Structure of the Vacuolar H + -ATPase Rotary Motor Reveals New Mechanistic Insights

Abstract

SummaryVacuolar H+-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases.

Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector

“…Upon reconstitution into DOPC lipid bilayers, we were able to generate two-dimensional crystals of the V o , to our knowledge the first two-dimensional crystals of any eukaryotic V-ATPase proton channel sector. Projection maps calculated at a resolution of ϳ24 Å show a ring-like structure with an asymmetric mass bound at the periphery of the ring, consistent with current structural models obtained for the holo V-ATPase from single-particle reconstructions (18,(31)(32)(33). Although the relatively small size of the crystals obtained so far has limited our ability to use cryo-electron crystallography for structural analysis, there are several features that are noteworthy at the current resolution.…”

Disorders of lysosomal acidification—The emerging role of v-ATPase in aging and neurodegenerative disease

“…Notably, this rate varies based on the pH of the compartment (Kettner et al, 2003). For more extensive information on the molecular biology of the v-ATPase, we refer readers to several comprehensive reviews (Cotter et al, 2015; Forgac, 2007; Marshansky et al, 2014; Masashi et al, 2010; Mindell, 2012; Muench et al, 2011; Rawson et al, 2015; Saroussi and Nelson, 2009a, b). Implicit in this complexity of the v-ATPase is the sizeable number of opportunities for v-ATPase function to be compromised in disease states.…”

Breaking up and making up: The secret life of the vacuolar H+‐ATPase

“…The early models not only confirmed the V‐ATPase's structural similarity to the related F‐ATP synthases, which was already evident from primary sequence analysis of the catalytic and proteolipid subunits, but the EM models also revealed the increased complexity of the V‐ATPase compared to its ATP synthase counterparts. Recently, advances in electron detector technology have allowed determination of cryoEM maps of insect and yeast V‐ATPase at subnanometer resolution . These maps revealed secondary structure elements in both the soluble V 1 and, for the first time, in the membrane embedded V o .…”