Phosphorylation of yeast plasma membrane H+-ATPase by casein kinase I

The plasma membrane H+-ATPase of Saccharomyces cerevisiae is subject to phosphorylation by a casein kinase I activity in vitro. We show this casein kinase I activity to result from the combined function of YCK1 and YCK2, two highly similar and plasma membrane-associated casein kinase I homologues. First, H+-ATPase phosphorylation is severely impaired in the plasma membrane of YCK-deficient yeast strains. Furthermore, the wild-type level of the phosphoprotein is restored by the addition of purified mammalian casein kinase I to the mutant membranes. We used the H+-ATPase as well as a synthetic peptide substrate that contains a phosphorylation site for casein kinase I to compare kinase activity in membranes prepared from yeast cells grown in the presence or absence of glucose. The addition of glucose results in increased H+-ATPase activity which is associated with a decline in the phosphorylation level of the enzyme. Mutations in both YCK1 and YCK2 affect this regulation, suggesting that H+-ATPase activity is modulated by glucose via a combination of a "down-regulating" casein kinase I activity and another, yet uncharacterized, "up-regulating" kinase activity. Biochemical mapping of phosphorylated H+-ATPase identifies a major phosphopeptide that contains a consensus phosphorylation site (Ser-507) for casein kinase I. Site-directed mutagenesis of this consensus sequence indicates that Glu-504 is important for glucose-induced decrease in the apparent Km for ATP.     

Three-dimensional map of the plasma membrane H+-ATPase in the open conformation

The H+-ATPase from the plasma membrane of Neurospora crassa is an integral membrane protein of relative molecular mass 100K, which belongs to the P-type ATPase family that includes the plasma membrane Na+/K+-ATPase and the sarcoplasmic reticulum Ca2+-ATPase. The H+-ATPase pumps protons across the cell's plasma membrane using ATP as an energy source, generating a membrane potential in excess of 200mV. Despite the importance of P-type ATPases in controlling membrane potential and intracellular ion concentrations, little is known about the molecular mechanism they use for ion transport. This is largely due to the difficulty in growing well ordered crystals and the resulting lack of detail in the three-dimensional structure of these large membrane proteins. We have now obtained a three-dimensional map of the H+-ATPase by electron crystallography of two-dimensional crystals grown directly on electron microscope grids. At an in-plane resolution of 8 A, this map reveals ten membrane-spanning alpha-helices in the membrane domain, and four major cytoplasmic domains in the open conformation of the enzyme without bound ligands.     

Complementation of the Saccharomyces cerevisiae plasma membrane H+-ATPase by a plant H+-ATPase generates a highly abundant fusicoccin binding site

Accumulating evidence suggests that the H+-ATPase of the plant plasma membrane is activated by a direct, reversible interaction with 14-3-3 proteins involving the displacement of the C-terminal autoinhibitory domain of the enzyme. The fungal phytotoxin fusicoccin (FC) appears to stabilize this H+-ATPase.14-3-3 complex, thus leading to a persistent activation of the H+-ATPase in vivo. In this study we show that functional replacement of the Saccharomyces cerevisiae H+-ATPase genes by a Nicotiana plumbaginifolia H+-ATPase (pma2) results in the generation of a high affinity fusicoccin binding site that is exceptionally abundant. Acquisition of FC binding capacity is accompanied by a significant increase in the amount of plasma membrane-associated yeast 14-3-3 homologs. The existence of a (plant) PMA2.(yeast)14-3-3 complex was demonstrated using two-dimensional gel systems (native/denaturing). After expression of PMA2 lacking most of its C-terminal region, neither H+-ATPase.14-3-3 complex formation nor FC binding activity could be observed. Furthermore, we obtained direct biochemical evidence for a minimal FC binding complex consisting of the C-terminal PMA2 domain and yeast 14-3-3 homologs. Thus we demonstrated unambiguously the relevance of this regulatory ATPase domain for 14-3-3 interaction as well as its requirement for FC binding.     

Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants

Hematopoiesis is viewed as a differentiating system emanating from a pluripotent hematopoietic stem cell capable of both self-renewal and differentiation. By identifying and characterizing a novel and highly specific in vitro mitogenic response to the N-acetyl glucosamyl/sialic acid specific, stem cell-binding lectin wheat germ agglutinin (WGA), we demonstrate the existance of a rare (0.1%), plastic adherent precursor in rat bone marrow capable of proliferation (two to seven divisions) in response to WGA. Stimulated cells possess a lineage (lin)low/- immunophenotype and immature blastoid morphology (WGA blasts). A subsequent proliferative response to stem cell factor (SCF), the ligand for the proto-oncogene receptor tyrosine kinase c-kit, is characterized by an initial maturation in immunophenotype and subsequent self-renewal of cells (SCF blasts) without differentiation for at least 50 generations. Although granulocyte colony-stimulating factor (G-CSF), interleukin (IL) -6, IL-7, and IL-11 synergize with SCF to increase blast colony formation, cytokines such as granulocyte-macrophage CSF or IL-3 are without significant effect. At all time points in culture, however, cells rapidly differentiate to mature neutrophils with dexamethasone or to mainly monocytes/macrophages in the presence of 1alpha,25-dihydroxyvitamin D3, characterized by cell morphology and cytochemistry. Removal of SCF during blast maturation, self-renewal, or induction of differentiation phases results in apoptotic cell death. Data indicate a pivotal role for SCF/c-kit interaction during antigenic maturation, self-renewal, and apoptotic protection of these lineage-restricted progenitors during non-CSF-mediated induction of differentiation. This approach provides a source of many normal, proliferating myelomonocytic precursor cells, and introduces possible clinical applications of ex vivo expanded myeloid stem cells.     

Inactivation of the Kluyveromyces lactis H+-ATPase by dicyclohexylcarbodiimide: binding stoichiometry and effect of nucleophiles

Dicyclohexylcarbodiimide (DCCD) inactivated the plasma membrane H+-ATPase (EC 3.6.1.35) from Kluyveromyces lactis, with a second-order rate constant of 420 M(-1) min(-1). The inhibition kinetics was apparently complex, due to degradation of DCCD with time. Neither Mg2+ nor Mg-ADP affected the inactivation of the ATPase by DCCD. In contrast, vanadate, a transition state analog of phosphate, partially protected the enzyme with a Kd of 14 microM, indicating a coupling between the DCCD-reactive site and the vanadate-binding site. The incubation of H+-ATPase with 14C-DCCD showed that the incorporation of 1.2 mol of DCCD/mol ATPase leads to complete inactivation. The hydrophobic carbodiimide reacted with the protonated form of the carboxylic group, which displayed a pKa of 7.4, strongly suggesting that the residue is in the hydrophobic environment of the membrane. Benzylamine increased the rate of inactivation by DCCD. In this case, full inactivation of the enzyme was associated with the incorporation of 2.4 mol of DCCD/mol of enzyme, indicating the opening of new reactive sites, resulting from a conformational change induced by benzylamine.     

Modulation of plant plasma membrane H+-ATPase by phytotoxic lipodepsipeptides produced by the plant pathogen Pseudomonas fuscovaginae

OBJECTIVE: Dienogest, a synthetic steroid with progestational activity, is used as a component of oral contraceptives and is currently being evaluated clinically for the treatment of endometriosis. The present study was conducted to confirm the effects of dienogest on experimental endometriosis in rats and to elucidate its mechanism of action. DESIGN: Experimental endometriosis induced by autotransplantation of endometrium in rats. METHODS: Endometrial implants, immune system, and bone mineral were investigated after 3 weeks of medication. RESULTS: Dienogest (0.1-1 mg/kg per day, p.o.) reduced the endometrial implant volume to the same extent as danazol (100 mg/kg per day, p.o.). Simultaneously, dienogest ameliorated the endometrial implant-induced alterations of the immune system: i.e. it increased the natural killer activity of peritoneal fluid cells and splenic cells, decreased the number of peritoneal fluid cells, and decreased interleukin-1beta production by peritoneal macrophages. In contrast, danazol (100 mg/kg per day, p.o.) and buserelin (30 microg/kg per day, s.c.) had none of these immunologic effects. Additionally, combined administration of dienogest (0.1 mg/kg per day) plus buserelin (0.3 microg/kg per day) suppressed the bone mineral loss induced by buserelin alone, with no reduction of the effect on endometrial implants. In vitro studies on dienogest revealed an antiproliferative effect on rat endometrial cells due to inhibition of protein kinase C activity plus a partial progestational effect. CONCLUSIONS: Dienogest appears to be a potent agent with mechanisms of action different from those of danazol and GnRH agonists currently available for the treatment of endometriosis.     

