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.     

The pma1 and pma2 H(+)-ATPases from Schizosaccharomyces pombe are functionally interchangeable.

The pma2 gene of Schizosaccharomyces pombe codes for a polypeptide having a predicted Mr of 110,126 and which is 79% identical to the plasma membrane H(+)-ATPase encoded by the pma1 gene. The pma2 gene, unlike pma1, is weakly expressed and not essential to mitotic growth. By constructing yeast strains in which the chromosomal pma2 gene is under control of the adh promoter, it has been possible to identify the overproduced ATPase in plasma membrane via formation of a phosphoenzyme. In a pma1-1 mutant strain whose ATPase activity is insensitive to vanadate, the overexpressed pma2 gene restores vanadate sensitivity. It also rescues a pma1 null mutant from lethality. These results demonstrate that the two H(+)-ATPases are functionally interchangeable in vivo but differently expressed.     

Primary structure of the Neurospora plasma membrane H+-ATPase deduced from the gene sequence. Homology to Na+/K+-, Ca2+-, and K+-ATPase

The gene for the Neurospora crassa plasma membrane H+-ATPase has been cloned and sequenced. The gene encodes for a protein of 920 amino acids with a molecular weight of 100,002. The coding region is interrupted by four introns: three near the amino terminus and one near the carboxyl terminus. The deduced amino acid sequence of the N. crassa plasma membrane H+-ATPase exhibits 75% homology to the amino acid sequence of the Saccharomyces cerevisiae plasma membrane H+-ATPase. Also, an amino acid comparison with the Na+/K+-ATPase from sheep kidney, Ca2+-ATPase from rabbit muscle, and K+-ATPase from Escherichia coli reveals that certain regions are highly conserved and suggest that these regions may serve essential functions which are common to the various cation-motive ATPases. This observation suggests that the phosphorylatable, cation-motive ATPases may function via a similar energy transduction mechanism.     

Structure of a plasma membrane H+-ATPase gene from the plant Arabidopsis thaliana

Physiological and biochemical studies have suggested that the plant plasma membrane H+-ATPase controls many important aspects of plant physiology, including growth, development, nutrient transport, and stomata movements. We have started the genetic analysis of this enzyme by isolating both genomic and cDNA clones of an H+-ATPase gene from Arabidopsis thaliana. The cloned gene is interrupted by 15 introns, and there is partial conservation of exon boundaries with respect to animal (Na+/K+)- and Ca2+-ATPases. In general, the relationship between exons and the predicted secondary and transmembrane structure of different ATPases with phosphorylated intermediate support a somewhat degenerate correspondence between exons and structural modules. The predicted amino acid sequence of the plant H+-ATPase is more closely related to fungal and protozoan H+-ATPases than to bacterial K+-ATPases or to animal (Na+/K+)-, (H+/K+)-, and Ca2+-ATPases. There is evidence for the existence of at least three isoforms of the plant H+-ATPase gene. These results open the way for a molecular approach to the structure and function of the plant proton pump.     

Post-translational modification of plant plasma membrane H(+)-ATPase as a requirement for functional complementation of a yeast transport mutant.

Many heterologous membrane proteins expressed in the yeast Saccharomyces cerevisiae fail to reach their normal cellular location and instead accumulate in stacked internal membranes. Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 (AHA2) is expressed predominantly in yeast internal membranes and fails to complement a yeast strain devoid of its endogenous H(+)-ATPase Pma1. We observed that phosphorylation of AHA2 in the heterologous host and subsequent binding of 14-3-3 protein is crucial for the ability of AHA2 to substitute for Pma1. Thus, mutants of AHA2, complementing pma1, showed increased phosphorylation at the penultimate residue (Thr(947)), which creates a binding site for endogenous 14-3-3 protein. Only a pool of ATPase in the plasma membrane is phosphorylated. Double mutants carrying in addition a T947A substitution lost their ability to complement pma1. However, mutants affected in both autoinhibitory regions of the C-terminal regulatory domain complemented pma1 irrespective of their ability to become phosphorylated at Thr(947). This demonstrates that it is the activity status of the mutant enzyme and neither redirection of trafficking nor 14-3-3 binding per se that determines the ability of H(+)-pumps to rescue pma1.     

