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Elucidation of rotary ion transport mechanism of V-ATPase by visualization and control of substrate

Research Project

Project/Area Number 16K14706
Research Category

Grant-in-Aid for Challenging Exploratory Research

Allocation TypeMulti-year Fund
Research Field Biophysics
Research InstitutionThe University of Tokyo

Principal Investigator

Ueno Hiroshi  東京大学, 大学院工学系研究科(工学部), 助教 (10546592)

Research Collaborator MURATA takeshi  
NOJI hiroyuki  
Project Period (FY) 2016-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000)
Fiscal Year 2018: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2016: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Keywords分子モーター / 1分子計測 / ATPase / 1分子計測・操作 / イオン輸送
Outline of Final Research Achievements

In this study, we optimized the expression and purification system of Enterococcus hirae V-ATPase (E. hirae VoV1) using sodium ion as a transport substrate to obtain the highly active E. hirae VoV1. We succeeded in constructing an expression system / purification system that can stably obtain highly active E. hirae VoV1 and also succeeded in conducting the structural analysis of whole E. hirae VoV1 by electron microscopy using this sample. Furthermore, we detected ATP-driven rotation of E. hirae VoV1 with high time resolution (up to microsecond). E. hirae VoV1 rotated slowly when the external sodium concentration was decreased and showed multiple rotation steps reflecting sodium binding at Vo portion.

Academic Significance and Societal Importance of the Research Achievements

V-ATPaseはATP加水分解のエネルギーを用いてイオン(プロトンやナトリウム)を輸送する分子機械である。このV-ATPaseは膜小胞の酸性化や破骨細胞による骨吸収や癌細胞の転移にも関わることから創薬のターゲットとしても注目されている。そのためこの分子の回転イオン輸送を理解することは医学的にも重要であり、今回の研究がきっかけで明らかになったこのV-ATPaseの全体構造はこの分子をターゲットとした創薬につながると期待される。さらに今回明らかになったナトリウムイオンの結合を反映した回転挙動の検出は、回転によってイオン輸送を行う分子機械の理解につながり学術的にも重要な結果だと言える。

Report

(4 results)
  • 2018 Annual Research Report   Final Research Report ( PDF )
  • 2017 Research-status Report
  • 2016 Research-status Report
  • Research Products

    (32 results)

All 2018 2017 2016

All Journal Article (6 results) (of which Int'l Joint Research: 3 results,  Peer Reviewed: 6 results,  Open Access: 6 results,  Acknowledgement Compliant: 1 results) Presentation (25 results) (of which Int'l Joint Research: 2 results,  Invited: 1 results) Patent(Industrial Property Rights) (1 results)

  • [Journal Article] Off-axis rotor in Enterococcus hirae V-ATPase visualized by Zernike phase plate single-particle cryo-electron microscopy2018

    • Author(s)
      Tsunoda Jun、Song Chihong、Imai Fabiana Lica、Takagi Junichi、Ueno Hiroshi、Murata Takeshi、Iino Ryota、Murata Kazuyoshi
    • Journal Title

      Scientific Reports

      Volume: 8 Issue: 1 Pages: 15632-15632

    • DOI

      10.1038/s41598-018-33977-9

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Structure and dynamics of rotary V1 motor2018

    • Author(s)
      Ueno Hiroshi、Suzuki Kano、Murata Takeshi
    • Journal Title

      Cellular and Molecular Life Sciences

      Volume: 75 Issue: 10 Pages: 1789-1802

    • DOI

      10.1007/s00018-018-2758-3

    • Related Report
      2018 Annual Research Report 2017 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Catalytic robustness and torque generation of the F1-ATPase2017

    • Author(s)
      Hiroyuki Noji, Hiroshi Ueno and Duncan G. G. McMillan
    • Journal Title

      Biophysical reviews

      Volume: 9 Issue: 2 Pages: 103-103

    • DOI

      10.1007/s12551-017-0262-x

    • Related Report
      2016 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Direct real-time detection of single proteins using silicon nanowire-based electrical circuits2016

    • Author(s)
      Jie Li, Gen He, Hiroshi Ueno, Chuancheng Jia, Hiroyuki Noji, Chuanmin Qi, Xuefeng Guo
    • Journal Title

      Nanoscale

      Volume: 8 Issue: 36 Pages: 16172-16176

    • DOI

      10.1039/c6nr04103e

    • Related Report
      2016 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Rotation of artificial rotor axles in rotary molecular motors2016

    • Author(s)
      Baba M, Iwamoto K, Iino R, Ueno H, Hara M, Nakanishia A, Kishikawa J, Noji H, Yokoyama K
    • Journal Title

      Proc Natl Acad Sci USA

      Volume: 113 Issue: 40 Pages: 11214-11219

    • DOI

      10.1073/pnas.1605640113

    • Related Report
      2016 Research-status Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Biophysical Characterization of a Thermoalkaliphilic Molecular Motor with a High Stepping Torque Gives Insight into Evolutionary ATP Synthase Adaptation2016