Phage mimotopes of monoclonal antibodies against plasma membrane Ca2+-ATPase

Libraries of random phage-displayed pentadeca- and hexapeptides were screened with the use of four monoclonal antibodies against the human plasma membrane Ca2(+)-ATPase. Bacteriophages specifically binding the antibodies were selected, and the amino acid sequences of the expressed peptides (mimotopes) were determined. Mimotopes for three antibodies (8B8, 2D8, F9) did not correspond to the Ca2(+)-ATPase sequence. Pentadecapeptides for the 7C8 antibodies displayed similarity to the fragment Glu1097-Arg1113 of the Ca2(+)-ATPase calmodulin-binding site. However, these antibodies failed to bind recombinant fragment Leu1069-Leu1220; therefore, the structure of this epitope remains obscure. This work opens a series of studies of the plasma membrane Ca2(+)-ATPase structure by means of monoclonal antibodies and the phage display method.     

Structure-function relationships in membrane segment 5 of the yeast Pma1 H+-ATPase

Membrane segment 5 (M5) is thought to play a direct role in cation transport by the sarcoplasmic reticulum Ca2+-ATPase and the Na+, K+-ATPase of animal cells. In this study, we have examined M5 of the yeast plasma membrane H+-ATPase by alanine-scanning mutagenesis. Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles as described previously (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940-7949). Three substitutions (R695A, H701A, and L706A) led to misfolding of the H+-ATPase as evidenced by extreme sensitivity to trypsin; the altered proteins were arrested in biogenesis, and the mutations behaved genetically as dominant lethals. The remaining mutants reached the secretory vesicles in sufficient amounts to be characterized in detail. One of them (Y691A) had no detectable ATPase activity and appeared, based on trypsinolysis in the presence and absence of ligands, to be blocked in the E1-to-E2 step of the reaction cycle. Alanine substitution at an adjacent position (V692A) had substantial ATPase activity (54%), but was likewise affected in the E1-to-E2 step, as evidenced by shifts in its apparent affinity for ATP, H+, and orthovanadate. Among the mutants that were sufficiently active to be assayed for ATP-dependent H+ transport by acridine orange fluorescence quenching, none showed an appreciable defect in the coupling of transport to ATP hydrolysis. The only residue for which the data pointed to a possible role in cation liganding was Ser-699, where removal of the hydroxyl group (S699A and S699C) led to a modest acid shift in the pH dependence of the ATPase. This change was substantially smaller than the 13-30-fold decrease in K+ affinity seen in corresponding mutants of the Na+, K+-ATPase (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). Taken together, the results do not give firm evidence for a transport site in M5 of the yeast H+-ATPase, but indicate a critical role for this membrane segment in protein folding and in the conformational changes that accompany the reaction cycle. It is therefore worth noting that the mutationally sensitive residues lie along one face of a putative alpha-helix.     

Structure of yeast plasma membrane H(+)-ATPase: comparison of activated and basal-level enzyme forms