A single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe

A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma-32P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors (Ulaszewski, S., Coddington, A., and Goffeau, A. (1986) Curr. Genet. 10, 359-364).     

Altered plasma membrane H(+)-ATPase from the Dio-9-resistant pma1-2 mutant of Schizosaccharomyces pombe.

The pma1-2 mutation affecting the plasma membrane H(+)-ATPase of Schizosaccharomyces pombe has been selected for resistance to the antibiotic Dio-9. In membrane fractions purified from glucose-starved cells, the mutant ATPase activity is reduced by 96%, is insensitive to inhibition by vanadate and has a pH profile displaced in the acidic pH range when compared to the wild type. The maximum velocity of the H(+)-ATPase activity of plasma membranes from glucose-activated pma1-2 cells is activated 20-fold. This is in striking contrast with the wild-type ATPase activity, the maximal velocity of which is not affected by glucose. However, similar to the wild-type enzyme, glucose activation of the pma1-2 mutant H(+)-ATPase reduces the Km for MgATP 9-2 mM and shifts the optimal pH from 4.8 to 6.0-6.5. The pma1-2 mutation modifies Lys250 to a threonine, which is highly conserved in fungal and plant H(+)-ATPases. These results, compared to those reported for mutations of neighbour residues in yeast or mammalian P-type ATPases, suggest that Lys250 could play a significant role, not only in phosphate binding and/or in the E1P-E2P conformational isomerisation, but also in glucose activation of the H(+)-ATPase.     

Defective H(+)-ATPase of hygromycin B-resistant pma1 mutants fromSaccharomyces cerevisiae

Mutations in the plasma membrane H(+)-ATPase gene (PMA1) of Saccharomyces cerevisiae that confer growth resistance to hygromycin B have been shown recently to cause a marked depolarization of whole cell membrane potential (Perlin, D. S., Brown, C. L., and Haber, J. E. (1988) J. Biol. Chem. 263, 18118-18122). In this report, the biochemical and genetic properties of H+-ATPases from four prominent hygromycin B-resistant pma1 mutants, pma1-105, pma1-114, pma1-147, and pma1-155, are described. Single base pair changes were identified in pma1-105, pma1-114, and pma1-147 that resulted in amino acid substitutions of Ser-368----Phe, Gly-158----Asp, Pro-640----Leu, respectively. An A----G transition mutation at -39 in the 5'-untranslated region of the mRNA of pma1-155 was also found. This mutation creates an out-of-Frame upstream AUG initiation codon that apparently reduces normal translation of PMA1. DNA sequence analysis of PMA1 from strain Y55 identified 9 base pair substitutions that resulted in 6 amino acid changes in nonconserved regions when compared to the published sequence for strain S288C. Plasma membranes of three of the four pma1 mutants contained normal amounts of H(+)-ATPase; membranes from pma1-155 contained enzyme at 62% of the wild-type level. The kinetics of ATP hydrolysis were most strongly altered for enzymes from pma1-105 and pma1-147 which showed changes in both Km and Vmax. A striking pH dependence for these parameters was found for enzyme from pma1-105 which resulted in a precipitous decline in Km and Vmax below pH 6.5. ATP hydrolysis by enzymes from pma1-105 and pma1-147 was insensitive to inhibition by vanadate. These enzymes, in contrast to wild-type and vanadate-sensitive mutant enzymes, were poorly protected from trypsin-induced inactivation by MgATP and vanadate or Pi alone. These results are pertinent to the mechanism of vanadate-induced enzyme inhibition and suggest that Ser-368 and Pro-640 influence the affinity of the phosphate-binding site for Pi. All mutant enzymes catalyzed ATP-induced pH gradient formation following purification and reconstitution into liposomes. Finally, these results further demonstrate the usefulness of hygromycin B as a generalized screening tool for isolating diverse plasma membrane ATPase mutants.     