    • Author(s)
      Duncan G. G. McMillan, Rikiya Watanabe, Hiroshi Ueno, Gregory M. Cook, and Hiroyuki Noji
    • Journal Title

      Journal of Biological Chemistry

      Volume: 291 Issue: 46 Pages: 23965-23977

    • DOI

      10.1074/jbc.m116.743633

    • Related Report
      2016 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] Rotation of Fusobacterium nucleatum F1-ATPase2018

    • Author(s)
      Hiroshi Ueno
    • Organizer
      Bilateral joint research project meeting in Otago
    • Related Report
      2018 Annual Research Report
  • [Presentation] Rotation of the engineered F1-ATPase with non-catalytic α-type P-loops2018

    • Author(s)
      Hiroshi Ueno, Rie Koga, Tomoko Masaike, Nobuyasu Koga, Hiroyuki Noji
    • Organizer
      20th European Bioenergetics Conference
    • Related Report
      2018 Annual Research Report
  • [Presentation] Single-molecule analysis of bovine mitochondrial F1-ATPase for direct assignment of crystal structures and rotational pausing states2018

    • Author(s)
      Ryohei Kobayashi, Hiroshi Ueno, Toshiharu Suzuki, Mayu Hara, Hiroyuki Noji
    • Organizer
      第56回日本生物物理学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Rotation of hybrid F1-ATPases between bacterial and mammalian ones2018

    • Author(s)
      Ryo Watanabe, Hiroshi Ueno, Toshiharu Suzuki, Ryohei Kobayashi, Hiroyuki Noji
    • Organizer
      第56回日本生物物理学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Rotational torque and kinetics of F1-ATPase containing the catalytic subunit with a non-catalytic hinge2018

    • Author(s)
      Tomoyasu Sato, Hiroshi Ueno, Kumiko Hayashi, Rie Koga, Nobuyasu Koga, Hiroyuki Noji, Tomoko Masaike
    • Organizer
      第56回日本生物物理学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Single Particle Analysis of EhV-ATPase by Phase-Plate electron cryo-microscopy2018

    • Author(s)
      Jun Tsunoda, Chihong Song, Yakushiji Fabiana Lica, Takeshi Murata, Hiroshi Ueno, Naoyuki Miyazaki, Kenji Iwasaki, Junichi Takagi, Ryota Iino, Kazuyoshi Murata
    • Organizer
      第56回日本生物物理学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Impact of the sequence difference of P-loop on the conformational changes of F1-ATPase2018

    • Author(s)
      Rie Koga, Hiroshi Ueno, Tomoko Masaike, Hiroyuki Noji, Nobuyasu Koga
    • Organizer
      第56回日本生物物理学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] On the timing of phosphate release in the ATPase reaction by TF12018

    • Author(s)
      Eiro Muneyuki, Yohei Nakayama, Shoichi Toyabe, Hiroshi Ueno
    • Organizer
      第56回日本生物物理学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Rotation of the engineered F1-ATPase with non-catalytic α-type P-loops2018

    • Author(s)
      Hiroshi Ueno, Rie Koga, Tomoko Masaike, Nobuyasu Koga, Hiroyuki Noji
    • Organizer
      第56回日本生物物理学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Chemomechanical Coupling of the Paracoccus denitrificans F1-ATPase2018

    • Author(s)
      Mariel Zarco - Zavala, Duncan G.G McMillan, Suzuki Toshiharu, Hiroshi Ueno, Rikiya Watanabe, Francisco Mendoza - Hoffmann, Jose J. Garcia-Trejo, Hiroyuki Noji
    • Organizer
      第56回日本生物物理学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Rotation mechanisms of rotary ATPases2018

    • Author(s)
      Hiroshi Ueno
    • Organizer
      Complex systems and Biological physics seminar
    • Related Report
      2018 Annual Research Report
  • [Presentation] Rotation of the engineered F1-ATPase with alpha-type P-loop on catalytic beta subunit2017

    • Author(s)
      Hiroshi Ueno
    • Organizer
      第55回日本生物物理学会年会
    • Related Report
      2017 Research-status Report
  • [Presentation] Single-molecule imaging of rotation of bovine mitochondrial F1-ATPase2017

    • Author(s)
      Hiroshi Ueno
    • Organizer
      Gordon Research Conference, Bioenergetics
    • Related Report
      2017 Research-status Report
    • Int'l Joint Research
  • [Presentation] Probing the biophysical properties of a Thermoalkaliphilic F1 ATPase gives insight into adaptation and regulation2016

    • Author(s)
      Duncan G. G. McMillan, Rikiya Watanabe, Hiroshi Ueno, Gregory M. Cook, Hiroyuki Noji
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場(茨城県つくば市)
    • Year and Date
      2016-11-27
    • Related Report
      2016 Research-status Report
  • [Presentation] Fluorescence detection of the flagellar type III protein export using the inverted membrane vesicles2016

