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

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Shirouzu, Mikako Saijo, Shinya Toyama, Mitsutoshi Yabuki-miyata, Yukako Unzai, Satoru Yamato, Ichiro Mizutani, Kenji Terada, Takaho Suno-ikeda, Chiyo Murata, Takeshi Kobayashi, Takuya Ito, Kazuki Yokoyama, Shigeyuki Iwata, So Kakinuma, Yoshimi Yamamoto, Misaki
Description	Author Affiliation: Yamamoto M ( Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan.)
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 (NtpI(Nterm)) 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. NtpI(Nterm) directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI(Nterm) bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI(Nterm) and NtpF. Purified NtpE-F-I(Nterm) 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.
ISSN	00219258
e-ISSN	1083351X
Journal	Journal of Biological Chemistry
Issue Number	28
Volume Number	283
Language	English
Publisher	American Society for Biochemistry and Molecular Biology (United States)
Publisher Date	2008-07-11
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Bacterial Proteins Metabolism Enterococcus Enzymology Vacuolar Proton-Translocating ATPases Chemistry Genetics Hydrophobic And Hydrophilic Interactions Protein Binding Physiology Protein Structure, Quaternary Protein Structure, Tertiary Protein Subunits Research Support, Non-U.S. Gov't Biochemistry Molecular Biology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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