Does plasma membrane H+-ATPase activation by fusicoccin involve protein kinase?

Sugar beet ( Beta vulgaris L.) root suspension-cultured cells were converted to protoplasts which responded to fusicoccin (FC) by a rise in cytoplasmic pH (pH_cyt) averaging 0.25 units in the fluorimetric assay. This effect was blocked by erythrosin B, a specific inhibitor of the plasma membrane H₊-ATPase. A protein kinase inhibitor, staurosporine also caused cytosolic alkalinization that was sensitive to H⁺-ATPase inhibitors. Most strikingly, the effect of staurosporine was suppressed by fusicoccin and vice versa. Addition of okadaic acid, entailing overall protein phosphorylation, also led to H⁺-ATPase activation, whereupon fusicoccin lost its effect on proton transport. In parallel, kinetic and inhibitor analyses demonstrated that FC binding to the protoplast plasma membrane involved two sites with dissociation constants of 1 n M and 0.2 μ M and was indifferent to phosphorylation and dephosphorylation inhibitors. Thus, it could be concluded that (1) the effect of FC on cytoplasmic pH probably depends on the phosphorylation state of plasma membrane proteins and may have either sign; (2) the activation of H⁺-ATPase by FC most likely proceeds directly through conformational receptor-enzyme interaction.     

Enhanced H+/ATP coupling ratio of H+-ATPase and increased 14-3-3 protein content in plasma membrane of tomato cells upon osmotic shock

Modulation of proton extrusion and ATP-dependent H⁺ transport through the plasma membrane in relation to the presence of 14-3-3 proteins in this membrane in response to osmotic shock was studied in tomato ( Lycopersicon esculentum Mill. cv. Pera) cell cultures. In vivo H⁺ extrusion by cells was activated rapidly and significantly after adding 100 m M NaCl, 100 m M KCl, 50 m M Na₂SO₄, 1.6% sorbitol or 2 µ M fusicoccin to the medium. The increase in H⁺ extrusion by cells treated with 100 m M NaCl was correlated with an increase of H⁺ transport by the plasma membrane H⁺-ATPase (EC 3.6.1.35), but not with changes in ATP hydrolytic activity of this enzyme, suggesting an increased coupling ratio of the enzyme. Immunoblot experiments showed increased amounts of 14-3-3 proteins in plasma membrane fractions isolated from tomato cells treated with 100 m M NaCl as compared to control cells without changing the amount of plasma membrane H⁺-ATPase. Together, these data indicate that in tomato cells an osmotic shock could enhance coupling between ATP hydrolysis and proton transport at the plasma membrane through the formation of a membrane 14-3-3/H⁺-ATPase complex.     

Synergistic activation of plasma membrane H+– ATPase in Arabidopsis thaliana cells by turgor decrease and by fusicoccin

The regulation of the H⁺-ATPase of plasma membrane is a crucial point in the integration of transport processes at this membrane. In this work the regulation of H⁺-ATPase activity induced by changes in turgor pressure was investigated and compared with the stimulating effect of fusicoccin (FC). The exposure of cultured cells of Arabidopsis thaliana L. (ecotype Landsberg 310–14-2) to media containing mannitol (0. 15 or 0. 3 M ) or polyethylene glycol 6000 (PEG) (15. 6% or 22% w/v) resulted in a decrease in the turgor pressure of the cells and in a strong stimulation of H⁺ extrusion in the incubation medium. The osmotica-induced H⁺ extrusion was (1) inhibited by the inhibitor of plasma membrane H⁺-ATPase, erythrosin B (EB), (2) dependent on the external K⁺ concentration, (3) associated with a net K⁺ influx, and (4) lead to an increase of cellular malate content. These results show that the reduction of external osmotic potential stimulates the activity of plasma membrane H⁺-ATPase
The effect of mannitol was only partially inhibited by treatments with cycloheximide (CH) and cordycepin, which block protein and mRNA synthesis, respectively. All the effects of osmotica were qualitatively and quantitatively similar to those induced by 5 μ M FC. However, when FC and mannitol (or PEG) were fed together, their effects on H⁺ extrusion appeared synergistic, irrespective of whether FC was present at suboptimal or optimal concentrations. This behaviour suggests that the modes of action of FC and of the osmotica on H⁺-ATPase activity differ at least in some step(s)     

Bacterial phytotoxins, syringomycin, syringostatin and syringotoxin, exert their effect on the plasma membrane H+-ATPase partly by a detergent-like action and partly by inhibition of the enzyme

