Subunit Positioning and Stator Filament Stiffness in Regulation and Power Transmission in the V-1 Motor of the Manduca sexta V-ATPase

Abstract

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.