Yeast ATPase Research Articles

Functional comparisons between plant plasma membrane H(+)-ATPase isoforms expressed in yeast.

To examine the functional properties of the three major isoforms of plasma membrane H(+)-ATPase expressed in Arabidopsis thaliana (AHA1, AHA2, and AHA3), we employed a system for the heterologous expression of functional plant plasma membrane H(+)-ATPase in yeast (Villalba, J. M., Palmgren, M. G., Berberian, G. E., Ferguson, C., and Serrano, R. (1992) J. Biol. Chem. 267, 12341-12349). Each isoform was expressed efficiently but appeared to be retained in the endoplasmic reticulum of yeast. All isoforms displayed qualitatively similar enzymatic properties, but quantitative differences were found. When compared with AHA3, AHA1 and AHA2 had an apparent higher turnover rate for ATP hydrolysis, exhibited a 10-fold higher apparent affinity for ATP, and a 3-fold higher sensitivity toward vanadate. In addition, AHA2 had a slightly lower apparent affinity for H+ and seemed to be more susceptible to activation by lysophosphatidylcholine than did AHA1 and AHA3. This study represents the first comparison of the functional properties of isoforms of the plant plasma membrane H(+)-ATPase.

Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65.

We have developed two independent assays to study the integration, folding, and intracellular transport of the polytopic plasma membrane H(+)-ATPase in yeast. To follow folding, controlled trypsinolysis was used to distinguish between the E1 conformation of the ATPase (favored in the presence of ADP) and the E2 conformation (favored in the presence of vanadate). By this criterion, wild-type ATPase appears to recognize its ligands and assume distinct conformations within a short time after its biosynthesis. To follow intracellular transport, we have exploited the fact that export of newly synthesized ATPase from the endoplasmic reticulum is accompanied by kinase-mediated phosphorylation, leading to a shift in electrophoretic mobility. Because proper folding is required for transport from the endoplasmic reticulum, the mobility shift also serves as a convenient bioassay for correct folding. As a first step toward identifying cell components important in folding of the nascent ATPase, we have used the dual assays to examine the role of KAR2, encoding the yeast homolog of immunoglobulin heavy chain binding protein/78-kDa glucose-regulated protein, and SEC65, encoding a subunit of the yeast signal recognition particle. Although mutation of KAR2 caused defective translocation of several secretory precursors into the endoplasmic reticulum lumen, ATPase folding and intracellular transport were unperturbed. By contrast, in a sec65 mutant, the folding and intracellular transport of newly synthesized ATPase were delayed. Our data suggest that conformational maturation of the ATPase is a rapid process in wild-type cells and that membrane integration mediated by signal recognition peptide is important for the proper folding of this polytopic protein.

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.

Cloning and characterization of the plasma membrane H(+)-ATPase from Candida albicans

The Candida albicans PMA1 gene was isolated from a genomic library by using a hybridization probe obtained from the PMA1 gene of Saccharomyces cerevisiae. The gene was localized to chromosome III of the Candida genome. An open reading frame of 2,685 nucleotides predicts an amino acid sequence of 895 amino acids that is 83% homologous at both the DNA and protein levels to its S. cerevisiae equivalent. A polyadenylated mRNA transcript of about 4,000 nucleotides contains a highly folded AU-rich leader of 242 nucleotides. The structure of the gene, codon bias, and levels of approximately 100-kDa H(+)-ATPase protein recovered in plasma membranes indicate a highly expressed gene. The plasma membrane ATPase was purified to about 90% homogeneity and appeared to be blocked at the amino terminus. Three hydrophobic membrane sector tryptic fragments from the partially digested ATPase provided internal sequence information for over 50 amino acids, which agrees with the sequence predicted by the cloned gene. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the C. albicans enzyme is about 3 kDa smaller than its Saccharomyces counterpart and was consistent with a predicted Mr of 97,398. Antibodies to the S. cerevisiae whole ATPase or its carboxyl terminus bound to the C. albicans enzyme but with lower avidity. Kinetic analysis showed that the Candida and Saccharomyces ATPases respond to glucose activation-starvation in nonidentical fashions. The amino-terminal domain of the C. albicans ATPase is marked by a net deletion of 23 amino acids in comparison with the S. cerevisiae ATPase. These differences maintain net charge, occur in nonconserved regions of fungal ATPases, and are sufficient to account for the observed difference in electrophoretic mobility between the two yeast ATPases.

The mgtB Mg2+ transport locus of Salmonella typhimurium encodes a P-type ATPase.