Syringostatin is a newly discovered phytotoxin produced by a phytopathogenic bacterium Pseudomonas syringae pv. syringae lilac isolate. The effects of syringostatin and the similar phytotoxins, syringomycin and syringotoxin, on H⁻-ATPase activity were investigated using cultured mung bean ( Vigna radiata L. cv. Ryokuto) cells or plasma membrane vesicles isolated from mung bean hypocotyls. ³¹P-NMR analysis of cultured cells treated with syringostatin revealed that the cytoplasmic pH was decreased. When plasma membrane was prepared by a two-step method (Dextran gradient followed by a sucrose gradient). syringostatin, syringomycin and syringotoxin inhibited the H⁺-ATPase activity in a dose-dependent manner. In contrast, these toxins stimulated H⁺-ATPase activity when plasma membrane was prepared by a one-step method (sucrose gradient). While these toxins inhibited the H⁺-ATPase activity of inside-out plasma membrane vesicles, the H⁺-ATPase activity of right-side-out vesicles was stimulated. The detergent. Triton X-100, abolished this stimulatory effect of the toxins on the H⁺-ATPase of right-side-out vesicles and of one-step purified plasma membrane. The toxins also inhibited the activity of the plasma membrane H⁺-ATPase solubilized with deoxycholate and Zwittergent 3–14. Taken together, these results indicate that these toxins exert their effects partly by a detergent-like action on the plasma membrane and partly by inhibition of the enzyme.     

Protein phosphatase 2A scaffolding subunit A interacts with plasma membrane H+-ATPase C-terminus in the same region as 14-3-3 protein

The activity of the plant plasma membrane H⁺-ATPase is tightly regulated via phosphorylation and binding of 14-3-3 protein to the C-terminus of the pump. Whereas the 14-3-3-binding mechanism has been described in detail, the identity of specific protein kinases and phosphatases involved in the control of 14-3-3 binding has remained elusive. Using the yeast two-hybrid system, GST pull-down assays and overlay experiments, we report that scaffolding subunit A of protein phosphatase 2A (PP2A-A) interacts with the C-terminus of the Arabidopsis plasma membrane H⁺-ATPase isoform 2. PP2A-A binding is inhibited in the presence of 14-3-3 protein and fusicoccin, a fungal toxin which induces binding of 14-3-3 protein to the C-terminal end of the plasma membrane H⁺-ATPase. This indicates that PP2A-A and 14-3-3 protein compete with each other for binding to the same region in the C-terminus of the H⁺-ATPase.     

Immunopurification of H+-ATPase-containing lipid-protein particles from the cytosol of carnation petals

Lipid-protein particles originating from the plasma membrane were immunopurified from the cytosol of carnation petal cells ( Dianthus caryophyllus L. cv. Improved White Sim) using antibodies raised against the central hydrophilic domain of the H⁺-ATPase. The immunopurified particles are enriched in lipid metabolites, in particular free fatty acids and steryl/wax esters, by comparison with corresponding microsomal membranes, and the lipids of the particles are more saturated than those of microsomal membranes. Proteolytic catabolites of the H⁺-ATPase, a protein associated with the plasma membrane, but not the native H⁺-ATPase protein, are also present in the immunopurified cytosolic particles. Osmiophilic particles were discernible in the cytosol of carnation petal cells by transmission electron microscopy, and the association of H⁺-ATPase catabolites with a subpopulation of these particles was confirmed by immunogold labelling with H⁺-ATPase antiserum. Cross-reaction of the H⁺-ATPase antiserum with elements of the cytosol was also evident by immunofluorescent light microscopy. These observations collectively indicate that lipid-protein particles of plasma membrane origin are present in the cytosol of carnation petal cells and that their formation may serve as a means of removing lipid and protein metabolites from the plasma membrane which would otherwise destabilize its structure.     

Aquaporin functionality in relation to H+-ATPase activity in root cells of Capsicum annuum grown under salinity

As water and nutrient uptake should be related in the response of plants to salinity, the aim of this paper is to establish whether or not aquaporin functionality is related to H⁺-ATPase activity in root cells of pepper ( Capsicum annuum L.) plants. Thus, H⁺-ATPase activity was measured in plasma membrane vesicles isolated from roots and aquaporin functionality was measured using a cell pressure probe in intact roots. Salinity was applied as 60 m M NaCl or 60 m M KCl, to determine which ion (Na⁺, K⁺ or Cl⁻) is producing the effects. We also investigated whether the effects of both salts were ameliorated by Ca²⁺. Similar results were obtained for cell hydraulic conductivity, Lp_c, and H⁺-ATPase activity, large reductions in the presence at NaCl or KCl and an ameliorative effect of Ca²⁺. However, fusicoccin (an activator of H⁺-ATPase) did not alter osmotic water permeability of protoplasts isolated from roots. Addition of Hg²⁺ inhibited both ATPase and aquaporins, but ATPase also contains Hg-binding sites. Therefore, the results indicate that H⁺-ATPase and aquaporin activities may not be related in pepper plants.     

