Structure of human V-ATPase with the a2-isoform reveals a proton-release gate in the luminal half-channel

V-ATPases are ATP-dependent proton pumps that acidify intracellular organelles in eukaryotic cells. In mammals, the V0 a-subunit has four isoforms, a1-a4, with a2 enriched in the Golgi and early endosomes. Mutations in a2 are implicated in cutis laxa, male infertility and cancer. Here, we have used cryo-EM to determine the structures of human V-ATPase with the a2-isoform in both the disassembled (a2V0) and assembled (a2V-ATPase) states. We found that in vitro, a2V-ATPase disassembles more readily than a4 containing V-ATPase. The structure of a2V0 allows visualization of proposed lipid binding motifs, a unique luminal extension hypothesized to sense pH in early endosomes, and the location of missense mutations that cause cutis laxa. Most strikingly, the structure of a2 reveals a salt bridge impeding the luminal half-channel that is conserved in vertebrates, but not in yeast. We propose that this salt bridge functions as a gating mechanism to regulate the flow of protons, and thereby V-ATPase activity in higher eukaryotes.