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

Organelles such as lysosomes and synaptic vesicles are acidified by V-ATPases, which consist of a cytosolically oriented V₁ complex that hydrolyzes ATP and a membrane-embedded V_O complex that pumps protons. In yeast, V₁-V_O association is facilitated by the RAVE (regulator of H⁺-ATPase of the vacuolar and endosomal membrane) 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 and DMXL2 interact with subunits A and D of the inactive, isolated V₁. On dissipation of proton gradients, mRAVE binds to V₁ and V_O, forming a supercomplex on the membrane. mRAVE then catalyzes V₁-V_O assembly, enabling lysosomal acidification, neurotransmitter loading into vesicles and ATG16L1 recruitment for LC3/ATG8 conjugation onto single membranes. Our findings provide a molecular basis for neurological disorders caused by mRAVE mutations.