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Vol. 15, Issue 11, 5075-5091, November 2004

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

Per Malkus ^*, Laurie A. Graham , Tom H. Stevens , and Randy Schekman ^* 

^* Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; Institute of Molecular Biology, University of Oregon, Eugene, OR 97403

Submitted June 23, 2004; Accepted August 20, 2004
Monitoring Editor: Juan S. Bonifacino

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 V₀ requires an endoplasmic reticulum (ER)-localized accessory factor, Vma21p. We found that in vma21 cells, the major proteolipid subunit of V₀ failed to interact with the 100-kDa V₀ subunit, Vph1p, indicating that Vma21p is necessary for V₀ assembly. Immunoprecipitation of Vma21p from wild-type membranes resulted in coimmunoprecipitation of all five V₀ subunits. Analysis of vma strains showed that binding of V₀ subunits 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 V₀ subunits, indicating that assembly of V₀ occurs 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 V₀ was transient and that Vma21p/V₀ dissociation was concomitant with V₀/V₁ assembly. Blocking ER export in vivo stabilized the interaction between Vma21p and V₀ and abrogated assembly of V₀/V₁. Although a Vma21p mutant lacking an ER-retrieval signal remained associated with V₀ in the vacuole, this interaction did not affect the assembly of vacuolar V₀/V₁ complexes. We conclude that Vma21p is not involved in regulating the interaction between V₀ and V₁ sectors, but that it has a crucial role in coordinating the assembly of V₀ subunits and in escorting the assembled V₀ complex into ER-derived transport vesicles.

Abbreviations used: COP, coat protein complex; CPY, carboxypeptidase Y; gp-factor, glycosylated pro--factor; SIC, semi-intact cell; V-ATPase, vacuolar-type proton-translocating ATPase; VMA, vacuolar membrane ATPase.

Corresponding author. E-mail address: schekman{at}berkeley.edu.

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