Plant proton pumping pyrophosphatase: the potential for its pyrophosphate synthesis activity to modulate plant growth

Cellular pyrophosphate (PPi) homeostasis is vital for normal plant growth and development. Plant proton‐pumping pyrophosphatases (H⁺‐PPases) are enzymes with different tissue‐specific functions related to the regulation of PPi homeostasis. Enhanced expression of plant H⁺‐PPases increases biomass and yield in different crop species. Here, we emphasise emerging studies utilising heterologous expression in yeast and plant vacuole electrophysiology approaches, as well as phylogenetic relationships and structural analysis, to showcase that the H⁺‐PPases possess a PPi synthesis function. We postulate this synthase activity contributes to modulating and promoting plant growth both in H⁺‐PPase‐engineered crops and in wild‐type plants. We propose a model where the PPi synthase activity of H⁺‐PPases maintains the PPi pool when cells adopt PPi‐dependent glycolysis during high energy demands and/or low oxygen environments. We conclude by proposing experiments to further investigate the H⁺‐PPase‐mediated PPi synthase role in plant growth.     

Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg

To clarify the kinetic characteristics and ionic requirements of the tonoplast H⁺-translocating inorganic pyrophosphatase (H⁺-PPiase), PPi hydrolysis and PPi-dependent H⁺ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H⁺-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H⁺-PPiase activity was maximal in the presence of K⁺ (K₅₀ approximately 3 millimolar), with PPi-dependent H⁺ transport being more selective for K⁺ than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H⁺-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg²⁺ for activation. At saturating concentrations of free Mg²⁺, H⁺-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi²⁻ but not Mg₂PPi, demonstrating that MgPPi²⁻ was the true substrate of the enzyme. The apparent K_m (MgPPi²⁻) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H⁺ transport (7 micromolar). Free Mg²⁺ was shown to be an allosteric activator of the H⁺-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H⁺ transport. Half-maximal H⁺-PPiase activity occurred at a free Mg²⁺ concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg²⁺. In contrast, cytosolic concentrations of K⁺ and MgPPi²⁻ appear to be saturating for H⁺-PPiase activity. We propose that one function of the H⁺-PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H⁺-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg²⁺ would be released from the MgATP²⁻ complex, thereby activating the tonoplast H⁺-PPiase.     

A Highly Temperature-sensitive Proton Current in Mouse Bone Marrow–derived Mast Cells

Proton (H⁺) conductive pathways are suggested to play roles in the regulation of intracellular pH. We characterized temperature-sensitive whole cell currents in mouse bone marrow–derived mast cells (BMMC), immature proliferating mast cells generated by in vitro culture. Heating from 24 to 36°C reversibly and repeatedly activated a voltage-dependent outward conductance with Q₁₀ of 9.9 ± 3.1 (mean ± SD) (n = 6). Either a decrease in intracellular pH or an increase in extracellular pH enhanced the amplitude and shifted the activation voltage to more negative potentials. With acidic intracellular solutions (pH 5.5), the outward current was detected in some cells at 24°C and Q₁₀ was 6.0 ± 2.6 (n = 9). The reversal potential was unaffected by changes in concentrations of major ionic constituents (K⁺, Cl⁻, and Na⁺), but depended on the pH gradient, suggesting that H⁺ (equivalents) is a major ion species carrying the current. The H⁺ current was featured by slow activation kinetics upon membrane depolarization, and the activation time course was accelerated by increases in depolarization, elevating temperature and extracellular alkalization. The current was recorded even when ATP was removed from the intracellular solution, but the mean amplitude was smaller than that in the presence of ATP. The H⁺ current was reversibly inhibited by Zn²⁺ but not by bafilomycin A₁, an inhibitor for a vacuolar type H⁺-ATPase. Macroscopic measurements of pH using a fluorescent dye (BCECF) revealed that a rapid recovery of intracellular pH from acid-load was attenuated by lowering temperature, addition of Zn²⁺, and depletion of extracellular K⁺, but not by bafilomycin A₁. These results suggest that the H⁺ conductive pathway contributes to intracellular pH homeostasis of BMMC and that the high activation energy may be involved in enhancement of the H⁺ conductance.     

Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots

A H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H⁺-PPase catalyzed the electrogenic transport of H⁺ into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb⁺ = K⁺ > Cs⁺; but it was inhibited by Na⁺ or Li⁺. Maximum rates of transport required at least 20 millimolar K⁺ and the K_m for this ion was 4 millimolar. Fluoride inhibited both ΔpH formation and K⁺-dependent PPase activity with an I₅₀ of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H⁺-ATPase (e.g. a disulfonic stilbene or NO₃⁻) had no effect on the PPase activity. Vanadate and azide were also ineffective. H⁺-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N′-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H⁺-PPase and the H⁺-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H⁺ pumping activity was strongly affected by Mg²⁺ and pyrophosphate (PPi) concentrations. At 5 millimolar Mg²⁺, H⁺ pumping showed a K_maPP for PPi of 15 micromolar. The rate of H⁺ pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H⁺-PPase.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Effects of colchicine on the accumulation of vacuolar H+-pyrophosphatase and H+-ATPase in germinating Acacia mangium seeds and the recovery effects by sucrose, indole butyric acid and 6-benzyladenine

Plant vacuoles were isolated from cotyledons of germinatingAcacia mangium seeds, which had been treated with or withoutcolchicine, to measure vacuolar membrane pyrophosphate (PPi)- andATP-dependent H⁺ transport activities, and enzymaticactivities of H⁺-pyrophosphatase(H⁺-PPase) and H⁺-ATPase. Innon-colchicine-treated seeds, activities of the two enzymes increasedrapidly after seed germination to almost a maximal level on the seventhday. A linear function relationship exists in magnitude between PPi- orATP-dependent H⁺transport activity and its correspondingenzymatic activity. The former regression equation is: PPi-dependentH⁺ transport activity(%A.min^–1.g^–1) =–0.039 + H⁺-PPase activity(units.mg^–1) × 1.574, the latter is:ATP-dependent H⁺ transport activity(%A.min^–1.g^–1) =–0.003 + H⁺-ATPase activity(units.mg^–1) × 0.549. In colchicine-treatedseeds, activities of the two enzymes increased very slowly during 8 daysof germination and the relationship to their respectiveH⁺ transport activities was not in agreement with theabove-mentioned regression equations. PPi- and ATP-dependentH⁺ transport activities were lower than thecorresponding values calculated from H⁺-PPase activityand H⁺-ATPase activity according to the two regressionequations, respectively. However, when sucrose, indole butyric acid(IBA), or 6-benzyladenine (6-BA) were applied exogenously to the seedsfollowing colchicine treatment for 3 days, activities ofH⁺-PPase, H⁺-ATPase, PPi- andATP-dependent H⁺ transport in the 6-day-old seedlingsall increased. By statistical analysis, it was concluded that colchicineinhibits cotyledon vacuolar membrane H⁺-PPase,H⁺-ATPase activities, PPi- and ATP-dependentH⁺ transport activities during seed germination andearly seedling growth of Acacia mangium. The inhibitory effectsof colchicine could be overcome by IBA, 6-BA and sucrose to varyingdegrees.     

Membrane-bound pyrophosphatase of human gut microbe Clostridium methylpentosum confers improved salt tolerance in Escherichia coli,Saccharomyces cerevisiae and tobacco

Membrane-bound pyrophosphatases (PPases) are involved in the adaption of organisms to stress conditions, which was substantiated by numerous plant transgenic studies with H⁺-PPase yet devoid of any correlated evidences for other two subfamilies, Na⁺-PPase and Na⁺,H⁺-PPase. Herein, we demonstrate the gene cloning and functional evaluation of the membrane-bound PPase (CmPP) of the human gut microbe Clostridium methylpentosum. The CmPP gene encodes a single polypeptide of 699 amino acids that was predicted as a multi-spanning membrane and K⁺-dependent Na⁺,H⁺-PPase. Heterologous expression of CmPP could significantly enhance the salt tolerance of both Escherichia coli and Saccharomyces cerevisiae, and this effect in yeast could be fortified by N-terminal addition of a vacuole-targeting signal peptide from the H⁺-PPase of Trypanosoma cruzi. Furthermore, introduction of CmPP could remarkably improve the salt tolerance of tobacco, implying its potential use in constructing salt-resistant transgenic crops. Consequently, the possible mechanisms of CmPP to underlie salt tolerance are discussed.     

