Subunit Positioning and Stator Filament Stiffness in Regulation and Power Transmission in the V1 Motor of the Manduca sexta V-ATPase

被引:18
作者
Muench, Stephen P. [1 ]
Scheres, Sjors H. W. [2 ]
Huss, Markus [3 ]
Phillips, Clair [1 ]
Vitavska, Olga [3 ]
Wieczorek, Helmut [3 ]
Trinick, John [4 ]
Harrison, Michael A. [1 ]
机构
[1] Univ Leeds, Fac Biol Sci, Sch Biomed Sci, Leeds LS2 9JT, W Yorkshire, England
[2] MRC, Mol Biol Lab, Cambridge CB2 0QH, England
[3] Univ Osnabruck, Fachbereich Biol Chem, Abt Tierphysiol, D-49069 Osnabruck, Germany
[4] Univ Leeds, Fac Biol Sci, Sch Mol & Cellular Biol, Leeds LS2 9JT, W Yorkshire, England
基金
英国生物技术与生命科学研究理事会;
关键词
vacuolar membrane; H plus -ATPase; cryo-electron microscopy; VACUOLAR H+-ATPASE; CRYSTAL-STRUCTURE; PERIPHERAL STALK; ELECTRON-MICROSCOPY; EPSILON-SUBUNIT; SYNTHASE; V-1-ATPASE; PURIFICATION; DISSOCIATION; GENERATION;
D O I
10.1016/j.jmb.2013.09.018
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V-1 domain is an ATPase, normally coupled to membrane-bound proton pump V, via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood. Low energy status can trigger release of V-1 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 V-1 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 V-1 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 V-1, both recombinant subunit C reconstituted with V-1 and its endogenous subunit H were poorly resolved in three-dimensional reconstructions, suggesting structural heterogeneity in the region at the base of V-1 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. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
引用
收藏
页码:286 / 300
页数:15
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