    • Author(s)
      Tsuyoshi Tono, Hiroyuki Terashima, Kazuhito Tabata, Hiroshi Ueno, Tomoki Matsuda, Takeharu Nagai, Hiroyuki Noji, Katsumi Imada
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場(茨城県つくば市)
    • Year and Date
      2016-11-27
    • Related Report
      2016 Research-status Report
  • [Presentation] Single Particle Analysis of EhV-ATPase by Phase-contrast cryo-Electron Microscopy2016

    • Author(s)
      Jun Tsunoda, Chihong Song, Fabiana Lica Yakushiji, Takeshi Murata, Hiroshi Ueno, Junichi Takagi, Ryota Iino, Kazuyoshi Murata
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場(茨城県つくば市)
    • Year and Date
      2016-11-27
    • Related Report
      2016 Research-status Report
  • [Presentation] Unveiling the chemomechanical coupling of F1 ATPase of Paracoccus denitrificans2016

    • Author(s)
      Mariel Zarco Zavala, Duncan G G McMillan, Toshiharu Suzuki, Hiroshi Ueno, Francisco Mendoza-Hoffmann, Jose J. Garcia-Trejo, Hiroyuki Noji
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場(茨城県つくば市)
    • Year and Date
      2016-11-27
    • Related Report
      2016 Research-status Report
  • [Presentation] Arginine finger mutant of Enterococcus hirae V1-ATPase shows unusual rotational behaviors2016

    • Author(s)
      Tatsuya Iida, Yoshihiro Minagawa, Hiroshi Ueno, Takeshi Murata, Ryota Iino
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場(茨城県つくば市)
    • Year and Date
      2016-11-27
    • Related Report
      2016 Research-status Report
  • [Presentation] Single-molecule simultaneous observation of rotation and nucleotide binding/release of Enterococcus hirae V1-ATPase2016

    • Author(s)
      Yoshihiro Minagawa, Hiroshi Ueno, Hiroyuki Noji, Takeshi Murata, Ryota Iino
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場(茨城県つくば市)
    • Year and Date
      2016-11-26
    • Related Report
      2016 Research-status Report
  • [Presentation] Measuring metabolic activities in single bacterial cells by Raman microspectroscopy2016

    • Author(s)
      Yota Kato, Hiroshi Ueno, Hiroyuki Noji
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場(茨城県つくば市)
    • Year and Date
      2016-11-26
    • Related Report
      2016 Research-status Report
  • [Presentation] Detection of enzymatic activity in femtliter droplets using micro-Raman spectroscopy2016

    • Author(s)
      Hironobu Yamashita, Kazuhito V Tabata, Hiroshi Ueno, Hiroyuki Noji
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場(茨城県つくば市)
    • Year and Date
      2016-11-26
    • Related Report
      2016 Research-status Report
  • [Presentation] The kinetics of Pi release in F1-ATPase investigated with P-loop mutations2016

    • Author(s)
      Hiroka Narita, Hitoshi Hoshina, Hikaru Yoshida, Yohei Nakayama, Shoichi Toyabe, Hiroshi Ueno, Eiro Muneyuki
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場(茨城県つくば市)
    • Year and Date
      2016-11-25
    • Related Report
      2016 Research-status Report
  • [Presentation] Identification of accelerators on Na+-depending ATPase activity of Enterococcus hirae V-ATPase2016

    • Author(s)
      Senka Gi, Lica Fabiana Yakushiji, Hiroshi Ueno, Hiroyuki Noji, Takayoshi Arai, Katsuhiko Moriyama, Hideo Togo, Takeshi Murata
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場(茨城県つくば市)
    • Year and Date
      2016-11-25
    • Related Report
      2016 Research-status Report
  • [Presentation] Measuring metabolic activities in single bacterial cells by Raman microspectroscopy2016

    • Author(s)
      Yota Kato, Hiroshi Ueno, Hiroyuki Noji
    • Organizer
      International Conference on Single Cell Research 2016
    • Place of Presentation
      Ito International Research Center, The University of Tokyo(東京都文京区)
    • Year and Date
      2016-11-17
    • Related Report
      2016 Research-status Report
    • Int'l Joint Research
  • [Presentation] 金ナノ粒子を用いた無負荷でのV1-ATPaseの回転とヌクレオチド結合解離の同時計測2016

    • Author(s)
      上野博史
    • Organizer
      蛋白研セミナー・第6回分子モーター討論会
    • Place of Presentation
      大阪大学蛋白質研究所(大阪府吹田市)
    • Year and Date
      2016-07-25
    • Related Report
      2016 Research-status Report
    • Invited
  • [Patent(Industrial Property Rights)] 蛍光観察装置2016

    • Inventor(s)
      野地博行・上野博史・皆川慶嘉
    • Industrial Property Rights Holder
      野地博行・上野博史・皆川慶嘉
    • Industrial Property Rights Type
      特許
    • Industrial Property Number
      2016-235614
    • Filing Date
      2016-12-05
    • Related Report
      2016 Research-status Report

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Published: 2016-04-21   Modified: 2020-03-30  

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