Functional analysis of conserved cysteine residues in the catalytic subunit of the yeast vacuolar H(+)-ATPase.

The A subunit of the yeast vacuolar ATPase contains three highly conserved cysteines: Cys-261, Cys-284, and Cys-538. Cys-261 is located within the nucleotide-binding P-loop. Each of the conserved cysteines, and one nonconserved cysteine, Cys-254, were altered to serine by site-directed mutagenesis, and the effects on growth at pH 7.5 were determined. The Cys-254-->Ser, Cys-261-->Ser and the double mutants all grew at pH 7.5 and contained nitrate- and bafilomycin-sensitive ATPase activity. However, the ATPase activities of the Cys-261-->Ser and the double mutants were insensitive to the sulfhydryl group inhibitor, N-ethylmaleimide, demonstrating that Cys-261 is the site of inhibition by N-ethylmaleimide. Changing either Cys-284 or Cys-538 to serine prevented growth at pH 7.5. Cys-284 and Cys-538 thus appear to be essential cysteine residues which are required either for assembly or catalysis.

Sulfite both stimulates and inhibits the yeast vacuolar H(+)-ATPase.

The yeast vacuolar H(+)-ATPase (V-ATPase) exhibits nonlinear hydrolysis kinetics, i.e. an initial rapid rate followed by a slower, steadily declining rate. Sulfite (50-100 mM) stimulates the yeast V-ATPase specifically during the latter period. Sulfite activation has been observed for the F-ATPases and archaebacterial ATPases and is thought to be caused by the release of tightly bound nucleotide at the catalytic site. However, turnover-dependent inactivation of the yeast V-ATPase, and sulfite stimulation, were only observed at MgATP concentrations > 1.0 mM. Below 1.0 mM MgATP, the hydrolysis time course was linear, and sulfite was inhibitory. The inhibition during the initial phase and the stimulation during the later phase of the time course could be accounted for by a 5.5-fold sulfite-induced increase in the apparent Km, and a small increase in the apparent Vmax. Sulfite also protected the enzyme against inhibition by cold inactivation and by dicyclohexylcarbodiimide but not by bafilomycin. Sulfite stimulation during the later phase was antagonized by delta mu H+, particularly by delta pH. In contrast to its effects on hydrolysis, sulfite inhibited the formation of a pH gradient at all times and failed to enhance the membrane potential even when delta pH was collapsed by nigericin. These results indicate that sulfite partially uncouples hydrolysis from proton transport in a way that preserves regulation by delta mu H+.

The use of antisense mRNA to inhibit the tonoplast H+ ATPase in carrot.

Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacuolar H+ ATPase A subunit cDNA cloned in the antisense orientation behind the cauliflower mosaic virus 35S promoter. Bafilomycin-sensitive ATPase, H(+)-pumping, and 14C-O-methyl-glucose uptake activities were specifically inhibited in the tonoplast fractions of mutant cell lines. Protein gel blotting confirmed that the expression of the A subunit was inhibited in the tonoplast fraction, but not in the Golgi fraction. Two-dimensional protein gel blots of total microsomes of wild-type and control transformant cell lines revealed two major immunoreactive polypeptides in the acidic pI range. In contrast, highly purified tonoplast membranes contained only the less acidic polypeptide. Because the less acidic polypeptide was preferentially diminished in the two antisense cell lines, we infer that the antisense constructs specifically blocked expression of a tonoplast-specific isoform of the V-ATPase A subunit in carrot. Regenerated plants containing the antisense constructs exhibited altered leaf morphologies and reduced cell expansion. The altered phenotype was correlated with the presence of the antisense construct.