Binding of phosphatidic acid to 14-3-3 proteins hampers their ability to activate the plant plasma membrane H+-ATPase

Phosphatidic acid is a phospholipid second messenger implicated in various cellular processes in eukaryotes. In plants, production of phosphatidic acid is triggered in response to a number of biotic and abiotic stresses. Here, we show that phosphatidic acid binds to 14-3-3 proteins, a family of regulatory proteins which bind client proteins in a phosphorylation-dependent manner. Binding of phosphatidic acid involves the same 14-3-3 region engaged in protein target binding. Consequently, micromolar phosphatidic acid concentrations significantly hamper the interaction of 14-3-3 proteins with the plasma membrane H(+)-ATPase, a well characterized plant 14-3-3 target, thus inhibiting the phosphohydrolitic enzyme activity. Moreover, the proton pump is inhibited when endogenous PA production is triggered by phospholipase D and the G protein agonist mastoparan-7. Hence, our data propose a possible mechanism involving PA that regulates 14-3-3-mediated cellular processes in response to stress.     

Mutational analysis of the interaction between 14-3-3 proteins and plant plasma membrane H+-ATPase

In this study, we report on mutational studies performed to investigate the mechanism of binding of 14-3-3 proteins to the plasma membrane H(+)-ATPase of plant cells. In fact, although the molecular basis of the interaction between 14-3-3 and the known mode-1 and mode-2 consensus sequences are well characterized, no information is available regarding the association with the H(+)-ATPase, which contains the novel binding site YTV totally unrelated to the 14-3-3 canonical motifs. To this purpose, different mutants of the maize 14-3-3 GF14-6 isoform were produced and used in interaction studies with the plasma membrane H(+)-ATPase and with a peptide reproducing the 14-3-3 binding site of the enzyme. The ability of 14-3-3 mutants to stimulate H(+)-ATPase activity was also tested. To investigate the mechanism of fusicoccin-dependent interaction, binding experiments between 14-3-3 proteins and mutants of the extreme portion of the H(+)-ATPase C terminus were also carried out. The results demonstrate that mutations of Lys(56) and Val(185) within the amphipathic groove disrupt the ability of GF14-6 to interact with H(+)-ATPase and to stimulate its activity. Moreover, substitution of Asp(938) and Asp(940) in the MHA2 H(+)-ATPase C terminus greatly decreased association with GF14-6, thereby demonstrating a crucial role of negatively charged residues in the fusicoccin-dependent interaction.     

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.     

Regulatory 14-3-3 proteins bind the atypical motif within the C terminus of the plant plasma membrane H(+)-ATPase via their typical amphipathic groove

The plant plasma membrane H(+)-ATPase contains a C-terminal autoinhibitory domain whose displacement from the catalytic site is caused by binding of regulatory 14-3-3 proteins. Members of the highly conserved 14-3-3 family bind their individual target proteins in a sequence-specific and phosphorylation-dependent manner within a central groove, the latter characterized by the presence of highly invariant residues. However, an atypical binding site for 14-3-3s within the H(+)-ATPase has been identified that does not resemble any other 14-3-3 binding motif. Combination of site-directed mutagenesis with glutathione S-transferase pull-down assays points to the importance of the central 14-3-3 groove for the interaction with the apparently unique site of the H(+)-ATPase. Furthermore, a 14-3-3 dimer is essential for binding such unusual motif.     

Phosphorylation-dependent interaction between plant plasma membrane H(+)-ATPase and 14-3-3 proteins

The H(+)-ATPase is a key enzyme for the establishment and maintenance of plasma membrane potential and energization of secondary active transport in the plant cell. The phytotoxin fusicoccin induces H(+)-ATPase activation by promoting the association of 14-3-3 proteins. It is still unclear whether 14-3-3 proteins can represent natural regulators of the proton pump, and factors regulating 14-3-3 binding to the H(+)-ATPase under physiological conditions are unknown as well. In the present study in vivo and in vitro evidence is provided that 14-3-3 proteins can associate with the H(+)-ATPase from maize roots also in a fusicoccin-independent manner and that the interaction depends on the phosphorylation status of the proton pump. Furthermore, results indicate that phosphorylation of H(+)-ATPase influences also the fusicoccin-dependent interaction of 14-3-3 proteins. Finally, a protein phosphatase 2A able to impair the interaction between H(+)-ATPase and 14-3-3 proteins was identified and partially purified from maize root.     