A Phosphopeptide Corresponding to the Cytosolic Stretch Connecting Transmembrane Segments 8 and 9 of the Plasma Membrane H+-ATPase Binds 14-3-3 Proteins and Inhibits Fusicoccin-Induced Activation of the H+-ATPase

Abstract: A putative consensus domain for binding of 14-3-3 proteins to the plasma membrane (PM) H⁺-ATPase was identified in the highly-conserved sequence RSR(p)SWSF [where (p)S is Ser776 of the maize isoform MHA2], localized in the cytosolic stretch connecting transmembrane segments 8 and 9. A 15 amino acid biotinylated phosphopeptide comprising this motif: i) bound a recombinant 14-3-3 protein, ii) inhibited fusicoccin-induced stimulation of the PM H⁺-ATPase activity both in PM isolated from germinating radish ( Raphanus sativus L.) seedlings and in ER isolated from Saccharomyces cerevisiae expressing AHA1 (an isoform of Arabidopsis thaliana PM H⁺-ATPase), and iii) inhibited fusicoccin binding to PM isolated from germinating radish seedlings. The corresponding non-phosphorylated peptide was inactive in all the performed assays. Together, these results suggest that the cytosolic strand connecting transmembrane segments 8 and 9 of the PM H⁺-ATPase is a 14-3-3 binding site which might cooperate with the C-terminal domain of the'enzyme in generating a stable association between the H⁺-ATPase and 14-3-3 protein.     

NaCl-induced accumulation of tonoplast and plasma membrane H+-ATPase message in tomato

NaCl-induced changes in the accumulation of message for the 70 kDa subunit of the tonoplast H⁺-ATPase and plasma membrane H⁺-ATPase were studied in hydroponically grown plants of Lycopersicon esculentum Mill. cv. Large Cherry Red. There was increased accumulation of message for the 70 kDa (catalytic) subunit of the tonoplast H⁺-ATPase in expanded leaves of tomato plants 24 h after final NaCl concentrations were attained. This was a tissue-specific response; levels of this message were not elevated in roots or in young, unexpanded leaves. The NaCl-induced accumulation of this message was transient in the expanded leaves and returned to control levels within 7 days. The temporal and spatial patterns of NaCl-induced accumulation of message for the plasma membrane H⁺-ATPase differed from the patterns associated with the 70 kDa subunit of the tonoplast H⁺-ATPase. NaCl-induced accumulation of the plasma membrane H⁺-ATPase message occurred in both roots and expanded leaves. Initially accumulation of the plasma membrane H⁺-ATPase message was greater in root tissue than in expanded leaves, but increased to higher levels in expanded leaves after 7 days. These results suggest that increased expression of the tonoplast H⁺-ATPase is an early response to salinity stress and may be associated with survival mechanisms, rather than with long-term adaptive processes.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Modified plant plasma membrane H+-ATPase with improved transport coupling efficiency identified by mutant selection in yeast

Transport across the plasma membrane is driven by an electrochemical gradient of H⁺ ions generated by the plasma membrane proton pump (H⁺-ATPase). Random mutants of Arabidopsis H⁺-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H⁺-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced by site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200–500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H⁺ extrusion by transformed yeast cells. The different species of aha1, however, displayed marked differences in initial rates of net H⁺ extrusion and in their ability to sustain an electrochemical H⁺ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H⁺-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H⁺ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plants transporters.     

Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH₄⁺ rather than NO₃^- is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH₄⁺ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H⁺-ATPase of rice root to low pH. Rice seedlings were grown either with NH₄⁺ or NO₃^-. For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H⁺-ATPase activity, V _max, K _m, H⁺-pumping activity, H⁺ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H⁺-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H⁺-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H⁺-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH₄⁺ nutrition.     

Substrate stabilization of lysophosphatidylcholine-solubilized plasma membrane H+-ATPase from oat roots

Plasma membrane vesicles with H⁺-ATPase activity were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots using an aqueous polymer two-phase system. Of several detergents tested, only lysophosphatidylcholine solubilized the H⁺-ATPase in an active form. Solubilization of the H⁺-ATPase with lysophosphatidylcholine was possible in the absence of glycerol, but the ATPase activity decreased about 4–5 times as rapidly in the absence as in the presence of 30% (w/v) glycerol. The solubilized enzyme was further stabilized by ATP and protons. Addition of 1 m M ATP to the plasma membranes halted inactivation of the H⁺-ATPase. Even in the absence of polyol compounds and ATP, the enzyme was stable for hours at relatively low pH with an optimum around pH 6.7 at room temperature. The curve for the stability of soluble H⁺-ATPase as a function of pH closely resembles the pH curve for the activity of the H⁺-ATPase. This suggests that binding of protons to transport sites may stabilize the soluble H⁺-ATPase in an enzymatically active form.     

