The plant Ca2+ -ATPase repertoire: biochemical features and physiological functions.

Ca(2+)-ATPases are P-type ATPases that use the energy of ATP hydrolysis to pump Ca(2+) from the cytoplasm into intracellular compartments or into the apoplast. Plant cells possess two types of Ca(2+) -pumping ATPase, named ECAs (for ER-type Ca(2+)-ATPase) and ACAs (for auto-inhibited Ca(2+)-ATPase). Each type comprises different isoforms, localised on different membranes. Here, we summarise available knowledge of the biochemical characteristics and the physiological role of plant Ca(2+)-ATPases, greatly improved after gene identification, which allows both biochemical analysis of single isoforms through heterologous expression in yeast and expression profiling and phenotypic analysis of single isoform knock-out mutants.

Intracellular localisation of PPI1 (proton pump interactor, isoform 1), a regulatory protein of the plasma membrane H(+)-ATPase of Arabidopsis thaliana.

PPI1 (proton pump interactor isoform 1) is a novel protein able to interact with the C-terminal autoinhibitory domain of the Arabidopsis thaliana plasma membrane (PM) H(+)-ATPase. In vitro, PPI1 binds the PM H(+)-ATPase in a site different from the known 14-3-3 binding site and stimulates its activity. In this study, we analysed the intracellular localisation of PPI1. The intracellular distribution was monitored in A. thaliana cultured cells by immunolocalisation using an antiserum against the PPI1 N-terminus and in Vicia faba guard cells and epidermal cells by transient expression of a GFP::PPI1 fusion. The results indicate that the bulk of PPI1 is localised at the endoplasmic reticulum, from which it might be recruited to the PM for interaction with the H(+)-ATPase in response to as yet unidentified signals.

Abscisic acid stimulates the expression of two isoforms of plasma membrane Ca2+-ATPase in Arabidopsis thaliana seedlings.

AT-ACA8 and AT-ACA9 are two plasma membrane (PM) Ca (2+)-ATPases of ARABIDOPSIS THALIANA. In this article the expression of AT-ACA8, AT-ACA9, and of AT-ACA10, a third isoform of Ca (2+)-ATPase closely related to PM Ca (2+)-ATPases, was analysed and the effect of the hormone abscisic acid (ABA) on the expression level of PM Ca (2+)-ATPase specific transcripts was investigated. In adult plants of A. THALIANA, AT-ACA8 and AT-ACA10 are expressed in all organs considered whereas AT-ACA9 is expressed only in flowers. All isoforms of PM Ca (2+)-ATPases can be detected in young seedlings but the amount of AT-ACA9 mRNA is much lower than those of AT-ACA8 and AT-ACA10. ABA markedly and rapidly stimulates the expression of both AT-ACA8 and AT-ACA9 genes in young seedlings but not that of AT-ACA10. ABA also increases the level of AT-ACA8 protein at the PM, suggesting a role for PM Ca (2+)-ATPases in ABA signalling.

Tyrosine phosphorylation inhibits the interaction of 14-3-3 proteins with the plant plasma membrane H+-ATPase.

Interaction of 14-3-3 proteins with their targets depends not only on the phosphorylation status of the target but also on that of 14-3-3 (Fu et al., 2000). In this work we demonstrated that the maize 14-3-3 isoform GF14-6 is a substrate of the tyrosine kinase insulin growth factor receptor 1. By means of site-directed mutants of GF14-6, we identified Tyr-137 as the specific tyrosine residue phosphorylated by the insulin growth factor receptor 1. Phosphorylation of GF14-6 on Tyr-137 lowered its affinity for a peptide mimicking the 14-3-3 binding site of the plant plasma membrane H+-ATPase. Moreover, phosphorylation in planta of 14-3-3 tyrosine residues, resulting from incubation with the tyrosine phosphatase inhibitor, phenylarsine oxide, decreased their association to the H+-ATPase.

Involvement of the plasma membrane Ca2+-ATPase in the short-term response of Arabidopsis thaliana cultured cells to oligogalacturonides.

