Interaction and stoichiometry of the peripheral stalk subunits NtpE and NtpF and the N-terminal hydrophilic domain of NtpI of Enterococcus hirae V-ATPase

DOI: 10.1074/jbc.M801772200

Authors: Misaki Yamamoto (Tokyo University) , Satoru Unzai (Yokohama City University) , Shinya Saijo (SPring-8) , Kazuki Ito (RIKEN SPring-8 Center) , Kenji Mizutani (Kyoto University) , Chiyo Suno-Ikeda (Iwata Human Receptor Crystallography Project,) , Yukako Yabuki-Miyata (Protein Research Group, RIKEN Genomic Sciences Center) , Takaho Terada (Protein Research Group, RIKEN Genomic Sciences Center) , Mitsutoshi Toyama (Protein Research Group, RIKEN Genomic Sciences Center) , Mikako Shirouzu (Protein Research Group, RIKEN Genomic Sciences Center) , Takuya Kobayashi (Kyoto University) , Yoshimi Kakinuma (Ehime University) , Ichiro Yamato (Tokyo University) , Shigeyuki Yokoyama (Protein Research Group, RIKEN Genomic Sciences Center) , Takeshi Murata (Protein Research Group, RIKEN Genomic Sciences Center) , So Iwata (Yokohama City University; Kyoto University; Japan Science and Technology Agency)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 283 , PAGES 19422-19431

State: Published (Approved)
Published: May 2008

Abstract: The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpINterm) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpINterm directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpINterm bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpINterm and NtpF. Purified NtpE-F-INterm complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated “L” shape.

Journal Keywords: Membrane Transport; Structure; Function; Biogenesis

Diamond Keywords: Bacteria

Subject Areas: Biology and Bio-materials


Instruments: NONE-No attached Diamond beamline

Added On: 19/08/2009 23:07

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PIIS0021925820813969.pdf

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Structural biology Life Sciences & Biotech

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