Structural basis of V-ATPase V_Oregion assembly by Vma12p, 21p, and 22p

AbstractVacuolar-type ATPases (V-ATPases) are rotary proton pumps that acidify specific intracellular compartments in almost all eukaryotic cells. These multi-subunit enzymes consist of a soluble catalytic V1region and a membrane-embedded proton-translocating VOregion. VOis assembled in the endoplasmic reticulum (ER) membrane and V1is assembled in the cytosol. However, V1binds VOonly after VOis transported to the Golgi membrane, thereby preventing acidification of the ER. We isolated VOcomplexes and subcomplexes fromSaccharomyces cerevisiaebound to V-ATPase assembly factors Vma12p, Vma21p, and Vma22p. Electron cryomicroscopy shows how the Vma12-22p complex recruits subunits a, e, and f to the rotor ring of VOwhile blocking premature binding of V1. Vma21p, which contains an ER-retrieval motif, binds the VO:Vma12-22p complex, ‘mature’ VO, and a complex that appears to contain a ring of loosely-packed rotor subunits and the proteins YAR027W and YAR028W. The structures suggest that Vma21p binds assembly intermediates that contain a rotor ring, and that activation of proton pumping following assembly of V1with VOremoves Vma21p, allowing V-ATPase to remain in the Golgi. Together, these structures show how Vma12-22p and Vma21p function in V-ATPase assembly and quality control, ensuring the enzyme acidifies only its intended cellular targets.