Phosphorylation-independent interaction between 14-3-3 protein and the plant plasma membrane H+-ATPase

14-3-3 proteins interact with a novel phosphothreonine motif (Y(946)pTV) at the extreme C-terminal end of the plant plasma membrane H(+)-ATPase molecule. Phosphorylation-independent binding of 14-3-3 protein to the YTV motif can be induced by the fungal phytotoxin fusicoccin. The molecular basis for the phosphorylation-independent interaction between 14-3-3 and H(+)-ATPase in the presence of fusicoccin has been investigated in more detail. Fusicoccin binds to a heteromeric receptor that involves both 14-3-3 protein and H(+)-ATPase. Binding of fusicoccin is dependent upon the YTV motif in the H(+)-ATPase and, in addition, requires residues further upstream of this motif. Apparently, 14-3-3 proteins interact with the unusual epitope in H(+)-ATPase via its conserved amphipathic groove. This implies that very diverse epitopes bind to a common structure in the 14-3-3 protein.     

Association of 14-3-3 proteins with the C-terminal autoinhibitory domain of the plant plasma-membrane H+-ATPase generates a fusicoccin-binding complex

The plant plasma-membrane H⁺-ATPase (EC 3.6.1.35) contains a C-terminal autoinhibitory domain whose displacement from the catalytic site is caused by treatment of intact plant tissue with the phytotoxin fusicoccin (FC). The FC-induced activation of the H⁺-ATPase was proposed to involve a direct interaction of 14-3-3 proteins with the H⁺-ATPase. By analysing plasma membranes derived from leaves of Commelina communis L., direct biochemical evidence has now been obtained for a complex between the C-terminus of the H⁺-ATPase and a 14-3-3 dimer. Stabilization of this complex was achieved by FC treatment in vivo or in vitro. Furthermore, the C-terminal domain of the H⁺-ATPase in association with a 14-3-3 dimer is essential for the creation of a functional FC-binding complex. Received: 1 August 1998 / Accepted: 15 September 1998     

A phosphorylation in the c-terminal auto-inhibitory domain of the plant plasma membrane H+-ATPase activates the enzyme with no requirement for regulatory 14-3-3 proteins

The plant plasma membrane H(+)-ATPase is regulated by an auto-inhibitory C-terminal domain that can be displaced by phosphorylation of the penultimate residue, a Thr, and the subsequent binding of 14-3-3 proteins. By mass spectrometric analysis of plasma membrane H(+)-ATPase isoform 2 (PMA2) isolated from Nicotiana tabacum plants and suspension cells, we identified a new phosphorylation site, Thr-889, in a region of the C-terminal domain upstream of the 14-3-3 protein binding site. This residue was mutated into aspartate or alanine, and the mutated H(+)-ATPases expressed in the yeast Saccharomyces cerevisiae. Unlike wild-type PMA2, which could replace the yeast H(+)-ATPases, the PMA2-Thr889Ala mutant did not allow yeast growth, whereas the PMA2-Thr889Asp mutant resulted in improved growth and increased H(+)-ATPase activity despite reduced phosphorylation of the PMA2 penultimate residue and reduced 14-3-3 protein binding. To determine whether the regulation taking place at Thr-889 was independent of phosphorylation of the penultimate residue and 14-3-3 protein binding, we examined the effect of combining the PMA2-Thr889Asp mutation with mutations of other residues that impair phosphorylation of the penultimate residue and/or binding of 14-3-3 proteins. The results showed that in yeast, PMA2 Thr-889 phosphorylation could activate H(+)-ATPase if PMA2 was also phosphorylated at its penultimate residue. However, binding of 14-3-3 proteins was not required, although 14-3-3 binding resulted in further activation. These results were confirmed in N. tabacum suspension cells. These data define a new H(+)-ATPase activation mechanism that can take place without 14-3-3 proteins.     

Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein

The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth.     

A plant plasma membrane H+-ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the absence of fusicoccin

The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified. Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2. This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants. When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form. Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region. The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated. In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin. Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase. These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H(+) pumping activity. Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth. We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme.     

Polyamines as physiological regulators of 14-3-3 interaction with the plant plasma membrane H+-ATPase

Polyamines are abundant polycationic compounds involved in many plant physiological processes such as cell division, dormancy breaking, plant morphogenesis and response to environmental stresses. In this study, we investigated the possible role of these polycations in modulating the association of 14-3-3 proteins with the H(+)-ATPase. In vivo experiments demonstrate that, among the different polyamines, spermine brings about 2-fold stimulation of the H(+)-ATPase activity and this effect is due to an increase in 14-3-3 levels associated with the enzyme. In vivo administration of polyamine synthesis inhibitors causes a small but statistically significant decrease of the H(+)-ATPase phosphohydrolytic activity, demonstrating a physiological role for the polyamines in regulating the enzyme activity. Spermine stimulates the activity of the H(+)-ATPase AHA1 expressed in yeast, in the presence of exogenous 14-3-3 proteins, with a calculated S(50) of 70 microM. Moreover, spermine enhances the in vitro interaction of 14-3-3 proteins with the H(+)-ATPase and notably induces 14-3-3 association with the unphosphorylated C-terminal domain of the proton pump. Comparison of spermine with Mg(2+), necessary for binding of 14-3-3 proteins to different target proteins, shows that the polyamine effect is stronger than and additive to that of the divalent cation.     

Involvement of 14-3-3 proteins in the osmotic regulation of H+-ATPase in plant plasma membranes

 Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H⁺-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H⁺ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes; the 14-3-3 content in the membranes increased 2- to 3-fold, while the H⁺-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H⁺-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H⁺ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins. Received: 10 February 2000 / Accepted: 31 March 2000     

The binding site for regulatory 14-3-3 protein in plant plasma membrane H+-ATPase: involvement of a region promoting phosphorylation-independent interaction in addition to the phosphorylation-dependent C-terminal end

14-3-3 proteins constitute a family of well conserved proteins interacting with a large number of phosphorylated binding partners in eukaryotic cells. The plant plasma membrane H+-ATPase is an unusual target in that a unique phosphothreonine motif (946YpTV, where pT represents phosphothreonine) in the extreme C-terminal end of the H+-ATPase interacts with the binding cleft of 14-3-3 protein (Wurtele, M., Jelich-Ottmann, C., Wittinghofer, A., and Oecking, C. (2003) EMBO J. 22, 987-994). We report binding of 14-3-3 protein to a nonphosphorylated peptide representing the 34 C-terminal residues of the Arabidopsis plasma membrane H+-ATPase isoform 2 (AHA2). Following site-directed mutagenesis within the 45 C-terminal residues of AHA2, we conclude that, in addition to the 946YpTV motif, a number of residues located further upstream are required for phosphorylation-independent binding of 14-3-3. Among these, Thr-924 is important for interaction with 14-3-3 protein even when Thr-947 is phosphorylated. We suggest that the role of phosphorylation, which is accentuated by fusicoccin, is to stabilize protein-protein interaction between 14-3-3 protein and several residues of the H+-ATPase C-terminal domain.     

The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase.

Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-ATPase isolated from fusicoccin-treated maize shoots was copurified with the 14-3-3 protein (as determined by protein gel blotting), and the H(+)-ATPase was recovered in an activated state. In the absence of fusicoccin treatment, H(+)-ATPase and the 14-3-3 protein were well separated, and the H(+)-ATPase was recovered in a nonactivated form. Trypsin treatment removed the 10-kD C-terminal region from the H(+)-ATPase as well as the 14-3-3 protein. Using the yeast two-hybrid system, we could show a direct interaction between Arabidopsis 14-3-3 GF14-phi and the last 98 C-terminal amino acids of the Arabidopsis AHA2 plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase.     

Adenosine 5'-monophosphate inhibits the association of 14-3-3 proteins with the plant plasma membrane H(+)-ATPase.

Although a well ascertained evidence proves that the activity of the plant plasma membrane H(+)-ATPase is regulated by 14-3-3 proteins, information about physiological factors modulating the phosphorylation-dependent association between 14-3-3 proteins and the proton pump is largely incomplete. In this paper we show that the 5'-AMP-mimetic, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), inhibits the fusicoccin-promoted proton extrusion in maize roots. We also demonstrate that 5'-AMP inhibits the association of 14-3-3 proteins with the C-terminal domain of the H(+)-ATPase in an overlay assay as well as the 14-3-3-dependent stimulation of the Arabidopsis thaliana H(+)-ATPase AHA1 isoform expressed in yeast membranes. Finally, by means of affinity chromatography with immobilized 5'-AMP and trinitrophenyl-AMP fluorescence analysis, we demonstrate that the 14-3-3 isoform GF14-6 from maize is able to bind 5'-AMP. The possible role of 5'-AMP as a general regulator of 14-3-3 functions in the plant cell is discussed.     

Identification of an autoinhibitory domain in the C-terminal region of the plant plasma membrane H(+)-ATPase.

Proteolytic (trypsin) treatment removes a small terminal segment from the 100-kDa plant plasma membrane H(+)-ATPase. This results in activation of H+ pumping across the plasma membrane, suggesting that an inhibitory domain is located in one of the terminal regions of the enzyme (Palmgren, M.G., Larsson, C., and Sommarin, M. (1990) J. Biol. Chem. 265, 13423-13426). In order to identify the origin of the fragment released by trypsin, polyclonal antibodies were raised against the first 55 amino acids (N-terminal region), the last 99 amino acids (C-terminal region), and a portion of 150 amino acids in the central part of the enzyme as deduced from one of the H(+)-ATPase genes (PMA2) of Arabidopsis thaliana. The native, 100-kDa H(+)-ATPase was recognized by all three antisera in Western blots. By contrast, the approximately 90-kDa polypeptide appearing after trypsin treatment was only recognized by the antisera against the N-terminal and central region, but not by the antiserum against the C-terminal region, suggesting that the inhibitory domain is located in this part of the enzyme. To more closely determine the position of the inhibitory domain, three peptides representing conserved parts of the C-terminal region were synthesized (residues 861-888, 912-943, and 936-949 of the Arabidopsis (PMA2) sequence). Only one of the peptides (residues 861-888) affected H+ pumping by the trypsin-activated (approximately 90-kDa) enzyme. This peptide of 28 amino acids inhibited H+ pumping with an IC50 of about 15 microM, suggesting that the auto-inhibitory domain is located within the corresponding part of the C-terminal region.     

Osmoregulation in Lilium pollen grains occurs via modulation of the plasma membrane H+ ATPase activity by 14-3-3 proteins

To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment. An acidification and alkalinization of the external medium was monitored after hyper- and hypoosmotic treatments, respectively, and pH changes were blocked by vanadate, indicating a putative role of the PM H(+) ATPase. Indeed, an increase in PM-associated 14-3-3 proteins and an increase in PM H(+) ATPase activity were detected in PGs challenged by hyperosmolar medium. We therefore suggest that in PGs the PM H(+) ATPase via modulation of its activity by 14-3-3 proteins is involved in the regulation of turgor pressure.     

