Search

The stalk segments of P-type ion-translocating enzymes are presumed to play important roles in energy coupling. In this work, stalk segments S4 and S5 of the yeast H(+)-ATPase were examined for helical character, optimal length, and segment orientation by a combination of proline substitution, insertion/deletion mutagenesis, and second-site suppressor analyses. The substitution of various residues for helix-disrupting proline in both S4 (L353P,L353G; A354P; and G371P) and S5 (D676P and I684P) resulted in highly defective or inactive enzymes supporting the importance of helical character and/or the maintenance of essential interactions. The contiguous helical nature of transmembrane segment M5 and stalk element S5 was explored and found to be favorable, although not essential. The deletion or addition of one or more amino acids at positions Ala(354) in S4 and Asp(676) in S5, which were intended to either rotate helical faces or extend/reduce the length of helical segments, resulted in enzyme destabilization that abolished most enzyme assembly. Second-site suppressor mutations were obtained to primary site mutations G371A (S4) and D676G (S5) and were analyzed with a molecular structure model of the H(+)-ATPase. Primary site mutations were predicted to alter the site of phosphorylation either directly or indirectly. The suppressor mutations either directly changed packing around the primary site or altered the environment of the site of phosphorylation. Overall, these data support the view that stalk segments S4 and S5 of the H(+)-ATPase are helical elements that are optimized for length and interactions with other stalk elements and can influence the phosphorylation domain.

The Cryptococcus neoformans PMA1 gene, encoding a plasma membrane H(+)-ATPase, was isolated from a genomic DNA library of serotype A strain ATCC 6352. An open reading frame of 3,380 nucleotides contains six introns and encodes a predicted protein consisting of 998 amino acids with a molecular mass of approximately 108 kDa. Plasma membranes were isolated, and the H(+)-ATPase was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiae H(+)-ATPase, consistent with its predicted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 optimum for ATP hydrolysis, K(m) and V(max) values of 0.5 mM and 3.1 micromol mg(-1) min(-1), respectively, and an apparent K(i) for vanadate inhibition of 1.6 microM. ATP hydrolysis in plasma membranes and medium acidification by whole cells were inhibited by ebselen, a nonspecific H(+)-ATPase antagonist which was also fungicidal. The predicted C. neoformans protein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits more than 50% identity to PMA1 genes from plants. Collectively, this study provides the basis for establishing the Cryptococcus H(+)-ATPase as a viable target for antifungal drug discovery.

The N-terminus of the H(+)-ATPase from Saccharomyces cerevisiae contains a serine-rich cluster of 11 serine residues in the first 17 amino acids, including a stretch of eight consecutive serine residues. This cluster is conserved in the weakly expressed PMA2 gene from the same organism, but it is not present in PMA genes from other organisms suggesting that it is not likely to represent a conserved functional motif. To better understand whether this region plays a regulatory role, a series of mutant enzymes were generated in which the serine tract was systematically converted to alanine or deleted. Conversion of the first six serine residues to alanine or deletion of the entire serine tract had little effect on cell growth phenotypes. However, when eight or more serines were converted, the mutant cells displayed prominent hygromycin B-resistant and low pH-sensitive phenotypes indicative of reduced H(+)-ATPase function. The mutant enzymes were found to display relatively normal kinetic properties for ATP hydrolysis, but showed significantly decreased abundance in the plasma membrane under stress conditions when eight or more serine residues were converted to alanine. The reduced abundance of the enzyme appeared to be due to degradative turnover, as mutant enzymes with multiple alanine substitutions showed an accelerated rate of turnover relative to wild-type. The polyserine tract in the H(+)-ATPase does not appear to be important for catalysis, but may contribute to overall protein stability.