The mgtB locus codes for one of three distinct Mg2+ transport systems of Salmonella typhimurium. The system encoded by the mgtB locus mediates Mg2+ influx only. The nucleotide sequence of a 4.6-kilobase fragment of DNA carrying mgtB has been determined. Two open reading frames were apparent. The most 5' (mgtC) could encode a hydrophobic protein of up to 25 kDa depending on which translation starts are used. A plasmid carrying this region downstream from a phage T7 promoter expresses a 22.5-kDa protein. The second open reading frame encoded a 101-kDa polypeptide (MgtB) consistent with our previous observation that a plasmid carrying the mgtB locus expresses a 102-kDa protein in maxicells. Insertions into either open reading frame abolished the ability of the plasmid to relieve the requirement for added Mg2+ and to restore Mg2+ uptake to a Mg2+ transport-deficient strain of S. typhimurium. The predicted amino acid sequence of MgtC showed no similarity to any other known protein. In contrast, the predicted sequence of MgtB indicated that it is a member of the family of cation transport P-type ATPases. Strikingly, however, MgtB was significantly more similar to eukaryotic Ca2(+)-ATPases than to prokaryotic P-type ATPases or other classes of eukaryotic P-type ATPases such as the Na+,K(+)-ATPase. MgtB is most closely related to Ca2(+)-ATPases of mammalian sarcoplasmic reticulum and yeast. A number of features of the Ca2(+)-ATPases thought to be important for cation transduction across the membrane are present in MgtB but not in other prokaryotic members of this enzyme family. Unlike the Ca2(+)-ATPases, however, which mediate efflux of cation from the cytosol, MgtB mediates influx of cation into the cytosol.

Transformation and pH homeostasis of fibroblasts expressing yeast H(+)-ATPase containing site-directed mutations.

Mouse fibroblasts expressing a yeast proton-pumping ATPase show tumorigenic transformation (R. Perona, and R. Serrano, Nature (London) 334:438-440, 1988). By expressing site-directed mutations of the yeast ATPase with different levels of activity, a close correlation has been found between enzyme activity, tumorigenic transformation, and intracellular pH measured by weak-acid distribution. Fibroblasts expressing the yeast proton-pumping ATPase showed increased capability to grow at acidic pH and to resist lethal acidification mediated by reversal of the Na(+)-H+ antiporter. Measurements with microelectrodes in individual cells demonstrated electrical hyperpolarization and confirmed the increased pH of cells expressing yeast ATPase. These results indicate that the yeast enzyme expressed in mouse fibroblasts has electrogenic proton-pumping activity and that this activity deregulates fibroblast growth. This suggests a connection between the biophysical phenomena of proton transport, intracellular pH, and membrane potential and the biochemical regulatory circuits based on protein kinases and transcription factors.

Transformation and pH Homeostasis of Fibroblasts Expressing Yeast H+-ATPase Containing Site-Directed Mutations

Mouse fibroblasts expressing a yeast proton-pumping ATPase show tumorigenic transformation (R. Perona, and R. Serrano, Nature (London) 334:438-440, 1988). By expressing site-directed mutations of the yeast ATPase with different levels of activity, a close correlation has been found between enzyme activity, tumorigenic transformation, and intracellular pH measured by weak-acid distribution. Fibroblasts expressing the yeast proton-pumping ATPase showed increased capability to grow at acidic pH and to resist lethal acidification mediated by reversal of the Na(+)-H+ antiporter. Measurements with microelectrodes in individual cells demonstrated electrical hyperpolarization and confirmed the increased pH of cells expressing yeast ATPase. These results indicate that the yeast enzyme expressed in mouse fibroblasts has electrogenic proton-pumping activity and that this activity deregulates fibroblast growth. This suggests a connection between the biophysical phenomena of proton transport, intracellular pH, and membrane potential and the biochemical regulatory circuits based on protein kinases and transcription factors.

Isolation of a gene for a regulatory 15‐kDa subunit of mitochondrial F1F0‐ATPase and construction of mutant yeast lacking the protein

A gene coding for yeast 15-kDa protein, a regulatory factor of mitochondrial F1F0-ATPase, was isolated. The cloned gene was disrupted in vitro and mutant strains that did not contain the 15-kDa protein were constructed by transformation of yeast cells with the disrupted gene. The ATP-synthesizing activity of the mutant mitochondria was the same as that of wild-type cells, suggesting that the 15-kDa protein is not required for mitochondrial oxidative phosphorylation. Collapse of the membrane potential induced ATP-hydrolyzing activity of F1F0-ATPase of the mutant mitochondria but not of normal mitochondria. Activation of the enzyme was also observed during incubation of submitochondrial particles from mutant cells, but not of those from wild-type cells. Thus, it is inferred that the 15-kDa protein supports the action of an intrinsic ATPase inhibitor of the ATP-hydrolyzing activity of the enzyme upon de-energization of mitochondrial membranes.