Experimental evidence suggests that the myocardial phospholipase D (PLD)-phosphatidate phosphohydrolase (PAP) signalling pathway may regulate Ca2+ movements and contractile performance of the heart. As abnormal Ca2+ homeostasis is associated with diabetic cardiomyopathy, we examined the functional status of the PLD/PAP pathway in sarcolemmal (SL) membranes isolated from insulin-dependent diabetic rat hearts at 8 weeks after a single i.v. injection of streptozotocin (65 mh/kg b.w.). Compared to age-matched controls, SL PLD hydrolytic (producing phosphatidic acid, PtdOH) and transphosphatidylation activities were significantly depressed in diabetic animals, while SL PAP was significantly augmented. The net effect of the altered enzyme activities in diabetic animals was a severely diminished (by 67% of controls) membrane level of PLD-derived PtdOH. Two weeks of insulin therapy to the 6 week diabetic animals normalized PLD, while PAP activity and PtdOH level were significantly modified, but had not completely reverted to control values. The observed changes were not due to hypothyroidism associated to the diabetic model as the induction of hypothyroidism in healthy non-diabetic animals did not affect SL PLD and PAP. The results suggest that the severe reduction of PLD-derived PtdOH and increased production of sn-1,2-diacylglycerol by phosphatidate phosphohydrolase may lead to an impairment of the bioprocesses mediated by these signalling lipids.     

Genetic probing of the stalk segments associated with M2 and M3 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae

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.     

Characterization of yeast plasma membrane H(+)-ATPase mutant pma1-A135V and its revertants

An A135V substitution in the first transmembrane segment of the yeast plasma membrane H(+)-ATPase (PMA1) confers cellular resistance to hygromycin B, exhibits growth sensitivity to low external pH, and results in a defective enzyme that hydrolyzes ATP at 33% of wild type level. The importance of the A135 residue was probed genetically by analysis involving both site-directed mutagenesis and randomly generated second-site intragenic suppressor mutations. No other amino acid at position 135 gave either the wild type phenotype or the normal enzyme activity of A135. Substitutions with the bulkier amino acid residues A135L, A135I, and A135F produced more severe cellular phenotypes than the original A135V mutation. The substitution of the smaller side chain residue Gly was also a mutant, although not as severe as the A135V mutant. The introduction of a bulky Trp or a polar Ser residue produced dominant lethality, while charged amino acids produced recessive lethality. Reduced rates of proton transport measured by acidification of the medium by whole cells correlate closely with the severity of cellular phenotype. Some of the mutant enzymes exhibit an apparent instability in vitro. Thus, the localized structure around A135 is highly constrained. The cellular sensitivity to low external pH of the A135V mutant was used to select intragenic revertants. Most full revertants (low pHR, HygS) restored A135, but second-site mutations in putative transmembrane segments 2 (V146I and V157F) and 4 (L327V) were also observed. Two partial revertants (low pHR, HygR) have secondary mutations at S660C or a double change at F611L-S660F in the putative ATP binding domain. These results provide additional evidence for functional coupling between the cytoplasmic domain catalyzing ATP hydrolysis and transmembrane helices 1 and 2.     

Protein kinase C and calmodulin effects on the plasma membrane Ca2+-ATPase from excitable and nonexcitable cells

We have purified Ca2+-ATPase from synaptosomal membranes (SM)1 from rat cerebellum by calmodulin affinity chromatography. The enzyme was identified as plasma membrane Ca2+-ATPase by its interaction with calmodulin and monoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, and by thapsigargin insensitivity. The purpose of the study was to establish whether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinase C (PKC), affect the Ca2+-ATPase isolated from excitable cells and whether their effects are comparable to those on the RBC Ca2+-ATPase. We found that calmodulin and PKC activated both enzymes. There were significant quantitative differences in the phosphorylation and activation of the SM versus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SM Ca2+-ATPase was approximately 3 fold lower and significantly less stimulated by calmodulin. The initial rate of PKC catalyzed phosphorylation (in the presence of 12-myristate 13-acetate phorbol) was approximately two times slower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approached maximum level at around 5 min, comparable level of phosphorylation of SM Ca2+-ATPase was observed only after 30 min. The PKC-catalyzed phosphorylation resulted in a statistically significant increase in Ca2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase. The differences may be associated with diversities in Ca2+-ATPase function in erythrocytes and neuronal cells and different isoforms composition.     