The stalk region of the H+-ATPase from Saccharomyces cerevisiae has been proposed to play a role in coupling ATP hydrolysis to proton transport. Genetic probing was used to examine the role of stalk segments S2 and S3, associated with M2 and M3, respectively. Saturation mutagenesis was used to explore the role of side group character at position Ile183 in S2, at which an alanine substitution was shown previously to partially uncouple the enzyme (Wang, G., Tamas, M. J., Hall, M. J., Pascual-Ahuir, A., and Perlin, D. S. (1996) J. Biol. Chem. 271, 25438-25445). Diverse side group substitutions were tolerated at this position, although three substitutions, I183N, I183R, and I183Y required second site mutations at the C terminus of the enzyme for stabilization. Substitution of glycine and proline at Ile183 resulted in lethal phenotypes, suggesting that the backbone may be more important than side group at this position. Proline/glycine mutagenesis was used to study additional sites in S2 and S3. The substitution of proline at Gly186 resulted in a lethal phenotype, whereas substitutions in S3 of proline or serine at Gly270 and proline or glycine at Thr287 resulted in viable mutants. Mutations G270P and T287P resulted in mutant enzymes that produced pronounced growth defects and ATP hydrolysis rates that were 35% and 60% lower than wild type enzyme, respectively. The mutant enzymes transported protons at rates consistent with their ATPase activity, suggesting that the growth defects observed were due to a reduced rate of ATP hydrolysis and not to uncoupling of proton transport. The prominent growth phenotypes produced by mutations G270P and T287P permitted the isolation of suppressor mutations, which restored wild type growth. Most of the suppressors either replaced the primary site mutation with alanine or restored the wild type residue by ectopic recombination with PMA2, both of which restore alpha-helical tendency. This study suggests that maintaining alpha-helical character is essential to S2 and may play an important role in S3.

Alkylation of Cys-89 of the gamma subunit of the coupling factor portion (CF1) of the chloroplast ATP synthase by N-ethylmaleimide was previously shown to inhibit ATP synthesis and hydrolysis. The gamma subunit interacts with the inhibitory epsilon subunit. Alkylation of gamma Cys-89 could cause changes that result in the strengthening of the interactions between the two subunits which would be inhibitory. We show that the inhibition of the ATPase activity of CF1 in solution persists after removal of the epsilon subunit. Additionally, epsilon rebinds to control and alkylated CF1 at very similar CF1 to epsilon concentration ratios. Although the bound nucleotide contents of control and alkylated CF1 were similar, the rate of exchange of nucleotide bound to one site with medium nucleotide was accelerated by more than two orders of magnitude by alkylation. We show that ATP-induced release of bound 2'(3')-O-trinitrophenyl-adenosine 5'-diphosphate can be monitored conveniently by stopped-flow fluorescence. The effects of N-ethylmaleimide modification are likely to be felt over long distances. As determined by fluorescence resonance energy transfer, no nucleotide binding site so far mapped is closer than 5 nm to Cys-89. The bulky maleimide probably causes structural changes that perturb subunit and catalytic site interactions.

The treatment of chloroplast coupling factor 1 (CF1) with dithiothreitol or with trypsin modifies the gamma subunit. Reduction of the gamma subunit disulfide bond in CF1 in solution with dithiothreitol enhances the dissociation of epsilon (Duhe, R. J., and Selman, B. R. (1990) Biochim. Biophys. Acta 1017, 70-78). The Ca(2+)-ATPase activity of either oxidized or reduced CF1 increases as the enzyme is diluted. Added epsilon subunit inhibits the Ca(2+)-ATPase activity of both forms of the diluted CF1, suggesting that epsilon dissociation is the cause of activation by dilution. Half-maximal activation occurred at much higher concentrations of the reduced CF1, indicating that reduction decreases the affinity for epsilon about 20-fold. Immunoblotting techniques show that there is only one epsilon subunit/CF1 in intact chloroplasts, in thylakoid membranes, and in solution. No epsilon is released from CF1 in thylakoids under conditions of ATP synthesis. The gamma subunit of CF1 in illuminated thylakoids is specifically cleaved by trypsin. CF1 purified from thylakoids treated with trypsin in the light is deficient in epsilon subunit, and has a high rate of ATP hydrolysis. Added epsilon neither inhibits the ATPase activity of, nor binds tightly to the cleaved enzyme.