Cloning and Expression of the Yeast Plasma Membrane ATPase in Escherichia coli

The yeast plasma membrane ATPase gene PMA1 was cloned into Escherichia coli using the high expression tac and T7 promoters. The gene product is toxic to the bacterial cell leading to very low expression levels and arrested growth of the host cell within minutes of induction. The expressed protein is immunologically cross-reactive with the yeast ATPase, comigrates with the original protein in sodium dodecyl sulfate-polyacrylamide gels, and is isolated in the E. coli membrane fraction. The partially purified protein exhibits ATPase activity.

DNA sequence analysis of bacterial toxic heavy metal resistances

Bacterial plasmids have genes that confer highly specific resistances to As, Bi, Cd, Cu, Cr, Hg, Pb, Te, Zn, and other toxic heavy metals. For each toxic cation or anion, generally a different resistance system exists, and these systems may be "linked" together on multiple resistance plasmids. For Cd2+, AsO2-, AsO4(3)-, Hg2+, and organomercurials, DNA sequence analysis has supplemented direct physiological and biochemical experiments to produce sophisticated understanding. The cadA ATPase of S. aureus plasmids is a 727 amino acid membrane ATPase that pumps Cd2+ from the cells as rapidly as it is accumulated. This polypeptide is related by sequence to other cation translocating ATPases, including the membrane K+ ATPases of Escherichia coli and Streptococcus faecalis, the H+ ATPases of yeast and Neurospora, the Na+/K+ ATPases of vertebrate animals, and the Ca2+ ATPases of rabbit muscle. The conserved residues include the aspartyl residue that is phosphorylated, the lysine involved in ATP binding, and the proline within a membrane translocating region. The arsenate and arsenite translocating ATPase consists of 3 polypeptides (from DNA sequence analysis), including a recognizable ATP binding protein (arsA), an integral membrane protein (arsB gene), and a substrate specificity subunit (arsC gene). Inorganic mercury and organomercurial degradation is carried out by a series of about 6 polypeptides, including 2 soluble intracellular enzymes (organomercurial lyase and mercuric reductase). The latter is related by sequence and function to glutathione reductase and lipoamide dehydrogenase of prokaryotes and eukaryotes. These enzymes are dimeric, FAD-containing, NAD(P)H-dependent oxidoreductases. Other recognizable polypeptides in the mer system include a DNA-binding regulatory protein from the merR gene and a Hg2+ transport system consisting of a periplasmic Hg2(+)-binding protein (merP gene) and a membrane protein (merT gene) in gram negative systems.

The progenitor of ATP synthases was closely related to the current vacuolar H +-ATPase

The gene encoding the proteolipid of the vacuolar H +-ATPase of yeast was cloned and sequenced. The deduced amino acid sequence of the yeast protein is highly homologous to that of the proteolipid from bovine chromaffin granules. In contrast to other membrane proteins the transmembrane segments of the bovine and yeast proteolipids were much more conserved than the hydrophilic parts. The fourth transmembrane segment, which contains the DCCD-binding site, was conserved 100%. Comparison of vacuolar and eubacterial proteolipids revealed a homology which pointed to a common ancestral gene that underwent gene duplication to form the vacuolar proteolipids. Additional support for this notion came from the amino acid sequences of subunits involved in the catalytic sectors of archaebacterial ATP synthase and plant and yeast vacuolar H +-ATPases, which reveal extensive sequence homology. Slight, but significant, homology between the archaebacterial and eubacterial ATP synthases was observed. These observations might suggest that the progenitor of ATP synthases was closely related to the present vacuolar H +-ATPases.

Increased pH and tumorigenicity of fibroblasts expressing a yeast proton pump.

A common early response of eukaryotic cells to stimuli which activate their proliferation is an increase in intracellular pH (ref. 1). In animal cells this is caused by the activation of an Na+/H+ exchange system; in fungi and plants an H+-pumping ATPase is involved. The critical question is whether this intracellular alkalinization is merely coincident with the activation of cell proliferation or whether it is a regulatory signal. To increase intracellular pH bypassing the usual physiological stimuli (growth factors, hormones etc.) alkaline media or ammonia have been used in the past. Both approaches suffer from long-term toxicity effects and cannot be used in tumorigenic assays with whole organisms. We introduce here a more specific approach which involves expressing the gene for the yeast plasma membrane H+-ATPase in fibroblasts. The resulting cells have an elevated intracellular pH and acquire tumorigenic properties, suggesting that the yeast ATPase gene behaves as an oncogene in mammalian cells. These experiments support a crucial role of intracellular pH in the growth control of animal cells.

