VemPがリボソームで合成された後、翻訳停止／解除の過程を経て、膜透過が完了するまでに生じる他のタンパク質因子との一過的な相互作用を、最近我々が開発したPiXie法（Pulse-chase and in vivo X-linking experiment)を用いて解析した。相互作用因子の同定、変異株を用いたin vivo解析から、i) VemPはSRP経路を介して膜透過装置まで標的化されること、ii) 翻訳アレストの解除は、シグナル配列が切断を受ける状態まで膜透過反応が進行した後に初めて起こることを見出した。これらの知見は、VemPの翻訳停止を解除する分子機構の解明の大きな手がかりになると期待される。

Research Progress Status

平成30年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

平成30年度が最終年度であるため、記入しない。

Research Products

• [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] 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] 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] 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] 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 on “Proteins; from the Cradle to the Grave”
  • Int'l Joint Research
• [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 on “Proteins; from the Cradle to the Grave”
  • Int'l Joint Research
• [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 on “Proteins; from the Cradle to the Grave”
  • Int'l Joint Research
• [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] 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
• [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