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Mutation of G250D in the glycine-rich loop 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 was unstable to detergent solubilization and isolation and was totally lacking in V-ATPase activity. By contrast, mutation of the acidic residue, which has been postulated to play a direct catalytic role in the homologous F-ATPases (E286Q), had no effect on stability or assembly of the V-ATPase complex, but also led to complete loss of VATPase activity. The E286Q mutant showed labeling by 2-azido-[ 32 P]ATP that was approximately 60% of that observed for wild type, suggesting that mutation of this glutamic acid residue affected primarily ATP hydrolysis rather than nucleotide binding.The vacuolar (H ϩ )-ATPases (or V-ATPases) are a class of ATP-dependent proton pumps that play an important role in a variety of cellular processes, including receptor-mediated endocytosis, intracellular targeting, macromolecular processing and degradation, and coupled transport (1-9). The V-ATPases are located in both intracellular compartments and in the plasma membrane of certain specialized cells (7, 10 -12). In yeast, acidification of the central vacuole by the V-ATPase serves to activate degradative enzymes and to drive uptake of solutes such as Ca 2ϩ and amino acids (5). The V-ATPases are composed of two domains, a 500-kDa peripheral V 1 domain with the structure A 3 B 3 (54) 1 C 1 D 1 E 1 F 1 G 1 (13-17) which is responsible for ATP hydrolysis and a 250-kDa V 0 domain with the structure 100 1 36 1 19 1 c 6 that is responsible for proton translocation (13,18,19). In Saccharomyces cerevisiae, the V-ATPase subunits are encoded by at least 14 genes, including VMA1 (encoding the 69-kDa A subunit) (20, 21), VMA2 (the 60-kDa B subunit) (22), VMA13 (the 54-kDa subunit) (17), VMA5 (the 42-kDa C subunit) (23, 24), VMA8 (the 32-kDa D subunit) (25), VMA4 (the 27-kDa E subunit) (23, 26), VMA10 (the 16-kDa G subunit) (17), VMA7 (the 14-kDa F subunit) (27, 28), VPH1 and STV1 (encoding isoforms of the 100-kDa subunit) (29, 30), VMA6 (encoding the 36-kDa V 0 subunit) (31), ppa1 (encoding the 19-kDa V 0 subunit) (32), and VMA3 and VMA11 (encoding the 17-kDa c subunits) (33, 34). Disruption of the VMA genes leads to a conditional lethal phenotype in which cells are unable to grow at neutral pH and in the presence of elevated Ca 2ϩ concentrations but are able to grow at acidic pH (11,35).Both the A and B subunits participate in nucleotide binding by the V-ATPases (36 -40) and show approximately 25% sequence identity with the ␤ and ␣ subunits of the F-ATPases (20,(41)(42)(43)(44)(45)(46). The F-ATPases normally function in ATP synthesis in mitochondria, chloroplasts, and bacteria (47-51), and a recent x-ray crystal structure of the F 1 domain has revealed a hexameric arrangement of ␣ and ␤ subunits, with the catalytic sites located primarily on ␤ and the noncatalytic sites located primarily on ␣ (52)....

We have isolated and analyzed the genes encoding the human and rat gastric H,K-ATPase catalytic subunits. The complete sequence of the human gene, including 2.2 kb of 5'-flanking sequence, and the 5' end of the rat gene, including exons 1-4 and 2.5 kb of 5'-flanking sequence, have been determined. The human gene contains 22 exons. Its intron-exon organization is identical to that of the Na,K-ATPase gene, except that exon 6 corresponds to a fusion of exons 6 and 7 of the Na,K-ATPase gene. The transcription initiation sites of both the human and rat genes were determined by primer extension and S1 nuclease protection analyses. Comparison of the 5'-flanking regions of the human and rat genes revealed three extended regions of high sequence similarity, one of which includes a potential TATA box and other basic promoter elements beginning about 30 nucleotides upstream of the transcription start site. Other conserved sequences, including possible response elements for Ca2+ and cAMP, which are known intracellular mediators of acid secretion, are located up to 2 kb 5' to the transcription initiation site.

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