Genetic approach on rotor and stator subunit interaction of sodium-tranlocating V-ATPase complex

• Project/Area Number: 24570162
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Functional biochemistry
• Research Institution: Ehime University
• Principal Investigator: KAKINUMA YOSHIMI 愛媛大学, 農学部, 教授 (80134394)
• Co-Investigator(Renkei-kenkyūsha): MURATA Takeshi 千葉大学, 理学部, 教授 (80415322)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000); Fiscal Year 2014: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2012: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: ナトリウム / ＶーＡＴＰアーゼ / 腸球菌 / イオン / サブユニット / 回転酵素 / 分子構造 / メカニズム / V-ATPアーゼ

Outline of Final Research Achievements

Enterococcus hirae V-ATPase is an ion-transporting rotary enzyme that consists of nine kinds of subunits. In order to understand the molecular mechanism of this enzyme, it is essential to clarify the overall architecture and the interaction among these subunits. In this study, we succeeded in X-ray crystal structure analysis of the catalytic headpiece (V1-ATPase) and present ATP-hydrolytic rotatory mechanism of V1-ATPase. Furthermore, based on biochemical and surface plasmon resonance analyses on reconstitution of V1-ATPase complex and mutagenesis of V0 rotor ring subunit, the features of interaction among rotor and stator subunits of V-ATPase were revealed.

Research Products

• [Journal Article] Mutant LV(476-7)AA of A-subunit of Enterococcus hirae V1-ATPase: High affinity of A3B3 complex to DF axis and low ATPase activity (2013)
  • Author(s): Alam J, Yamato I, Arai S, Saijo S, Mizutani K, Ishizuka-Katsura Y, Ohsawa N, Terada T, Shirouzu M, Yokoyama S, Iwata S, Kakinuma Y, Murata T.
  • Journal Title: Springerplus Volume: 2 Issue: 1 Pages: 689-695
  • DOI: 10.1186/2193-1801-2-689
  • Peer Reviewed
• [Journal Article] Loose Binding of the DF Axis with the A 3B3 Complex Stimulates the Initial Activity of Enterococcus hirae V 1-ATPase (2013)
  • Author(s): Md. Jahangir Alam, Satoshi Arai, Shinya Saijo, Kano Suzuki, Kenji Mizutani, Yoshiko Ishizuka-Katsura, Noboru Ohsawa, Takaho Terada, Mikako Shirouzu, Shigeyuki Yokoyama, So Iwata, Yoshimi Kakinuma, Ichiro Yamato and Takeshi Murata
  • Journal Title: PLoS ONE Volume: 8 Issue: 9 Pages: e74291-e74291
  • DOI: 10.1371/journal.pone.0074291
  • Peer Reviewed
• [Journal Article] Rotation mechanism of Enterococcus hirae V 1-ATPase based on asymmetric crystal structures (2013)
  • Author(s): Satoshi Arai, Shinya Saijo, Kano Suzuki, Kenji Mizutani, Yoshimi Kakinuma, Yoshiko Ishizuka-Katsura, Noboru, Ohsawa, Takaho Terada, Mikako Shirouzu, Shigeyuki Yokoyama, So Iwata, Ichiro Yamato and Takeshi Murata
  • Journal Title: Nature Volume: 493 Issue: 7434 Pages: 703-707
  • DOI: 10.1038/nature11778
  • Peer Reviewed
• [Journal Article] Mutagenesis of the residues forming an ion binding pocket of the NtpK subunit of Enterococcus hirae V-ATPase (2012)
  • Author(s): Miyuki Kawano-Kawada
  • Journal Title: J. Bacteriol. Volume: 194 Issue: 17 Pages: 4546-4549
  • DOI: 10.1128/jb.00714-12
  • Peer Reviewed
• [Presentation] 腸内連鎖球菌V型ATPase遺伝子群による大腸菌Na+/H+アンチポーター変異、F-ATPase変異の相補 (2014)
  • Author(s): 稲辺 宏輔, 山本 三沙岐, 柿沼 喜己, 村田 武士, 柿沼 喜己, 山登 一郎
  • Organizer: 第40回日本生体エネルギー研究会
  • Place of Presentation: 愛媛大学
  • Year and Date: 2014-12-11 – 2014-12-13
• [Presentation] 腸内連鎖球菌V型ATPaseの膜内在性サブユニットの精製条件の検討 (2014)
  • Author(s): 稲辺 宏輔, 山本 三沙岐, 柿沼 喜己, 村田 武士, 山登 一郎
  • Organizer: 第87回日本生化学会大会
  • Place of Presentation: 国立京都国際会館
  • Year and Date: 2014-10-15 – 2014-10-18
• [Presentation] 腸球菌Na+輸送性V-ATPaseのイオン結合部位NtpK(E139D)抑圧変異株の単離 (2014)
  • Author(s): 河田 美幸, 高橋 寛子, 五十嵐 一衛, 山登 一郎, 村田 武士, 柿沼 喜己
  • Organizer: 第87回日本生化学会大会発表
  • Place of Presentation: 国立京都国際会館
  • Year and Date: 2014-10-15 – 2014-10-18
• [Presentation] リン酸結合型V1-ATPaseのX線結晶構造解析 (2013)
  • Author(s): 鈴木 花野, 水谷 健二, 石塚 芳子, 寺田 貴帆, 白水 美香子, 柿沼 喜己, 岩田 想, 横山 茂之, 山登 一郎, 村田 武士
  • Organizer: 第86回日本生化学会大会発表
  • Place of Presentation: パシフィコ横浜
• [Presentation] 腸内連鎖球菌V-ATPase全体の大腸菌内発現と精製 (2013)
  • Author(s): 松戸 翔平, Suhaila Rahman, 西條 慎也, 山本 三沙岐, 柿沼 喜己, 水谷 健二, 村田 武士, 山登 一郎
  • Organizer: 第86回日本生化学会大会発表
  • Place of Presentation: パシフィコ横浜