Activity of plasma membrane H+-ATPase and expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cells grown at optimal and low pH

Cells of Saccharomyces cerevisiae grown in media with an initial pH of 2.5-6.0, acidified with a strong acid (HCl), exhibited the highest plasma membrane H+-ATPase-specific activity at an initial pH of 6.0. At a lower pH (above pH 2.5) ATPase activity (62-83% of the maximum level) still allowed optimal growth. At pH 2.5, ATPase activity was about 30% of the maximum value and growth was impaired. Quantitative immunoassays showed that the content of ATPase protein in the plasma membrane was similar across the entire pH range tested, although slightly lower at pH 2.5. The decrease of plasma membrane ATPase activity in cells grown at low pH was partially accounted for by its in vitro stability, which decreased sharply at pH below 5.5, although the reduction of activity was far below the values expected from in vitro measurements. Yeast growth under acid stress changed the pattern of gene expression observed at optimal pH. The level of mRNA from the essential plasma-membrane-ATPase-encoding gene PMA1 was reduced by 50% in cells grown at pH 2.5 as compared with cells grown at the optimal pH 5.0, although the content of ATPase in the plasma membrane was only modestly reduced. As observed in response to other kinds of stress, the PMA2 promoter at the optimal pH was up to eightfold more efficient in cells grown at pH 2.5, although it remained several hundred times less efficient than that of the PMA1 gene.     

Effects of ADP, DTT, and Mg2+ on the ion-conductive property of chloroplast H+-ATPase(CF0-CF1) reconstituted into bilayer membrane

The purified CF0-CF1 complex of spinach was incorporated into planar lipid bilayer membranes (LBMs) formed with soybean phospholipid, and the transmembrane ion-transmission properties were studied under voltage-clamp mode. The results showed that the presence of both ADP and Pi decreased the membrane current while Dithiothreitol could evoke a stronger conductive change of CF0-CF1 containing LBMs when Ca2+ or Mg2+ exists. Mg2+ can dramatically increase the CF0-CF1 conductance in various conditions. These results indicated that the H(+)-transitive function of CF0-CF1 reconstituted in bilayer is sensitive to those factors which can affect its ATP synthase activity in vivo.     

The PLC1 encoded phospholipase C in the yeast Saccharomyces cerevisiae is essential for glucose-induced phosphatidylinositol turnover and activation of plasma membrane H+-ATPase

Addition of glucose to glucose-deprived cells of the yeast Saccharomyces cerevisiae triggers rapid turnover of phosphatidylinositol, phosphatidylinositol-phosphate and phosphatidylinositol 4,5-bisphosphate. Glucose stimulation of PI turnover was measured both as an increase in the specific ratio of 32P-labeling and as an increase in the level of diacylglycerol after addition of glucose. Glucose also causes rapid activation of plasma membrane H+-ATPase. We show that in a mutant lacking the PLC1 encoded phospholipase C, both processes were strongly reduced. Compound 48/80, a known inhibitor of mammalian phospholipase C, inhibits both processes. However, activation of the plasma membrane H+-ATPase is only inhibited by concentrations of compound 48/80 that strongly inhibit phospholipid turnover. Growth was inhibited by even lower concentrations. Our data suggest that in yeast cells, glucose triggers through activation of the PLC1 gene product a signaling pathway initiated by phosphatidylinositol turnover and involved in activation of the plasma membrane H+-ATPase.     

Intracellular sequestration of sodium by a novel Na+/H+ exchanger in yeast is enhanced by mutations in the plasma membrane H+-ATPase. Insights into mechanisms of sodium tolerance

Sodium tolerance in yeast is disrupted by mutations in calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, which is required for modulation of Na+ uptake and efflux mechanisms. Five Na+-tolerant mutants were isolated by selecting for suppressors of calcineurin mutations, and mapped to the PMA1 gene, encoding the plasma membrane H+-ATPase. One mutant, pma1-alpha4, which has the single amino acid change Glu367 --> Lys at a highly conserved site within the catalytic domain of the ATPase, was analyzed in detail to determine the mechanism of Na+ tolerance. After exposure to Na+ in the culture medium, 22Na influx in the pma1 mutant was reduced 2-fold relative to control, consistent with a similar decrease in ATPase activity. Efflux of 22Na from intact cells was relatively unchanged in the pma1 mutant. However, selective permeabilization of the plasma membrane revealed that mutant cells retained up to 80% of intracellular Na+ within a slowly exchanging pool. We show that NHX1, a novel gene homologous to the mammalian NHE family of Na+/H+ exchangers, is required for Na+ sequestration in yeast and contributes to the Na+-tolerant phenotype of pma1-alpha4.     