Influence of exogenous application of glycinebetaine on antioxidative system and growth of salt-stressed soybean seedlings (Glycine max L.)

Glycinebetaine is one of the most competitive compounds which play an important role in salt stress in plants. In this study, the enhanced salt tolerance in soybean (Glycine max L.) by exogenous application of glycinebetaine was evaluated. To improve salt tolerance at the seedling stage, GB was applied in four different concentrations (0, 5, 25 and 50 mM) as a pre-sowing seed treatment. Salinity stress in the form of a final concentration of 150 mM sodium chloride (NaCl) over a 15 day period drastically affected the plants as indicated by increased proline, MDA and Na⁺ content of soybean plants. In contrast, supplementation with 50 mM GB improved growth of soybean plants under NaCl as evidenced by a decrease in proline, MDA and Na⁺ content of soybean plants. Further analysis showed that treatments with GB, resulted in increasing of CAT and SOD activity of soybean seedlings in salt stress. We propose that the role of GB in increasing tolerance to salinity stress in soybean may result from either its antioxidant capacity by direct scavenging of H₂O₂ or its role in activating CAT activity which is mandatory in scavenging H₂O₂.     

Kinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors

The responses of the vacuolar membrane (tonoplast) proton-pumping inorganic pyrophosphatase (H⁺-PPase) from oat (Avena sativa L.) roots to changes in Mg²⁺ and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the various PPi complexes were responsible for the observed responses. The results indicate that the substrate for the oat root vacuolar H⁺-PPase is Mg₂PPi and that this complex is also a non-competitive inhibitor. In addition, the enzyme is activated by free Mg²⁺ and competitively inhibited by free PPi. This conclusion differs from that reached in previous studies, in which it was proposed that MgPPi is the substrate for plant vacuolar H⁺-PPases. However, models incorporating MgPPi as a substrate were unable to describe the kinetics of the oat H⁺-PPase. It is demonstrated that models incorporating Mg₂PPi as the substrate can describe some of the published kinetics of the Kalanchoë daigremontiana vacuolar H⁺-PPase. Calculations of the likely concentrations of Mg₂PPi in plant cytoplasm suggest that the substrate binding site of the oat vacuolar H⁺-PPase would be about 70% saturated in vivo.     

Crystal structure of the cytoplasmic N-terminal domain of subunit I, a homolog of subunit a, of V-ATPase

Subunit “a” is associated with the membrane-bound (V_O) complex of eukaryotic vacuolar H⁺-ATPase acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit “I” homolog of subunit a. The structure is composed of a curved long central α-helix bundle capped on both ends by two lobes with similar α/β architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstructions from electron microscopy of prokaryotic and eukaryotic vacuolar H⁺-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.     

Evaluation of isotope discrimination in (13)C-based metabolic flux analysis

This paper describes a modified noninvasive microtest electrophysiological technology (NMT) for vacuolar H⁺ flux detection. In this NMT system, the vacuole isolation procedure and buffer slope were modified, and the measuring errors from small spherical geometry were corrected. The trends in changes of vacuolar H⁺ flux (ΔH⁺ flux) after ATP or PP_i supply calculated by NMT were consistent with the activities of V-ATPase and PPase measured by traditional methods. These findings indicate that our modified NMT is an appropriate method for vacuolar H⁺ flux detection.     