Arginine 328 of the beta-subunit of the mitochondrial ATPase in yeast is essential for protein stability.

The mitochondrial ATPase is rapidly inactivated by the arginine selective reagent phenylglyoxal. Recently, the purported major reacting residue has been reported for the chloroplast enzyme (Viale, A. M., and Vallejos, R. H. (1985) J. Biol. Chem. 260, 4958-4962) corresponding to Arg-328 in the beta-subunit of the yeast Saccharomyces cerevisiae mitochondrial ATPase, a highly conserved residue in the ATPase. This arginine residue was concluded to be in the active site of the ATPase and possibly involved in the binding of nucleotides. To test this hypothesis, site-directed mutagenesis of the yeast enzyme has been used to replace Arg-328 with alanine and lysine. The modified genes were transformed into a yeast strain, DMY111, which contained a null mutation in the gene coding for the beta-subunit of the ATPase. Both of the substitutions were functional in vivo as demonstrated by the ability of yeast transformants to grow on a nonfermentable carbon source. The water soluble F1-ATPase with Ala-328 and Lys-328 were extremely unstable, but could be stabilized with glycerol. The rate of enzymatic decay followed first order kinetics with half-lives of 1.1 and 4.0 min for the mutants with Ala-328 and Lys-328 in 10% and 5% glycerol, respectively, while the wild type enzyme was stable even in the absence of glycerol. Kinetic analysis of both ATPase and GTPase has been determined. The wild type enzyme had two observable apparent Km and Vmax values for ATPase which were 0.056 mM-1 and 67 units/min/mg and 0.140 mM-1 and 100 units/min/mg. The mutant enzyme containing Lys-328 showed similar kinetic values of 0.066 mM-1 and 23 units/min/mg and 0.300 mM-1 and 43 units/min/mg. The mutant enzyme containing Ala-328, however, only demonstrated a single site with values of 0.121 mM-1 and 45 units/min/mg. In contrast to ATPase activity, kinetic values for GTPase were nearly identical for the wild type and mutant enzymes. Opposite to predicted results, the mutant enzymes were more sensitive to the reagent phenylglyoxal. These results indicate that Arg-328 is important for protein stability, but not involved in catalysis.

Secretory vesicles externalize the major plasma membrane ATPase in yeast.

Yeast cell surface growth is accomplished by constitutive secretion and plasma membrane assembly, culminating in the fusion of vesicles with the bud membrane. Coordination of secretion and membrane assembly has been investigated by examining the biogenesis of plasma membrane ATPase (PM ATPase) in secretion-defective (sec) strains of Saccharomyces cerevisiae. PM ATPase is synthesized as a approximately 106-kD polypeptide that is not detectably modified by asparagine-linked glycosylation or proteolysis during transit to the plasma membrane. Export of the PM ATPase requires the secretory pathway. In sec1, a mutant defective in the last step of secretion, large amounts of Golgi-derived vesicles are accumulated. Biochemical characterization of this organelle has demonstrated that PM ATPase and the secretory enzyme, acid phosphatase, are transported in a single vesicle species.

51] Genetics of Kdp, the K+-transport ATPase of Escherichia coli

This chapter describes the application of a variety of widely used techniques of bacterial genetics to the study of Kdp. Analogous genetic methods are already available for lower eukaryotes, such as yeast, but at present only a few are readily applied to higher eukaryotes. The rapid progress of genetics gives promise that the ease and extent of genetic dissection in bacteria and yeast will soon be possible in higher eukaryotes. Kdp is a complex of three-membrane proteins that form a K + -transport ATPase in Escherichia coli . Kdp belongs to the E l -E 2 class of transport ATPases, as shown by the formation of an acyl phosphate intermediate, and by homology to the Ca 2+ -ATPase and the Na + , K + -ATPase of animal cells, and to H + -ATPases of yeast and fungi. Genetic analysis has been important from the outset in the identification and characterization of the Kdp transport ATPase, in contrast to eukaryotic transport ATPases, where genetics has only recently added to the extensive information obtained by biochemical techniques. To analyze the kdp genotype of a strain and to construct strains with particular kdp genotypes for mapping, complementation analysis, or cloning, it is useful to be able to move kdp mutations from one strain to another. A convenient way to move them is cotransduction by bacteriophage P1 with a nearby marker. Three useful markers close to kdp are nagA, gltA, and nadA.