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.     

Gene Conversions and Crossing over during Homologous and Homeologous Ectopic Recombination in Saccharomyces Cerevisiae

The pma1-105 mutation reduces the activity of the yeast plasma membrane H(+)-ATPase and causes cells to be both low pH and ammonium ion sensitive and resistant to the antibiotic hygromycin B. Revertants that can grow at pH 3.0 and on ammonium-containing plates frequently arise by ectopic recombination between pma1-105 and PMA2, a diverged gene that shares 85% DNA sequence identity with PMA1. The gene conversion tracts of revertants of pma1-105 were determined by DNA sequencing the hybrid PMA1::PMA2 genes. Gene conversion tracts ranged from 18-774 bp. The boundaries of these replacements were short (3-26 bp) regions of sequences that were identical between PMA1 and PMA2. These boundaries were not located at the regions of greatest shared identity between the two PMA genes. Similar results were obtained among low pH-resistant revertants of another mutation, pma1-147. One gene conversion was obtained in which the resulting PMA1::PMA2 hybrid was low pH-resistant but still hygromycin B-resistant. This partially active gene differs from a wild-type revertant only by the presence of two PMA2-encoded amino acid substitutions. Thus, some regions of PMA2 are not fully interchangeable with PMA1. We have also compared the efficiency of recombination between pma1-105 and either homeologous PMA2 sequence or homologous PMA1 donor sequences inserted at the same location. PMA2 X pma1-105 recombination occurred at a rate approximately 75-fold less than PMA1 X pma1-105 events. The difference in homology between the interacting sequences did not affect the proportion of gene conversion events associated with a cross-over, as in both cases approximately 5% of the Pma(+) recombinants had undergone reciprocal translocations between the two chromosomes carrying pma1-105 and the donor PMA sequences. Reciprocal translocations were identified by a simple and generally useful nutritional test.     

Activity of the plasma membrane H(+)-ATPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid.

The weak acid sorbic acid transiently inhibited the growth of Saccharomyces cerevisiae in media at low pH. During a lag period, the length of which depended on the severity of this weak-acid stress, yeast cells appeared to adapt to this stress, eventually recovering and growing normally. This adaptation to weak-acid stress was not due to metabolism and removal of the sorbic acid. A pma1-205 mutant, with about half the normal membrane H+-ATPase activity, was shown to be more sensitive to sorbic acid than its parent. Sorbic acid appeared to stimulate plasma membrane H+-ATPase activity in both PMA1 and pma1-205. Consistent with this, cellular ATP levels showed drastic reductions, the extent of which depended on the severity of weak-acid stress. The weak acid did not appear to affect the synthesis of ATP because CO2 production and O2 consumption were not affected significantly in PMA1 and pma1-205 cells. However, a glycolytic mutant, with about one-third the normal pyruvate kinase and phosphofructokinase activity and hence a reduced capacity to generate ATP, was more sensitive to sorbic acid than its isogenic parent. These data are consistent with the idea that adaptation by yeast cells to sorbic acid is dependent on (i) the restoration of internal pH via the export of protons by the membrane H+-ATPase in an energy-demanding process and (ii) the generation of sufficient ATP to drive this process and still allow growth.     

C-terminal deletion analysis of plant plasma membrane H(+)-ATPase: yeast as a model system for solute transport across the plant plasma membrane.