Treatment of Arabidopsis thaliana cells with oligogalacturonides (OG) initiates a transient production of reactive oxygen species (ROS), the concentration of which in the medium peaks after about 20 min of treatment. The analysis of OG effects on Ca (2+) fluxes shows that OG influence both Ca (2+) influx and Ca (2+) efflux (measured as (45)Ca (2+) fluxes) in a complex way. During the first 10 - 15 min, OG stimulate Ca (2+) influx and decrease its efflux, while at successive times of treatment, OG cause an increase of Ca (2+) efflux and a slight decrease of its influx. Treatment with sub- micro M concentrations of eosin yellow (EY), which selectively inhibits the Ca (2+)-ATPase of plasma membrane (PM), completely prevents the OG-induced increase in Ca (2+) efflux. EY also suppresses the transient feature of OG-induced ROS accumulation, keeping the level of ROS in the medium high. The biochemical analysis of PM purified from OG-treated cells indicates that treatment with OG for 15 to 45 min induces a significant decrease in Ca (2+)-ATPase activation by exogenous calmodulin (CaM), and markedly increases the amount of CaM associated with the PM. During the same time span, OG do not influence the expression of At-ACA8, the main isoform of PM Ca (2+)-ATPase in suspension-cultured A. thaliana cells, and of CaM genes. Overall, the reported results demonstrate that the PM Ca (2+)-ATPase is involved in the response of plant cells to OG and is essential in regulation of the oxidative burst.

H(+)/Ca(2+) exchange driven by the plasma membrane Ca(2+)-ATPase of Arabidopsis thaliana reconstituted in proteoliposomes after calmodulin-affinity purification.

The plasma membrane Ca(2+)-ATPase was purified from Arabidopsis thaliana cultured cells by calmodulin (CaM)-affinity chromatography and reconstituted in proteoliposomes by the freeze-thaw sonication procedure. The reconstituted enzyme catalyzed CaM-stimulated 45Ca(2+) accumulation and H(+) ejection, monitored by the increase of fluorescence of the pH probe pyranine entrapped in the liposomal lumen during reconstitution. Proton ejection was immediately reversed by the protonophore FCCP, indicating that it is not electrically coupled to Ca(2+) uptake, but it is a primary event linked to Ca(2+) uptake in the form of countertransport.

At-ACA8 encodes a plasma membrane-localized calcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus.

A Ca(2+)-ATPase was purified from plasma membranes (PM) isolated from Arabidopsis cultured cells by calmodulin (CaM)-affinity chromatography. Three tryptic fragments from the protein were microsequenced and the corresponding cDNA was amplified by polymerase chain reaction using primers designed from the microsequences of the tryptic fragments. At-ACA8 (Arabidopsis-autoinhibited Ca(2+)-ATPase, isoform 8, accession no. AJ249352) encodes a 1,074 amino acid protein with 10 putative transmembrane domains, which contains all of the characteristic motifs of Ca(2+)-transporting P-type Ca(2+)-ATPases. The identity of At-ACA8p as the PM Ca(2+)-ATPase was confirmed by immunodetection with an antiserum raised against a sequence (valine-17 through threonine-31) that is not found in other plant CaM-stimulated Ca(2+)-ATPases. Confocal fluorescence microscopy of protoplasts immunodecorated with the same antiserum confirmed the PM localization of At-ACA8. At-ACA8 is the first plant PM localized Ca(2+)-ATPase to be cloned and is clearly distinct from animal PM Ca(2+)-ATPases due to the localization of its CaM-binding domain. CaM overlay assays localized the CaM-binding domain of At-ACA8p to a region of the N terminus of the enzyme around tryptophan-47, in contrast to a C-terminal localization for its animal counterparts. Comparison between the sequence of At-ACA8p and those of endomembrane-localized type IIB Ca(2+)-ATPases of plants suggests that At-ACA8 is a representative of a new subfamily of plant type IIB Ca(2+)-ATPases.

Phenylarsine oxide inhibits the fusicoccin-induced activation of plasma membrane H(+)-ATPase.

To investigate the mechanism by which fusicoccin (FC) induces the activation of the plasma membrane (PM) H(+)-ATPase, we used phenylarsine oxide (PAO), a known inhibitor of protein tyrosine-phosphatases. PAO was supplied in vivo in the absence or presence of FC to radish (Raphanus sativus L.) seedlings and cultured Arabidopsis cells prior to PM extraction. Treatment with PAO alone caused a slight decrease of PM H(+)-ATPase activity and, in radish, a decrease of PM-associated 14-3-3 proteins. When supplied prior to FC, PAO drastically inhibited FC-induced activation of PM H(+)-ATPase, FC binding to the PM, and the FC-induced increase of the amount of 14-3-3 associated with the PM. On the contrary, PAO was completely ineffective on all of the above-mentioned parameters when supplied after FC. The H(+)-ATPase isolated from PAO-treated Arabidopsis cells maintained the ability to respond to FC if supplied with exogenous, nonphosphorylated 14-3-3 proteins. Altogether, these results are consistent with a model in which the dephosphorylated state of tyrosine residues of a protein(s), such as 14-3-3 protein, is required to permit FC-induced association between the 14-3-3 protein and the PM H(+)-ATPase.

