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

Vacuolar-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 V ₁ region and a membrane-embedded proton-translocating V _O region. V _O is assembled in the endoplasmic reticulum (ER) membrane and V ₁ is assembled in the cytosol. However, V ₁ binds V _O only after V _O is transported to the Golgi membrane, thereby preventing acidification of the ER. We isolated V _O complexes and subcomplexes from Saccharomyces cerevisiae bound 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 V _O while blocking premature binding of V ₁ . Vma21p, which contains an ER-retrieval motif, binds the V _O :Vma12-22p complex, ‘mature’ V _O , 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 V ₁ with V _O removes 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.