A genome-guided analysis of energy conservation in the thermophilic,cytochrome-free acetogenic bacterium Thermoanaerobacter?kivui

Background

Acetogenic bacteria are able to use CO₂ as terminal electron acceptor of an anaerobic respiration, thereby producing acetate with electrons coming from H₂. Due to this feature, acetogens came into focus as platforms to produce biocommodities from waste gases such as H₂ + CO₂ and/or CO. A prerequisite for metabolic engineering is a detailed understanding of the mechanisms of ATP synthesis and electron-transfer reactions to ensure redox homeostasis. Acetogenesis involves the reduction of CO₂ to acetate via soluble enzymes and is coupled to energy conservation by a chemiosmotic mechanism. The membrane-bound module, acting as an ion pump, was of special interest for decades and recently, an Rnf complex was shown to couple electron flow from reduced ferredoxin to NAD⁺ with the export of Na⁺ in Acetobacterium woodii. However, not all acetogens have rnf genes in their genome. In order to gain further insights into energy conservation of non-Rnf-containing, thermophilic acetogens, we sequenced the genome of Thermoanaerobacter kivui.

Results

The genome of Thermoanaerobacter kivui comprises 2.9 Mbp with a G + C content of 35% and 2,378 protein encoding orfs. Neither autotrophic growth nor acetate formation from H₂ + CO₂ was dependent on Na⁺ and acetate formation was inhibited by a protonophore, indicating that H⁺ is used as coupling ion for primary bioenergetics. This is consistent with the finding that the c subunit of the F₁F_O ATP synthase does not have the conserved Na⁺ binding motif. A search for potential H⁺-translocating, membrane-bound protein complexes revealed genes potentially encoding two different proton-reducing, energy-conserving hydrogenases (Ech).

Conclusions

The thermophilic acetogen T. kivui does not use Na⁺ but H⁺ for chemiosmotic ATP synthesis. It does not contain cytochromes and the electrochemical proton gradient is most likely established by an energy-conserving hydrogenase (Ech). Its thermophilic nature and the efficient conversion of H₂ + CO₂ make T. kivui an interesting acetogen to be used for the production of biocommodities in industrial micobiology. Furthermore, our experimental data as well as the increasing number of sequenced genomes of acetogenic bacteria supported the new classification of acetogens into two groups: Rnf- and Ech-containing acetogens.     

Proton Pump Activity of Mitochondria-rich Cells : The Interpretation of External Proton-concentration Gradients

We have hypothesized that a major role of the apical H⁺-pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl⁻ via an apical Cl⁻/HCO₃ ⁻-exchanger. The activity of the H⁺ pump was studied by monitoring mucosal [H⁺]-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 ± 0.07 (mean ± SEM) pH-units below that of the lightly buffered bulk solution (pH = 7.40). The average distance at which the pH-gradient is dissipated was 382 ± 18 μm, corresponding to an estimated “unstirred layer” thickness of 329 ± 29 μm. Mucosal acidification was dependent on serosal pCO₂, and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H⁺. The [H⁺] was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate [H⁺]-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H⁺ distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace''s equation. Model calculations predicted that with a physiological density of MR cells, the [H⁺] profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing [H⁺]–gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external [H⁺]-gradient depended on the mucosal anion such as to indicate that base (HCO₃ ⁻) is excreted in exchange not only for Cl ⁻, but also for Br⁻ and I⁻, indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells.     

Uptake of Phenylalanine into Isolated Barley Vacuoles Is Driven by Both Tonoplast Adenosine Triphosphatase and Pyrophosphatase : Evidence for a Hydrophobic l-Amino Acid Carrier System

The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent K_m-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; V_{max app} was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 × 10⁶ vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H⁺-translocating enzyme activities revealed K_{m app} values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Δ μH⁺ while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, l-Leu, and l-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. l-Ala, l-Val, d-Phe, and d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity.     

ATP Dependence of Na+/H+ Exchange : Nucleotide Specificity and Assessment of the Role of Phospholipids

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na⁺/H⁺ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pH_i) by microfluorimetry. Na⁺/H⁺ exchange activity was measured as the Na⁺-driven pH_i recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na⁺/H⁺ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na⁺/H⁺ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca²⁺ exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na⁺/H⁺ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na⁺/H⁺ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTPγS was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg²⁺ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the γ-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.     