Inhibitory interaction of the plasma membrane Na+/Ca2+ exchangers with the 14-3-3 proteins

The three Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, contain a large cytoplasmic loop that is responsible for the regulation of activity. We have used 347 residues of the loop of NCX2 as the bait in a yeast two-hybrid approach to identify proteins that could interact with the exchanger and regulate its activity. Screening of a human brain cDNA library identified the epsilon and zeta isoforms of the 14-3-3 protein family as interacting partners of the exchanger. The interaction was confirmed by immunoprecipitation and in vitro binding experiments. The effect of the interaction on the homeostasis of Ca2+ was investigated by co-expressing NCX2 and 14-3-3epsilon in HeLa cells together with the recombinant Ca2+ probe aequorin; the ability of cells expressing both NCX2 and 14-3-3epsilon to dispose of a Ca2+ transient induced by an InsP3-producing agonist was substantially decreased, indicating a reduction of NCX2 activity. The 14-3-3epsilon protein also inhibited the NCX1 and NCX3 isoforms. In vitro binding experiments revealed that all three NCX isoforms interacted with multiple 14-3-3 isoforms. 14-3-3 was bound by both phosphorylated and nonphosphorylated NCX, but the phosphorylated form had much higher binding affinity.     

Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+ -ATPase by combining X-ray crystallography and electron cryomicroscopy

Regulatory 14-3-3 proteins activate the plant plasma membrane H(+)-ATPase by binding to its C-terminal autoinhibitory domain. This interaction requires phosphorylation of a C-terminal, mode III, recognition motif as well as an adjacent span of approximately 50 amino acids. Here we report the X-ray crystal structure of 14-3-3 in complex with the entire binding motif, revealing a previously unidentified mode of interaction. A 14-3-3 dimer simultaneously binds two H(+)-ATPase peptides, each of which forms a loop within the typical 14-3-3 binding groove and therefore exits from the center of the dimer. Several H(+)-ATPase mutants support this structure determination. Accordingly, 14-3-3 binding could result in H(+)-ATPase oligomerization. Indeed, by using single-particle electron cryomicroscopy, the 3D reconstruction of the purified H(+)-ATPase/14-3-3 complex demonstrates a hexameric arrangement. Fitting of 14-3-3 and H(+)-ATPase atomic structures into the 3D reconstruction map suggests the spatial arrangement of the holocomplex.     

The two major plant plasma membrane H+-ATPases display different regulatory properties

The major plant plasma membrane H(+)-ATPases fall into two gene categories, subfamilies I and II. However, in many plant tissues, expression of the two subfamilies overlaps, thus precluding individual characterization. Yeast expression of PMA2 and PMA4, representatives of the two plasma membrane H(+)-ATPase subfamilies in Nicotiana plumbaginifolia, has previously shown that (i) the isoforms have distinct enzymatic properties and that (ii) PMA2 is regulated by phosphorylation of its penultimate residue (Thr) and binds regulatory 14-3-3 proteins, resulting in the displacement of the autoinhibitory C-terminal domain. To obtain insights into regulatory differences between the two subfamilies, we have constructed various chimeric proteins in which the 110-residue C-terminal-encoding region of PMA2 was progressively substituted by the corresponding sequence from PMA4. The PMA2 autoinhibitory domain was localized to a region between residues 851 and 915 and could not be substituted by the corresponding region of PMA4. In contrast to PMA2, PMA4 was poorly phosphorylated at its penultimate residue (Thr) and bound 14-3-3 proteins weakly. The only sequence difference around the phosphorylation site is located two residues upstream of the phosphorylated Thr. It is Ser in PMA2 (as in most members of subfamily I) and His in PMA4 (as in most members of subfamily II). Substitution of His by Ser in PMA4 resulted in an enzyme showing increased phosphorylation status, 14-13-3 binding, and ATPase activity, as well as improved yeast growth. The reverse substitution of Ser by His in PMA2 resulted in the failure of this enzyme to complement the absence of yeast H(+)-ATPases. These results show that the two plant H(+)-ATPase subfamilies differ functionally in their regulatory properties.