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

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Abstract
The structure of the VO subcomplex of yeast V-ATPase, solved by electron cryomicroscopy, reveals a new subunit and suggests a mechanism for the translocation of protons across membranes. The cell powers many cellular processes by pumping protons through membrane-embedded rotary ATPases. It has been difficult to determine the structure of a physiological ATPase complex, given the membrane association. John Rubinstein and colleagues have now solved this problem, and determine the structure of the VO complex of the yeast vacuolar ATPase within a membrane. At approximately 3.9 Å, the resolution of the structure is sufficient to permit the authors to propose a previously unidentified subunit. 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 V1 region drives proton translocation through the membrane-embedded VO 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 ATPase1,2,3,4,5. We induced dissociation and auto-inhibition of the V1 and VO regions of the V-ATPase by starving the yeast Saccharomyces cerevisiae6,7, allowing us to obtain a ~3.9-Å 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 ac8c′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.

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