Subunit positioning and stator filament stiffness in regulation and power transmission in the V1 motor of the Manduca sexta V-ATPase.
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.
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Stephen P
SP
Muench
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
Sjors H W
SH
Scheres
Abteilung Tierphysiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, 49069 Osnabrück, Germany.
Markus
M
Huss
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Clair
C
Phillips
Abteilung Tierphysiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, 49069 Osnabrück, Germany.
Olga
O
Vitavska
Abteilung Tierphysiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, 49069 Osnabrück, Germany.
Helmut
H
Wieczorek
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
John
J
Trinick
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Electronic address: M.A.Harrison@leeds.ac.uk.
Michael A
MA
Harrison
D000818:Animals; D020285:Cryoelectron Microscopy; D021621:Imaging, Three-Dimensional; D019476:Insect Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D018402:Manduca / Q000201:enzymology*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism* / Q000648:ultrastructure*
D019476:Insect Proteins; D021122:Protein Subunits; D025262:Vacuolar Proton-Translocating ATPases
3D; EM; H(+)-ATPase; cryo-electron microscopy; electron microscopy; three dimensional; vacuolar membrane