Role of Vma21p in Assembly and Transport of the Yeast Vacuolar ATPase

Abstract

The Saccharomyces cerevisiae vacuolar H+-ATPase (V-ATPase) is a multisubunit complex composed of a peripheral membrane sector (V1) responsible for ATP hydrolysis and an integral membrane sector (V0) required for proton translocation. Biogenesis of V0requires an endoplasmic reticulum (ER)-localized accessory factor, Vma21p. We found that in vma21Δ cells, the major proteolipid subunit of V0failed to interact with the 100-kDa V0subunit, Vph1p, indicating that Vma21p is necessary for V0assembly. Immunoprecipitation of Vma21p from wild-type membranes resulted in coimmunoprecipitation of all five V0subunits. Analysis of vmaΔ strains showed that binding of V0subunits to Vma21p was mediated by the proteolipid subunit Vma11p. Although Vma21p/proteolipid interactions were independent of Vph1p, Vma21p/Vph1p association was dependent on all other V0subunits, indicating that assembly of V0occurs in a defined sequence, with Vph1p recruitment into a Vma21p/proteolipid/Vma6p complex representing the final step. An in vitro assay for ER export was used to demonstrate preferential packaging of the fully assembled Vma21p/proteolipid/Vma6p/Vph1p complex into COPII-coated transport vesicles. Pulse-chase experiments showed that the interaction between Vma21p and V0was transient and that Vma21p/V0dissociation was concomitant with V0/V1assembly. Blocking ER export in vivo stabilized the interaction between Vma21p and V0and abrogated assembly of V0/V1. Although a Vma21p mutant lacking an ER-retrieval signal remained associated with V0in the vacuole, this interaction did not affect the assembly of vacuolar V0/V1complexes. We conclude that Vma21p is not involved in regulating the interaction between V0and V1sectors, but that it has a crucial role in coordinating the assembly of V0subunits and in escorting the assembled V0complex into ER-derived transport vesicles.