Effect of H+ extracellular and intracellular buffering capacity on the activity Ca2+/H+ exchange in the lymphocyte plasma membrane

Formation of delta pH is registered when studying Ca2+ passive transport through lymphocytes' plasma membrane (PM). The pHi values strongly depended on pH0. Changes of pH0 lead to unidirectional changes of pHi and affect Ca2+ concentration in cytoplasm of the intact cells. The presence of Ca(2+)-channels antagonists does not affect this phenomenon. Ca2+/H+ exchange is supposed to exist in PM. It is also of great interest that cytoplasmic Ca2+ and H+ activities are some equal in physiological range. Besides, H(+)-buffering as well as Ca(2+)-buffering systems are present in the cell and have their maximal capacity about 7.2 in the intact cells. The spectrofluorimetric study of internal lymphocytes' H(+)-buffering capacity with titration technique using weak base, acid or other buffer addition has demonstrated maximal value of 9.0-1.1 mM depending on the substance to be added.     

Irreversible inhibition of plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+ transport by 4-OH-2,3-trans-nonenal

4-OH-2,3-trans-nonenal (HNE), a major aldehydic lipid peroxidation product, has been shown to cause cellular toxicities and has been linked to a number of pathophysiological processes including atherogenesis. Specifically, in vitro exposure of erythrocyte plasma membrane preparations to HNE resulted in the inhibition of membrane transport function and integrity. To characterize the nature of the inhibitory effects of HNE on plasma membrane regulatory mechanisms, we investigated its effects on substrate and calmodulin (CaM) stimulation on erythrocyte Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities. Concentration-effect relationship analysis in erythrocyte membrane "ghosts" and inside-out vesicles (IOVs) yielded purely noncompetitive kinetics for Ca2+, ATP, and CaM activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport. Reductions of Vmax from direct addition of 0.1 mM HNE to the assay incubation mixtures ranged from 23 to 41%. Similarly, pretreatment with HNE of both membrane ghosts and IOVs resulted in a concentration-dependent inactivation of ATPase and transport activities without changes in affinity for Ca2+, ATP, or CaM. Conversely, pretreatment of CaM itself did not impair its ability to stimulate (Ca2+ + Mg2+)-ATPase activity threefold. Moreover, HNE-pretreated membranes exhibited unaltered acetylcholinesterase activity compared to sham-pretreated membranes. Together, these results suggest that HNE may structurally, and thus irreversibly, modify one or more functionally important sites on the transport protein itself.     

Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region

The Nicotiana plumbaginifolia pma2 (plasma membrane H+-ATPase) gene is capable of functionally replacing the H+-ATPase genes of the yeast Saccharomyces cerevisiae, provided that the external pH is kept above 5.0. Single point mutations within the pma2 gene were previously identified that improved H+-ATPase activity and allowed yeast growth at pH 4.0. The aim of the present study was to identify most of the PMA2 positions, the mutation of which would lead to improved growth and to determine whether all these mutations result in similar enzymatic and structural modifications. We selected additional mutants in total 42 distinct point mutations localized in 30 codons. They were distributed in 10 soluble and membrane regions of the enzyme. Most mutant PMA2 H+-ATPases were characterized by a higher specific activity, lower inhibition by ADP, and lower stimulation by lysophosphatidylcholine than wild-type PMA2. The mutants thus seem to be constitutively activated. Partial tryptic digestion and immunodetection showed that the PMA2 mutants had a conformational change making the C-terminal region more accessible. These data therefore support the hypothesis that point mutations in various H+-ATPase parts displace the inhibitory C-terminal region, resulting in enzyme activation. The high density of mutations within the first half of the C-terminal region suggests that this part is involved in the interaction between the inhibitory C-terminal region and the rest of the enzyme.