Plasma membrane H-ATPase activity is involved in adaptation of tomato calli to NaCl

A tomato ( Lycopersicon esculentum Mill. cv. Pera) callus culture tolerant to NaCl was obtained by successive subcultures of NaCl-sensitive calli in medium supplemented with 50 m M NaCl. NaCl-tolerant calli grew better than NaCl-sensitive calli in media supplemented with 50 and 100 m M NaCl. Analysis of callus ion content showed a strong increase in Na⁺ and Cl⁻ both in NaCl-tolerant and -sensitive calli grown in media containing NaCl for one subculture. Cells from NaCl-tolerant calli showed a higher H⁺ extrusion activity than those from NaCl-sensitive calli grown for one subculture in the presence of NaCl. The inhibition of H⁺ extrusion by NaCl-sensitive cells was correlated with an inhibition of microsomal vanadate-sensitive H⁺-ATPase (EC 3.6.1.35) and ATP-dependent H⁺ transport, while the stimulation of H⁺ extrusion by cells tolerant to 50 m M NaCl was correlated with an increase in plasma membrane ATP-dependent H⁺ transport. The increase of ATP-dependent H⁺ extrusion in plasma membranes isolated from 50 m M NaCl-tolerant calli was not a result of stimulation of a vanadate-sensitive ATP hydrolytic activity or an increase in passive permeability to H⁺. Relative to NaCl-sensitive calli, plasma membrane H⁺-ATPase from calli tolerant to 50 m M NaCl showed a lower K_m for Mg²⁺-ATP. Our results indicate that tolerance of tomato calli to 50 m M NaCl increases the affinity of plasma membrane H⁺-ATPase for the substrate ATP and stimulates the H⁺-pumping activity of this enzyme without modifying its phosphohydrolytic activity.     

Plasma membrane ATPase activity during pepino (Solanum muricatum) ripening

Plasma membrane-enriched samples were extracted from pepino fruit (cv. El Camino) by phase partitioning. H⁺-ATPase (EC 3.6.1.35) activity in these samples increased during late fruit development (immediately before the onset of ripening) and western blotting confirmed there was an increase in enzyme abundance at this time. H⁺-ATPase activity decreased during early ripening and then increased again in the final phase of ripening. Immunolocalisation showed the plasma membrane H⁺-ATPase was most abundant in the outer cell layers of the fruit, which are considered to have a major role in determining fruit texture. Fruit softening was not accelerated by harvest and there was no stimulation of H⁺-ATPase activity by harvest. An in vitro tensile test using fruit rings showed tissue softening proceeded faster at low apoplastic pH (4.5) than at pH 6.5; and tissue buffered at pH 6.5 softened less than unbuffered rings. Erythrosin B, an inhibitor of the plasma membrane H⁺-ATPase, also retarded softening in vitro. These data suggest that plasma membrane H⁺-ATPase activity may contribute to the onset of pepino softening through a reduction in apoplastic pH.     

An electrogenic pump in the xylem parenchyma of barley roots

Analysis of an electrogenic pump in the plasma membrane of xylem-parenchyma protoplasts from barley roots was performed using the patch-clamp technique in the whole-cell configuration. Particularly with regard to understanding xylem loading and unloading, the study of the electrogenic pump from this cell type is important; its functional confirmation was lacking to date. About one-half of the investigated protoplasts displayed current responses with reversal potentials between −80 and −200 mV. The application of fusicoccin, an H⁺-pump stimulator, caused an increase in currents recorded at a membrane potential of 0 mV and a shift of the reversal potential by about −50 mV. Treatment with dicylohexylcarbodiimid, an H⁺-pump inhibitor, resulted in the reduction of the current at 0 mV. The Ca²⁺-pump inhibitor, erythrosin B, showed no effect on current density at 0 mV and on the polarisation of the membrane potential. Enlarging the transmembrane pH gradient by raising the pH of the extracellular solution from 5.8 to 8.8 stimulated the currents. These are strong indications that the electrogenic pump was an H⁺-pump. Neither intracellular pH nor the intracellular Ca²⁺ concentration affected its activity. Simultaneous activity of the electrogenic pump and anion conductances could produce states in which protoplasts exhibited 'intermediate' reversal potentials. It was concluded that the electrogenic pump was not directly involved in the loading of KCl and KNO₃ into the xylem but, in combination with anion channel activities, contributed to the establishment of membrane potentials at which electroneutral salt transport and acid release can proceed.