Structure of yeast RAVE bound to a partial V1complex

Abstract

AbstractVacuolar-type ATPases (V-ATPases) are membrane-embedded proton pumps that acidify intracellular compartments in almost all eukaryotic cells. Homologous with ATP synthases, these multi-subunit enzymes consist of a soluble catalytic V1subcomplex and a membrane-embedded proton-translocating VOsubcomplex. The V1and VOsubcomplexes can undergo reversible dissociation to regulate proton pumping, with reassociation of V1and VOrequiring the protein complex known as RAVE (regulator of theATPase ofvacuoles andendosomes). In the yeastSaccharomyces cerevisiae, RAVE consists of subunits Rav1p, Rav2p, and Skp1p. We used electron cryomicroscopy (cryo-EM) to determine a structure of yeast RAVE bound to V1. In the structure, RAVE is a L-shaped complex with Rav2p pointing toward the membrane and Skp1p distant from both the membrane and V1. Only Rav1p interacts with V1, binding to a region of subunit A not found in the corresponding ATP synthase subunit. When bound to RAVE, V1is in a rotational state suitable for binding the free VOcomplex, but it is partially disrupted in the structure, missing five of its 16 subunits. Other than these missing subunits and the conformation of the inhibitory subunit H, the V1complex with RAVE appears poised for reassembly with VO.