Assembly of the Yeast Vacuolar H+-ATPase Occurs in the Endoplasmic Reticulum and Requires a Vma12p/Vma22p Assembly Complex

doi:10.1083/jcb.142.1.39

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© The Rockefeller University Press, 0021-9525/1998//39 $5.00
The Journal of Cell Biology, Volume 142, Number 1, , 1998 39-49

Articles

Assembly of the Yeast Vacuolar H⁺-ATPase Occurs in the Endoplasmic Reticulum and Requires a Vma12p/Vma22p Assembly Complex

Laurie A. Graham, Kathryn J. Hill, and Tom H. Stevens

Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

Three previously identified genes from Saccharomyces cerevisiae,VMA12, VMA21, and VMA22, encode proteins localized to the endoplasmicreticulum (ER). These three proteins are required for the biogenesisof a functional vacuolar ATPase (V-ATPase), but are not partof the final enzyme complex. Subcellular fractionation andchemical cross-linking studies have revealed that Vma12p andVma22p form a stable membrane associated complex. Cross-linkinganalysis also revealed a direct physical interaction betweenthe Vma12p/Vma22p assembly complex and Vph1p, the 100-kD integralmembrane subunit of the V-ATPase. The interaction of the Vma12p/Vma22pcomplex with Vph1p was transient (half-life of $~$ 5 min), reflecting trafficking of this V-ATPase subunit through the ER en routeto the vacuolar membrane. Analysis of these protein–proteininteractions in ER-blocked sec12 mutant cells indicated thatthe Vph1p-Vma12p/Vma22p interactions are quite stable when transportof the V-ATPase out of the ER is blocked. Fractionation of solubilizedmembrane proteins on a density gradient revealed comigrationof Vma22p and Vma12p, indicating that they form a complex evenin the absence of cross-linker. Vma12p and Vma22p migrated tofractions separate from Vma21p. Loss of Vph1p caused the Vma12p/Vma22pcomplex to sediment to less dense fractions, consistent withassociation of Vma12p/ Vma22p with nascent Vph1p in ER membranes.This is the first evidence for a dedicated assembly complexin the ER required for the assembly of an integral membraneprotein complex (V-ATPase) as it is transported through thesecretory pathway.

Key Words: vacuoles • vacuolar ATPase • endoplasmic reticulum • molecular chaperones • membrane proteins

Abbreviations used in this paper: ALP, alkaline phosphatase; CPY, carboxypeptidase Y; DAPI, 4',6'-diamidino-2-phenylindole; DSP, dithio-bis(succinimidylpropionate); V-ATPase, vacuolar proton-translocating ATPase; V₁, peripheral catalytic sector of the V-ATPase; V_o, integral membrane sector of the V-ATPase; VMA, vacuolar membrane ATPase; VPH, vacuolar pH; YEPD, 1% yeast extract, 2% peptone, and 2% dextrose.

Dedicated to the memory of Wesley C. Graham.

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