A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients

Organelles like lysosomes and synaptic vesicles are acidified by V-ATPases, which consist of a cytosolically-oriented V1 complex that hydrolyzes ATP and a membrane-embedded VO complex that pumps protons. In yeast, V1-VO association is facilitated by the RAVE complex, but how higher eukaryotes assemble V-ATPases remains unclear. Here, we identify a metazoan RAVE complex (mRAVE) whose structure and composition are notably divergent from the ancestral counterpart. mRAVE consists of DMXL1 or DMXL2, WDR7, and the central linker ROGDI. DMXL1/2 interacts with subunits A and D of the inactive, isolated V1. Upon dissipation of proton gradients, mRAVE binds to V1 and VO, forming a supercomplex on the membrane. mRAVE then catalyzes V1-VO assembly, enabling lysosomal acidification, neurotransmitter loading into vesicles, and ATG16L1 recruitment for LC3/ATG8 conjugation onto single-membranes (CASM). Our findings provide a molecular basis for neurological disorders caused by mRAVE mutations.