Purification of the Plasma Membrane Ca2+-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography

The Ca2+-ATPase of the plasma membrane (PM) of germinating radish (Raphanus sativus L.) seeds was purified by calmodulin (CaM)-affinity chromatography using a batch procedure. PM purified by aqueous two-phase partitioning was solubilized with n-dodecyl beta-d-maltoside and applied to a CaM-agarose matrix. After various washings with decreasing Ca2+ concentrations, the Ca2+-ATPase was eluted with 5 mm ethylenediaminetetraacetate (EDTA). The EDTA-eluted fraction contained about 25% of the loaded Ca2+-ATPase activity, with a specific activity 70-fold higher than that of the starting PM fraction. The EDTA-eluted fraction was highly enriched in a 133-kD polypeptide, which was identified as the PM Ca2+-ATPase by 125I-CaM overlay and fluorescein-isothiocyanate labeling. The PM Ca2+-ATPase cross-reacted with an antiserum against a putative Ca2+-ATPase of the Arabidopsis thaliana chloroplast envelope.

Fusicoccin binding to its plasma membrane receptor and the activation of the plasma membrane H(+)-ATPase. IV. Fusicoccin induces the association between the plasma membrane H(+)-ATPase and the fusicoccin receptor.

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H(+)-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H(+)-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H(+)-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H(+)-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H(+)-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H(+)-ATPase antiserum and with an anti-14-3-3 antiserum indicated an FC-induced association of FCBP with the PM H(+)-ATPase. Analysis of the activation state of PM H(+)-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.

Changes in the level and activation state of the plasma membrane H(+)-ATPase during aging of red beet slices.

The effect of aging on the plasma membrane (PM) H(+)-ATPase of red beet (Beta vulgaris L.) parenchyma discs was analyzed in PM purified by aqueous two-phase partitioning. Aging increased both the activity in the amount of immunodetectable H(+)-ATPase in the PM. The activity assayed at slightly alkaline pH values increased earlier and more strongly than that assayed at acidic pH values, so that the pH curve of the enzyme from aged beet discs was shifted toward more alkaline values. Aging decreased the stimulation of the PM H(+)-ATPase activity by controlled trypsin treatments or by lysophosphatidylcholine. After trypsin treatment the pH dependence of H(+)-ATPase from dormant or aged beet discs became equal. These results indicate that aging not only increases the level of H(+)-ATPase in the PM, but also determines its activation, most likely by modifying the interaction between the autoinhibitory carboxyl-terminal domain and the catalytic site. When the PM H(+)-ATPase activity was assayed at a slightly alkaline pH, the tyrosine modifier N-acetylimidazole inhibited the H(+)-ATPase in the PM from dormant beet discs much less than in the PM from aged discs, suggesting that modification of a tyrosine residue may be involved in the activation of the PM H(+)-ATPase induced by aging. The results are discussed with regard to aging-induced development of transmembrane transport activities.

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase (III. Is There a Direct Interaction between the Fusicoccin Receptor and the Plasma Membrane H+-ATPase?).

A radioimmunoassay using antibodies raised against bovine serum albumin-conjugated fusicoccin (FC) was applied to measure FC bound to the plasma membrane (PM) isolated from seedlings of radish (Raphanus sativus L.) and of Arabidopsis thaliana treated in vivo plus or minus the toxin. FC bound to the PM from seedlings treated with 5 [mu]M FC was 2-fold (radish) to 7-fold (A. thaliana) higher than the binding capacity of control PM. FC binding depended on the duration of the in vivo treatment but was unaffected by cycloheximide. When FC binding and the PM H+-ATPase activity were compared under different conditions (in vivo or in vitro treatment of different lengths or with different concentrations of FC), a strict linear relation between FC binding and the activation of the PM H+-ATPase was observed in both plant materials under all the conditions tested. Comparison between the maximum binding capacity and the amount of H+-ATPase observed in PM from the two plant materials suggest a one-to-one stoichiometry between the FC receptor and the PM H+-ATPase.

Identification of the Plasma Membrane Ca2+-ATPase and of Its Autoinhibitory Domain.

