SITE-DIRECTED MUTAGENESIS OF THE YEAST V-ATPASE-A SUBUNIT

To investigate the function of residues at the catalytic nucleotide bi nding site of the V-ATPase, we have carried out site-directed mutagene sis of the VMA1 gene encoding the A subunit of the V-ATPase in yeast. Of the three cysteine residues that are conserved in all A subunits se quenced thus far, two (Cys(284) and Cys(539)) appear essential for cor rect folding or stability of the A subunit. Mutation of the third cyst eine (Cys(261)), located in the glycine-rich loop, to valine, generate d an enzyme that was fully active but resistant to inhibition by N-eth ylmalemide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and oxidation. To test the role of disulfide bond formation in regulation of vacuolar ac idification in vivo, we have also determined the effect of the C261V m utant on targeting and processing of the soluble vacuolar protein carb oxypeptidase Y. No difference in carboxypeptidase Y targeting or proce ssing is observed between the wild type and C261V mutant, suggesting t hat disulfide bond formation in the V-ATPase A subunit is not essentia l for controlling vacuolar acidification in the Golgi. In addition, fl uid phase endocytosis of Lucifer Yellow, quinacrine staining of acidic intracellular compartments and cell growth are indistinguishable in t he C261V and wild type cells. Mutation of G250D in the glycine-rich lo op also resulted in destabilization of the A subunit, whereas mutation of the lysine residue in this region (K263Q) gave a V-ATPase complex which showed normal levels of A subunit on the vacuolar membrane but w as unstable to detergent solubilization and isolation and was totally lacking in V-ATPase activity. By contrast, mutation of the acidic resi due, which has been postulated to play a direct catalytic role in the homologous F-ATPases (E286Q), had no effect on stability or assembly o f the V-ATPase complex, but also led to complete loss of V-ATPase acti vity. The E286Q mutant showed labeling by 2-azido-[P-32]ATP that was a pproximately 60% of that observed for wild type, suggesting that mutat ion of this glutamic acid residue affected primarily ATP hydrolysis ra ther than nucleotide binding.