THE SACCHAROMYCES-CEREVISIAE VMA6 GENE ENCODES THE 36-KDA SUBUNIT OF THE VACUOLAR H-ATPASE MEMBRANE SECTOR()

The yeast vacuolar membrane proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral catalytic, and integral membrane domains. At least eight proteins cofractionate with purified preparations of the enzyme including 100-, 69-, 60-, 42-, 36-, 32-, 2 7-, and 17-kDa polypeptides (Kane, P. M., Yamashiro, C. T., and Steven s, T. H. (1989a) J. Biol. Chem. 264, 19236-19244). We took a reverse g enetic approach to clone the structural gene for the 36-kDa subunit of the V-ATPase, VMA6. vma6 null mutants displayed growth characteristic s typical of other vma mutants including sensitivity to media buffered at neutral pH or media containing 100 mM Ca2+. Vacuolar acidification was defective in vma6 cells and isolated vacuolar membrane preparatio ns contained no detectable V-ATPase activity. The VMA6 gene encodes a hydrophilic polypeptide of 345 amino acids (predicted molecular mass 3 9.8-kDa). We present evidence that the VMA6 gene product (Vma6p) is a non-integral membrane component of the membrane pore domain and is req uired for V-ATPase complex assembly. Vma6p was removed from wild type vacuolar membranes by strong chaotropic agents such as alkaline Na2CO3 or 5M urea, which did not remove integral membrane polypeptides. In y east cells lacking the integral membrane portion of the V-ATPase compl ex, Vma6p was unable to stably associate with vacuolar membranes. Conv ersely, in mutants specifically lacking Vma6p, components of the V-ATP ase integral membrane domain were destabilized, and peripheral subunit s failed to assemble onto vacuolar membranes. These results are discus sed in the context of a developing model for V-ATPase assembly in yeas t.