The effect of controlled proteolysis on the plasma membrane (PM)Ca2+-ATPase was studied at the molecular level in PM purified from radish (Raphanus sativus L.) seedlings. Two new methods for labeling the PM Ca2+-ATPase are described. The PM Ca2+-ATPase can be selectively labeled by treatment with micromolar fluorescein isothiocyanate (FITC), a strong inhibitor of enzyme activity. Both inhibition of activity and FITC binding to the PM Ca2+-ATPase are suppressed by millimolar MgITP. The PM Ca2+-ATPase maintains the capability to bind calmodulin also after sodium dodecyl sulfate gel electrophoresis and blotting; therefore, it can be conveniently identified by 125l-calmodulin overlay in the presence of calcium. With both methods a molecular mass of 133 kD can be calculated for the PM Ca2+-ATPase. FITC-labeled PM Ca2+-ATPase co-migrates with the phosphorylated intermediate of the enzyme[mdash]labeled by incubation with [[gamma]-32P]GTP in the presence of calcium[mdash]on acidic sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Controlled trypsin treatment of purified PM determines a reduction of the molecular mass of the PM Ca2+-ATPase from 133 to 118 kD parallel to the increase of enzyme activity. Only the 133-kD but not the 118-kD PM Ca2+-ATPase binds calmodulin. These results indicate that trypsin removes from the PM Ca2+-ATPase an autoinhibitory domain that contains the calmodulin-binding domain of the enzyme.

Controlled Proteolysis Activates the Plasma Membrane Ca2+ Pump of Higher Plants (A Comparison with the Effect of Calmodulin in Plasma Membrane from Radish Seedlings).

The effects of calmodulin and of controlled trypsin treatments on the activity of the Ca2+ pump were investigated in plasma membrane purified from radish (Raphanus sativus L.) seedlings. Treatment of the plasma membrane with ethylenediaminetetra-acetate (EDTA), which removed about two-thirds of the plasma membrane-associated calmodulin, markedly increased the stimulation of the Ca2+ pump by calmodulin. In EDTA-treated plasma membrane, stimulation by calmodulin of the Ca2+ pump activity was maximal at low free Ca2+ (2-5 [mu]M) and decreased with the increase of free Ca2+ concentration. The Ca2+ pump activity was stimulated also by a controlled treatment of the plasma membrane with trypsin: the effect of trypsin treatment depended on the concentration of both trypsin and plasma membrane proteins and on the duration of incubation. Stimulation of the Ca2+ pump activity by trypsin treatment of the plasma membrane was similar to that induced by calmodulin both in extent and in dependence on the free Ca2+ concentration in the assay medium. Moreover, the Ca2+ pump of trypsin-treated plasma membrane was insensitive to further stimulation by calmodulin, suggesting that limited proteolysis preferentially cleaves a regulatory domain of the enzyme that is involved in its activation by calmodulin.

Controlled Proteolysis Mimics the Effect of Fusicoccin on the Plasma Membrane H+-ATPase.

We analyzed the effects of controlled treatments with trypsin of plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings on the activity of the PM H+-ATPase, and we compared them with those of fusicoccin (FC). Mild treatments of the PM with trypsin, which led to a decrease of the molecular mass of the peptide of about 10 kD, markedly increased the H+-ATPase activity. The effect strongly increased with the increase of pH of the assay medium from 6.1 to 7.5, so the pH optimum of the enzyme activity shifted from 6.8 in untreated PM to 7.1 in trypsin-treated PM. The proteolytic treatment activated only the portion of PM H+-ATPase activity that is stable to preincubation in assay medium in the absence of ATP and determined a strong increase of Vmax and a less marked decrease of the apparent Km for Mg-ATP. All of these effects were very similar to those determined by FC, which activated the PM H+-ATPase without promoting its proteolytic cleavage. FC did not further activate the H+-ATPase activity of trypsin-treated PM under conditions in which the FC receptor was protected from the attack of trypsin. Conversely, trypsin treatment had little effect on the PM H+-ATPase preactivated with FC. Moreover, the activity of the PM H+-ATPase preactivated with FC was not further activated by Iysolecithin. These results indicate that the modification of the PM H+-ATPase of higher plants triggered by the FC-receptor complex hinders the inhibitory interaction of the regulatory C-terminal domain with the active site.

Plasma Membrane Ca-ATPase of Radish Seedlings : I. Biochemical Characterization Using ITP as a Substrate.