The plasma membrane proton pump (H(+)-ATPase) energizes solute uptake by secondary transporters. Wild-type Arabidopsis plasma membrane H(+)-ATPase (AHA2) and truncated H(+)-ATPase lacking 38, 51, 61, 66, 77, 92, 96, and 104 C-terminal amino acids were produced in yeast. All AHA2 species were correctly targeted to the yeast plasma membrane and, in addition, accumulated in internal membranes. Removal of 38 C-terminal residues from AHA2 produced a high-affinity state of plant H(+)-ATPase with a low Km value (0.1 mM) for ATP. Removal of an additional 12 amino acids from the C terminus resulted in a significant increase in molecular activity of the enzyme. There was a close correlation between molecular activity of the various plant H(+)-ATPase species and their ability to complement mutants of the endogenous yeast plasma membrane H(+)-ATPase (pma1). This correlation demonstrates that, at least in this heterologous host, activation of H(+)-ATPase is a prerequisite for proper energization of the plasma membrane.     

Effects of phosphate and hydrophobic molecules on two mutations in the beta-strand sector of the H(+)-ATPase from the yeast plasma membrane

At concentrations from 10 to 100 mM, inorganic phosphate and sulfate stimulate the activity of the H(+)-ATPase purified from the wild type Schizosaccharomyces pombe plasma membranes. Compared to the wild type ATPase, the stimulation by phosphate is more pronounced in the mutant pma1-1 (Gly-268----Asp) and is much reduced in the mutant pma1-2 (Lys-250----Thr) enzymes. In contrast, the inhibition by trifluoperazine is less pronounced in the pma1-1 mutant than in the wild type or pma1-2 mutant. The mutant pma1-2 ATPase activity is markedly stimulated by 10-20% dimethyl sulfoxide, which has a limited effect on the wild type and pma1-1 enzymes. These data indicate that the protein domain located in the beta-strand sector, including Lys-250 and Gly-268, is located in the active site and that its hydrophobic character influences the interactions of the yeast H(+)-ATPase with inorganic phosphate, as well as with the hydrophobic inhibitor trifluoperazine or the hydrophobic solvent dimethyl sulfoxide.     

Membrane potential defect in hygromycin B-resistant pma1 mutants of Saccharomyces cerevisiae

The proton transport properties of hygromycin B-resistant pma1 mutants which show kinetic defects in the plasma membrane H+-ATPase were examined. It was found that net proton efflux, as measured by whole cell medium acidification in the presence of 25 mM KCl, was similar for normal and pma1 mutant cells. However, in the absence of added KCl, the extent of net proton efflux was considerably less in wild type than in pma1 mutant cells. The cellular membrane potential was implicated as an important factor in regulating net proton transport and was determined from [14C]tetraphenylphosphonium uptake studies to be considerably depolarized in the pma1 mutants. The growth of wild type cells, which is normally inhibited by hygromycin B at 200 micrograms/ml, was found to be resistant to the antibiotic by the addition of 50 mM KCl to the growth medium. These results suggest that the electrogenic behavior of proton transport by the H+-ATPase may be altered in pma1 mutants and that resistance to hygromycin B may be mediated via depolarization of the cellular membrane potential.     

Tissue-specific expression of the gene for a putative plasma membrane H(+)-ATPase in a seagrass.

A cDNA clone corresponding to the gene (ZHA1) for a putative plasma membrane H(+)-ATPase of a seagrass (Zostera marina L.) was isolated and sequenced. Comparison of the amino acid predicted sequence from the nucleotide sequence of ZHA1 with those encoded by known genes for plasma membrane H(+)-ATPases from other plants indicated that ZHA1 is most similar to the gene (PMA4) for a plasma membrane H(+)-ATPase in a tobacco (84.4%). Northern hybridization indicated that ZHA1 was strongly expressed in mature leaves, which are exposed to seawater and have the ability of tolerate salinity; ZHA1 was weakly expressed in immature leaves, which are protected from seawater by tightly enveloping sheaths and are sensitive to salinity. In mature leaves, in situ hybridization revealed that ZHA1 was expressed specifically in epidermal cells, the plasma membranes of which were highly invaginated and morphologically similar to those of typical transfer cells. Therefore, the differentiation of the transfer cell-like structures, accompanied by the high-level expression of ZHA1, in the epidermal cells of mature leaves in particular may be important for the excretion of salt by these cells.