SITE-DIRECTED MUTAGENESIS OF THE YEAST V-ATPASE B-SUBUNIT (VMA2P)

The B subunit of the vacuolar (H+)-ATPase (V-ATPase) has previously be en shown to participate in nucleotide binding and to possess significa nt sequence homology with the alpha subunit of the mitochondrial F-ATP ase, which forms the major portion of the noncatalytic nucleotide bind ing sites and contributes several residues to the catalytic sites of t his complex. Based upon the recent x-ray structure of the mitochondria l F-1 ATPase (Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), site-directed mutagenesis of the ye ast VMA2 gene has been carried out in a strain containing a deletion o f this gene. VMA2 encodes the yeast V-ATPase B subunit (Vma2p). Mutati ons at two residues postulated to be contributed by Vma2p to the catal ytic site (R381S and Y352S) resulted in a complete loss of ATPase acti vity and proton transport, with the former having a partial effect on V-ATPase assembly. Interestingly, substitution of Phe for Tyr-352 had only minor effects on activity (15-30% inhibition), suggesting the req uirement for an aromatic ring at this position. Alteration of Tyr-370, which is postulated to be near the adenine binding pocket; at the non catalytic sites, to Arg, Phe, or Ser caused a 30-50% inhibition of pro ton transport and ATPase activity, suggesting that an aromatic ring is not essential at this position. Finally, mutagenesis of residues in t he region corresponding to the P-loop of the alpha subunit (H180K; H18 0G, H180D, N181V) also inhibited proton transport and ATPase activity by approximately 30-50%. None of the mutations in either the putative adenine binding pocket nor the P-loop region had any effect on the abi lity of Vma2p to correctly fold nor on the V-ATPase to correctly assem ble. The significance of these results for the structure and function of the nucleotide binding sites on the B subunit is discussed.