Atomic model for the membrane-embedded VO motor of a eukaryotic V-ATPase

Vacuolar-type ATPases (V-ATPases) are ATP-powered proton pumps involved in processes such as endocytosis, lysosomal degradation, secondary transport, TOR signalling, and osteoclast and kidney function. ATP hydrolysis in the soluble catalytic V-1 region drives proton translocation through the membrane-embedded V-O region via rotation of a rotor subcomplex. Variability in the structure of the intact enzyme has prevented construction of an atomic model for the membrane-embedded motor of any rotary ATPase(1-5). We induced dissociation and auto-inhibition of the V1 and VO regions of the V-ATPase by starving the yeast Saccharomyces cerevisiae(6,7), allowing us to obtain a resolution electron cryomicroscopy map of the VO complex and build atomic models for the majority of its subunits. The analysis reveals the structures of subunits ac(8)c'c '' de and a protein that we identify and propose to be a new subunit (subunit f). A large cavity between subunit a and the c-ring creates a cytoplasmic half-channel for protons. The c-ring has an asymmetric distribution of proton-carrying Glu residues, with the Glu residue of subunit c '' interacting with Arg735 of subunit a. The structure suggests sequential protonation and deprotonation of the c-ring, with ATP-hydrolysis-driven rotation causing protonation of a Glu residue at the cytoplasmic half-channel and subsequent deprotonation of a Glu residue at a luminal half-channel.