A Novel Plant Vacuolar Na+/H+ Antiporter Gene Evolved by DNA Shuffling Confers Improved Salt Tolerance in Yeast

Plant vacuolar Na⁺/H⁺ antiporters play important roles in maintaining cellular ion homeostasis and mediating the transport of Na⁺ out of the cytosol and into the vacuole. Vacuolar antiporters have been shown to play significant roles in salt tolerance; however the relatively low V_max of the Na⁺/H⁺ exchange of the Na⁺/H⁺ antiporters identified could limit its application in the molecular breeding of salt tolerant crops. In this study, we applied DNA shuffling methodology to generate and recombine the mutations of Arabidopsis thaliana vacuolar Na⁺/H⁺ antiporter gene AtNHX1. Screening using a large scale yeast complementation system identified AtNHXS1, a novel Na⁺/H⁺ antiporter. Expression of AtNHXS1 in yeast showed that the antiporter localized to the vacuolar membrane and that its expression improved the tolerance of yeast to NaCl, KCl, LiCl, and hygromycin B. Measurements of the ion transport activity across the intact yeast vacuole demonstrated that the AtNHXS1 protein showed higher Na⁺/H⁺ exchange activity and a slightly improved K⁺/H⁺ exchange activity.     

Essential amino acid residues in the central transmembrane domains and loops for energy coupling of Streptomyces coelicolor A3(2) H-pyrophosphatase

The H⁺-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14−17 transmembrane domains, and is found in a range of organisms. We focused on the second quarter region of Streptomyces coelicolor A3(2) H⁺-pyrophosphatase, which contains long conserved cytoplasmic loops. We prepared a library of 1536 mutants that were assayed for pyrophosphate hydrolysis and proton translocation. Mutant enzymes with low substrate hydrolysis and proton-pump activities were selected and their DNAs sequenced. Of these, 34 were single-residue substitution mutants. We generated 29 site-directed mutant enzymes and assayed their activity. The mutation of 10 residues in the fifth transmembrane domain resulted in low coupling efficiencies, and a mutation of Gly¹⁹⁸ showed neither hydrolysis nor pumping activity. Four residues in cytoplasmic loop e were essential for substrate hydrolysis and efficient H⁺ translocation. Pro¹⁸⁹, Asp²⁸¹, and Val³⁵¹ in the periplasmic loops were critical for enzyme function. Mutation of Ala³⁵⁷ in periplasmic loop h caused a selective reduction of proton-pump activity. These low-efficiency mutants reflect dysfunction of the energy-conversion and/or proton-translocation activities of H⁺-pyrophosphatase. Four critical residues were also found in transmembrane domain 6, three in transmembrane domain 7, and five in transmembrane domains 8 and 9. These results suggest that transmembrane domain 5 is involved in enzyme function, and that energy coupling is affected by several residues in the transmembrane domains, as well as in the cytoplasmic and periplasmic loops. H⁺-pyrophosphatase activity might involve dynamic linkage between the hydrophilic and transmembrane domains.     

Chloroplast-generated ROS dominate NaCl- induced K+ efflux in wheat leaf mesophyll

Mesophyll K⁺ retention ability has been recently reported as an important component of salinity stress tolerance in wheat. In order to investigate the role of ROS in regulating NaCl^-induced K⁺ efflux in wheat leaf mesophyll, a series of pharmacological experiments was conducted using MV (methyl viologen, superoxide radical inducer), DPI (an inhibitor of NADPH oxidase), H₂O₂ (to mimic apoplastic ROS), and EGCG ((−)-Epigallocatechin gallate, ROS scavenger). Mesophyll pre-treatment with 10 μM MV resulted in a significantly higher NaCl^-induced K⁺ efflux in leaf mesophyll, while 50 μM EGCG pre-treatment alleviated K⁺ leakage under salt stress. No significant change in NaCl^-induced K⁺ efflux in leaf mesophyll was found in specimens pre-treated by H₂O₂ and DPI, compared with the control. The highest NaCl^-induced H⁺ efflux in leaf mesophyll was also found in samples pre-treated with MV, suggesting a futile cycle between increased H⁺-ATPase activity and ROS-induced K⁺ leak. Overall, it is suggested that, under saline stress, K⁺ efflux from wheat mesophyll is mediated predominantly by non-selective cation channels (NSCC) regulated by ROS produced in chloroplasts, at least in bread wheat.