In this work, we exploited the capability of the plasma membrane Ca-ATPase to utilize ITP as a substrate to study its characteristics in plasma membrane vesicles purified from radish (Raphanus sativus L.) seedlings. The majority of the ITPase activity of plasma membrane was Ca(2+)-dependent. The Ca(2+)-dependent ITPase activity was Mg(2+)-dependent and was stimulated by the calcium ionophore A23187. It was inhibited by erythrosin B (concentration giving 50% inhibition, 50 nanomolar) and by vanadate (concentration giving 50% inhibition, 3 micromolar) and displayed a broad pH optimum around pH 7.2 to 7.5. Both the hydrolytic and the transport activity of the plasma membrane Ca-ATPase were half-saturated by Ca(2+) in the micromolar concentration range. No major effect of EGTA on the saturation kinetics of the enzyme was observed. The affinity of the plasma membrane Ca-ATPase for Ca(2+) was about fourfold higher at pH 7.5 than at pH 6.9. The Ca(2+)-dependent ITPase activity was stimulated about twofold by polyoxyethylene 20 cetyl ether, although it was inhibited by Triton X-100 and by lysolecithin.

Plasma Membrane Ca-ATPase of Radish Seedlings : II. Regulation by Calmodulin.

The effect of calmodulin on the activity of the plasma membrane Ca-ATPase was investigated on plasma membranes purified from radish (Raphanus sativus L.) seedlings. Calmodulin stimulated the hydrolytic activity and the transport activity of the plasma membrane Ca-ATPase to comparable extents in a manner dependent on the free Ca(2+) concentration. Stimulation was marked at low, nonsaturating Ca(2+) concentrations and decreased increasing Ca(2+), so that the effect of calmodulin resulted in an increase of the apparent affinity of the enzyme for free Ca(2+). The pattern of calmodulin stimulation of the plasma membrane Ca-ATPase activity was substantially the same at pH 6.9 and 7.5, in the presence of ATP or ITP, and when calmodulin from radish seeds was used rather than that from bovine brain. At pH 6.9 in the presence of 5 micromolar free Ca(2+), stimulation of the plasma membrane Ca-ATPase was saturated by 30 to 50 micrograms per milliliter bovine brain calmodulin. The calmodulin antagonist calmidazolium inhibited both basal and calmodulin-stimulated plasma membrane Ca-ATPase activity to comparable extents.

Identification and Characterization of the Ca-ATPase which Drives Active Transport of Ca at the Plasma Membrane of Radish Seedlings.

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca(2+) uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MI De Michelis [1987] Plant Physiol 83: 994-1000). A Ca(2+)-dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca(2+)-dependent ATPase is stimulated by the Ca(2+) ionophore A(23187) and is localized at the plasma membrane. Ca(2+)-dependent ATPase activity and ATP-dependent Ca(2+) uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca(2+) (half-maximal rate at about 70 nanomolar), and MgATP (K(m) 15-20 micromolar). Ca(2+) uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca(2+) uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca(2+)-ATPase described in this paper is the enzyme which drives active transport of Ca(2+) at the plasma membrane of higher plants.

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H-ATPase: I. Characteristics and Intracellular Localization of the Fusicoccin Receptor in Microsomes from Radish Seedlings.

The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a K(d) of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (K(d) 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H(+)-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H(+)-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H(+)-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange.

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca(2+) upon addition of MgATP. MgATP-dependent Ca(2+) uptake co-migrates with the plasma membrane H(+)-ATPase on a sucrose gradient. Ca(2+) uptake is insensitive to oligomycin, inhibited by vanadate (IC(50) 40 micromolar) and erythrosin B (IC(50) 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca(2+) uptake is insensitive to protonophores. These results indicate that Ca(2+) transport in these microsomal vesicles is catalyzed by a Mg(2+)-dependent ATPase localized on the plasma membrane. Ca(2+) strongly reduces DeltapH generation by the plasma membrane H(+)-ATPase and increases MgATP-dependent membrane potential difference (Deltapsi) generation. These effects of Ca(2+) on DeltapH and Deltapsi generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca(2+) uptake into plasma membrane vesicles. The Ca(2+)-induced increase of Deltapsi is collapsed by permeant anions, which do not affect Ca(2+)-induced decrease of DeltapH generation by the plasma membrane H(+)-ATPase. The rate of decay of MgATP-dependent DeltapH, upon inhibition of the plasma membrane H(+)-ATPase, is accelerated by MgATP-dependent Ca(2+) uptake, indicating that the decrease of DeltapH generation induced by Ca(2+) reflects the efflux of H(+) coupled to Ca(2+) uptake into plasma membrane vesicles. It is therefore proposed that Ca(2+) transport at the plasma membrane is mediated by a Mg(2+)-dependent ATPase which catalyzes a nH(+)/Ca(2+) exchange.