An alignment of 17 deduced protein sequences from plant, fungi, and ciliate H(+)-ATPase genes.

Seventeen protein sequences of H(+)-ATPases from plants (Arabidopsis thaliana, Nicotiana plumbaginifolia, Lycopersicum esculentum), fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Zygosaccharomyces rouxii, Neuropora crassa, Candida albicans), and a parasitic ciliate (Leishmania donovani) have been aligned. Twenty sequence fragments were identified which were conserved in H(+)-, Na+/K(+)-, and Ca++ plasma membrane-ATPases. In addition, a total of 118 residues not located in these fragments were found to be conserved in all H(+)-ATPases. Among those, 38 amino acid residues were screened out as being priority targets for site-directed mutagenesis experiments aimed at the identification of the amino acid residues specifically involved in cation specificity.

Molecular and biochemical characterization of the Dio-9-resistant pma1-1 mutation of the H(+)-ATPase from Saccharomyces cerevisiae.

The plasma-membrane H(+)-ATPase gene PMA1 was sequenced in four Dio-9-resistant strains of Saccharomyces cerevisiae, isolated independently. The same amino acid substitution Ala608----Thr was found in the four mutated strains. The mutant ATPase activity was decreased while the Km value for MgATP was increased. The ATPase efficiency (V/Km) of the mutant was reduced by a factor of 25 under acid conditions (pH 5.5), and by a factor of 10 at physiological pH (pH 6.6). The mutation also strongly reduces the inhibition by vanadate of ATPase activity, suggesting that the altered amino acid is involved in phosphate binding and/or in the E1-E2 transition.

A second transport ATPase gene in Saccharomyces cerevisiae.

A second transport ATPase gene from Saccharomyces cerevisiae has been identified by hybridization to a PMA1 probe and sequenced. The gene called PMA2 encodes a polypeptide of Mr = 102,157, which, with the exception of the 144 amino-terminal residues, is highly homologous to the structural gene PMA1 for the H+-ATPase. It is localized on the chromosome XVI at 16.7 centimorgan from gal4 and is not essential for haploid growth. Comparison between the upstream, noncoding DNA regions of PMA1 and PMA2 indicates that the two genes are controlled differently. The extensive amino acid sequence homology with the fungal H+-ATPases described so far indicates that the PMA2-encoded protein is also able to function as a H+ pump. This is supported by the observation that in pma1 mutants with reduced plasma membrane ATPase activity, disruption of the PMA2 gene confers the ability to grow under alkaline pH conditions. Slower development of diploids is also observed on normal minimal medium after bilateral disruption of PMA2 in the two parents.

Mutation of a conserved glycine residue modifies the vanadate sensitivity of the plasma membrane H+-ATPase from Schizosaccharomyces pombe.

The structural gene pma+1 for the H+-ATPase from the fission yeast Schizosaccharomyces pombe has been isolated and sequenced. The intron-less gene encodes for a protein of Mr = 99,769 which is 75% homologous to those of Saccharomyces cerevisiae and Neurospora crassa. The S. pombe pma+1 gene complements not only S. pombe pma-1-1 but also S. cerevisiae pma-1-4 mutants selected for in vitro vanadate-resistant ATPase activity. The sequence of the S. pombe mutant pma-1-1 allele reveals that the glycine residue 268, which is perfectly conserved in the transduction domain of all animal and fungal transport ATPases sequenced so far, is modified into an aspartate residue by the mutation. Replacement of glycine 268 by aspartate has been monitored by the appearance of a new PvuI restriction site in the mutant DNA. Mitotic cosegregation has been observed between the PvuI site and vanadate-resistant ATPase activity in a growing population of S. pombe transformants.