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MBC in Press, published online ahead of print September 8, 2004
Mol. Biol. Cell 10.1091/mbc.E04-06-0514

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Submitted on June 23, 2004
Accepted on August 20, 2004

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

Per Malkus,* Laurie A. Graham,{dagger} Tom H. Stevens,{dagger} and Randy Schekman*{ddagger}

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

Monitoring Editor: Juan S. Bonifacino

The S. 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 ER-localized accessory factor, Vma21p. We found that in vma21{Delta} 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{Delta} strains showed that binding of V0 subunits to Vma21p was mediated by the proteolipid subunit Vma11p. Whereas 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.

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






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