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J Mol Biol. 2014 Jan 23;426(2):286-300. doi: 10.1016/j.jmb.2013.09.018. Epub 2013 Sep 27.

Subunit positioning and stator filament stiffness in regulation and power transmission in the V1 motor of the Manduca sexta V-ATPase.

Author information

  • 1School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
  • 2MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
  • 3Abteilung Tierphysiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, 49069 Osnabrück, Germany.
  • 4School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
  • 5School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Electronic address: M.A.Harrison@leeds.ac.uk.

Abstract

The vacuolar H(+)-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V1 domain is an ATPase, normally coupled to membrane-bound proton pump Vo via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood. Low energy status can trigger release of V1 from the membrane and curtail ATP hydrolysis. To investigate the molecular basis for these processes, we have carried out cryo-electron microscopy three-dimensional reconstruction of deactivated V1 from Manduca sexta. In the resulting model, three peripheral stalks that are parts of the mechanical stator of the V-ATPase are clearly resolved as unsupported filaments in the same conformations as in the holoenzyme. They are likely therefore to have inherent stiffness consistent with a role as flexible rods in buffering elastic power transmission between the domains of the V-ATPase. Inactivated V1 adopted a homogeneous resting state with one open active site adjacent to the stator filament normally linked to the H subunit. Although present at 1:1 stoichiometry with V1, both recombinant subunit C reconstituted with V1 and its endogenous subunit H were poorly resolved in three-dimensional reconstructions, suggesting structural heterogeneity in the region at the base of V1 that could indicate positional variability. If the position of H can vary, existing mechanistic models of deactivation in which it binds to and locks the axle of the V-ATPase rotary motor would need to be re-evaluated.

© 2013. Published by Elsevier Ltd. All rights reserved.

KEYWORDS:

3D; EM; H(+)-ATPase; cryo-electron microscopy; electron microscopy; three dimensional; vacuolar membrane

PMID:
24075871
[PubMed - indexed for MEDLINE]
PMCID:
PMC3899036
Free PMC Article
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