膜タンパク質複合体の成熟化過程をin vivo活写する

• Project Area: Novel measurement techniques for visualizing 'live' protein molecules at work
• Project/Area Number: 17H05879
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Complex systems
• 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: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2018: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2017: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
• Keywords: In vivo光架橋実験 / PiXie / VemP / SecDF / LptDE / pBPA / タンパク質間相互作用 / in vivo光架橋法 / パルスチェイス実験 / 新生鎖 / pulse-chase / SecD/F / LtD/E / PhoA / 蛋白質

Outline of Annual Research Achievements

細胞質外で働くタンパク質は、リボソームによる合成→細胞質膜へのtargeting→膜透過→folding/assemblyと言った複数の過程を経て成熟化する。生細胞内では、個々の反応は独立にシークエンシャルに起こるのではなく、複数の過程が部分的に重複しながら協調的に進む。反応を素過程に分けて解析するin vitro実験系では、タンパク質成熟化の全体像を正しく理解することは難しい。それ故、タンパク質の成熟過程をin vivoで直接追跡し評価できる実験系を構築する必要がある。我々は、膜タンパク質SecYを用いた解析を通して、「部位特異的in vivo光架橋法が、一過的かつ動的なタンパク質間相互作用を高い空間分解能で検出できる優れた方法である。」ことを明らかにした。本研究では、「部位特異的in vivo光架橋実験」を「pulse-chase実験」と組み合わせ、生細胞内で秒単位のタンパク質間相互作用変化を検出・追跡できる高時間・高空間分解能を持つ実験システム (PiXie法と命名）を構築し、in vivoでのタンパク質の成熟化を含めた動態解析を行うことを目指す。昨年の研究により、超強力UV照射装置SP-9（ウシオ電機製）を用いることにより、僅か１秒のUV照射で効率良い架橋形成が可能であることを明らかにし、PiXie法を用いた解析により膜タンパク質のfolding/assembly過程を追跡できることを示した。本年度は、分泌タンパク質VemP新生鎖の成熟化と局在化を本手法を用いて追跡し、成熟化の過程で生じる細胞内因子との架橋形成に成功した。本実験手法の有効性が強く示唆された。更に、部位特異的in vivo光架橋法とPiXie法を用いた研究をまとめたreviewを執筆した。

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] 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] 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] Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of a marine bacterium, Vibrio alginolyticus. (2018)
  • 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] 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] 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