Lipid requirements of the plasma membrane ATPases from oat roots and yeast

The lipid specificity of the plasma membrane ATPases from oat roots and yeast has been investigated by reconstituting delipidated enzyme with phospholipid vesicles and with micelles of lysophospholipids and other detergents. The plant ATPase is activated by Triton X-100 and by all phospholipid and lysophospholipid species, exhibiting only a slight preference for zwitterionic polar heads (phosphorylcholine and phosphorylethanolamine). No unsaturation is required on the hydrophobic chain. On the other hand, the yeast ATPase requires a negatively charged polar head (with preference for phosphorylglycerol and phosphorylinositol) and an unsaturated hydrophobic chain.

Inhibition of the proton pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide

The inhibition of the proton-pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide (DCCD) can be correlated with the covalent incorporation of the inhibitor. Full inhibition of the yeast enzyme required the incorporation of about 1 mol DCCD/mol of the ATPase polypeptide of 100 kDa. A kinetic study of the interaction of DCCD with the yeast and oat ATPases indicates a second-order rate constant of about 500 m −1 min −1 and a stoichiometry of 1 mol DCCD/mol of enzyme, in agreement with the amount of DCCD incorporated by the yeast enzyme. It is proposed that DCCD reacts with a single carboxylic group present in a hydrophobic region of these proton-pumping ATPases and which could participate in proton binding and transport.

Complete amino-acid sequence of the natural ATPase inhibitor from the mitochondria of the yeast Candida utilis.

The complete alignment of the 63 residues of the mitochondrial ATPase inhibitor from the yeast Candida utilis has been determined. The sequence study was carried out mainly by automatic (liquid and solid-phase) methods. Peptides were obtained by enzymatic digestion with clostripain and purified by reverse-phase high-performance liquid chromatography. The ATPase inhibitor contains three sets of repetitive sequences and eight clusters of charged residues, as also found in the inhibitor of Saccharomyces cerevisiae, with which it shares 58.7% homology of conserved residues. When the two yeast ATPase inhibitor sequences were compared to that of beef heart, 20 residues remained common to the three alignments, although the latter protein contained a long histidine-rich insertion, only found in this inhibitor. Most of the homologous residues were clustered near the center of the protein, which by partial proteolytic digestion of the beef heart ATPase inhibitor [Dianoux, A.C. et al. (1982) FEBS Lett. 140, 223-228] has already been shown to be involved in the biological function.

Nucleotide Sequence of ATPase Subunit 6 Gene of Maize Mitochondria

The ATPase subunit 6, located in the inner mitochondrial membrane, is encoded by mitochondrial genomes in animals and fungi. We have isolated and characterized a mitochondrial gene, designated atp 6, that encodes the subunit 6 polypeptide of Zea mays. Nucleotide and predicted amino acid sequence comparisons have revealed a homology of 44.6 and 33.2% with the yeast ATPase subunit 6 gene and polypeptide, respectively. The predicted protein in maize contains 291 amino acids with a molecular weight of 31,721. Hydropathy profiles generated for the maize and yeast polypeptides are very similar and contain large hydrophobic domains, characteristic of membrane bound proteins. RNA transfer blot analysis indicates that atp 6 is actively transcribed. Interestingly, 122 base pairs of nucleotide sequence interior to atp 6 have extensive homology with the 5' end of the cytochrome oxidase subunit II gene of maize mitochondria, suggesting recombination between the two genes.

What family of ATPases does the vacuolar H+-ATPase belong to?

Polyacrylamide gel electrophoresis (PAGE) of partially purified ATPase from vacuoles of Saccharomyces carlsbergensis under non-dissociating conditions revealed 3 bands with ATPase activity. Further PAGE in dissociating conditions showed the similarity in composition of these 3 ATPase preparations. They were assumed to contain the same vacuolar ATPase exhibiting, however, different electrophoretic mobility due to the formation of enzyme complexes with different proteins and phospholipids. The ATPase preparation with the highest electrophoretic mobility contained 6 subunits of 75, 62, 16, 14, 12 and 9 kDa. Inhibitors of vacuolar ATPase [ 14C]DCCD and [ 14]NEM bound to a 9 kDa polypeptide, while [ 14C]DES associated with a polypeptide of 75 kDa. A partially purified preparation of the vacuolar ATPase was not phosphorylated by [γ- 32P]ATP under conditions when plasma membrane ATPase formed a phosphorylated intermediate. Our results show that vacuolar H +-ATPase consists of several polypeptides, does not form the phosphorylated intermediate, and evidently represents a new type of H +-ATPase of yeast.