Some Properties of a Functional Reconstituted Plasmalemma H-ATPase Activated by Fusicoccin

Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H⁺-translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V_max of the enzyme. Fusicoccin stimulation of maize H⁺-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.).     

H-pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots

The initial rate of quenching of quinacrine fluorescence was used to monitor Mg:ATP-dependent H⁺-pumping in membrane vesicles from corn (Zea mays L. cv WF9 × MO17) roots and obtain a preparation in which vanadate-sensitive H⁺-pumping could be observed. Separation of membranes on a linear sucrose density gradient resulted in two distinct peaks of H⁺-pumping activity: a major one, at density 1.11 grams per cubic centimeter, was sensitive to NO₃⁻ and resistant to vanadate, while a minor one, at density 1.17 grams per cubic centimeter, was substantially resistant to NO₃⁻ and sensitive to vanadate. A membrane fraction enriched in the vanadate-sensitive H⁺-pump could be obtained by washing microsomes prepared in the presence of 10% glycerol with 0.25 molar KI. The kinetics of inhibition of H⁺-pumping by vanadate in this membrane preparation indicated that most of the H⁺-pumping activity in this fraction is sensitive to inhibition by vanadate, 50% inhibition being reached at about 60 micromolar vanadate. This value is fairly close to that observed for inhibition by vanadate of the ATPase activity in similar experimental conditions (40 micromolar). The inhibitor sensitivity, divalent cation dependence, pH optimum (6.5), and K_m for ATP (0.7 millimolar) of the H⁺-pumping activity match quite closely those reported for the plasma membrane ATPase of corn roots and other plant materials.     

Effect of Mg2+, tritorn X-100 and temperature on basal and FC- stimulated plasma membrane ATPase activity

H⁺-pumping driven by the plasma membrane H⁺-ATPase in membrane vesicles from 24-hour-old radish seedlings is stimulated by pretreatment of the membranes with fusicoccin (FC) (Rasi-Caldogno et al., Plant Physiol., 82 (1986) 121).FC-pretreatment stimulates also the ATPase activity, but to a lesser extentthan H⁺-pumping. More than 80% of the ATPase activity is inhibited by 100 μM vanadate or by 3 mM Ca²⁺.Preincubation of diluted membranes in the presence of 5 mM MgSO₄ without ATP lowers both ATPase and H⁺-pumping activity by 20—30% without affecting FC-stimulated activities (i.e. the differences between FC-treated samples and the controls).After preincubation with MgSO₄, ATPase activity of membranes pretreatedwith or without FC is delivery affected by Triton X-100 and by temperature: Triton X-100 activates FC-stimulated ATPase more than that of the controls and an increase of temperature (between 13 and 33°C) enhances ATPase activity of the controls more than the FC-stimulated one.These results have been interpreted as suggesting that, while H⁺-pumping in this membrane fraction is driven only by the plasma membrane H⁺-ATPase, ATP-hydrolysis is catalyzed by two different enzymes (or forms of the same enzxxyme) diversely sensitive to FC, Triton X-100 and temperature and possibly diversely involved in H⁺-pumping.     

Anion-sensitive, h-pumping ATPase in membrane vesicles from oat roots

H⁺-pumping ATPases were detected in microsomal vesicles of oat (Avena sativa L. var Lang) roots using [¹⁴C]methylamine distribution or quinacrine fluorescent quenching. Methylamine (MeA) accumulation into vesicles and quinacrine quench were specifically dependent on Mg,ATP. Both activities reflected formation of a proton gradient (ΔpH) (acid inside) as carbonyl cyanide m-chlorophenylhydrazone, nigericin (in the presence of K⁺), or gramicidin decreased MeA uptake or increased quinacrine fluorescence. The properties of H⁺ pumping as measured by MeA uptake were characterized. The K_mapp for ATP was about 0.1 millimolar. Mg,GTP and Mg, pyrophosphate were 19% and 30% as effective as Mg,ATP. MeA uptake was inhibited by N,N′-dicyclohexylcarbodiimide and was mostly insensitive to oligomycin, vanadate, or copper. ATP-dependent MeA was stimulated by anions with decreasing order of potency of Cl⁻ > Br⁻ > NO₃⁻ > SO₄²⁻, iminodiacetate, benzene sulfonate. Anion stimulation of H⁺ pumping was caused in part by the ability of permeant anions to dissipate the electrical potential and in part by a specific requirement of Cl⁻ by a H⁺ -pumping ATPase. A pH gradient, probably caused by a Donnan potential, could be dissipated by K⁺ in the presence or absence of ATP. MeA uptake was enriched in vesicles of relatively low density and showed a parallel distribution with vanadate-insensitive ATPase activity on a continuous dextran gradient. ΔpH as measured by quinacrine quench was partially vanadate-sensitive. These results show that plant membranes have at least two types of H⁺ -pumping ATPases. One is vanadate-sensitive and probably enriched in the plasma membrane. One is vanadate-resistant, anion-sensitive and has many properties characteristic of a vacuolar ATPase. These results are consistent with the presence of electrogenic H⁺ pumps at the plasma membrane and tonoplast of higher plant cells.     

Fusicoccin stimulates the H+-ATPase of plasmalemma in isolated membrane vesicles from radish

The effect of fusicoccin on the plasmalemma H+-ATPase has been investigated in a membrane fraction from 24 h old radish seedlings, in which Mg:ATP-dependent H+-transport is mediated only by the plasmalemma H+-ATPase. Fusicoccin stimulated the plasmalemma H+-ATPase - i.e. Mg:ATP-dependent intravesicular acidification, hyperpolaryzation of delta psi and ATPase activity -, when these activities were measured at the physiologically relevant pHs of 7.3 to 7.6. No effect of FC on the plasmalemma H+-ATPase was evident at pH 6.6.     

Proton-pumping adenosine triphosphatase in membrane vesicles of tobacco callus: Sensitivity to vanadate and K+

H⁺-pumping adenosinetriphosphatases (ATPases, EC 3.6.1.3) were demonstrated in sealed microsomal vesicles of tobacco callus. Quinacrine fluorescence quenching was induced specifically by MgATP and stimulated by EGTA and Cl^?. Fluorescence quenching reflected a relative measure of pH gradient formation (inside acid), as it could be reversed by gramicidin (an H⁺/cation conductor) or 10 mM NH₄Cl (an uncoupler). H⁺ pumping was inhibited by tributyltin (an ATPase inhibitor) and sodium vanadate, but it was insensitive to oligomycin or fusicoccin. The vanadate concentration required to inhibit pH gradient formation was similar to that needed to inhibit KCl-stimulated Mg²⁺-ATPase activity and generation of a membrane potential (measured by ATP-dependent ³⁵SCN^? uptake). About 45% of all three activities (ATPase, pH gradient, membrane potential generation) were vanadate-insensitive, supporting the idea that non-mitochondrial membranes of plants have at least two types of electrogenic H⁺ pump.A vanadate-insensitive, H⁺-pumping ATPase previously shown by methylamine accumulation was characterized to be anion-sensitive and possibly enriched in vacuolar membranes (Churchill, K.A. and Sze, H. (1983) Plant Physiol. 71, 610–617). Yet, pH gradient formation determined by quinacrine fluorescence quenching was decreased by monovalent cations with a sequence K⁺, Rb⁺, Na⁺ > Cs⁺,Li⁺> choline, bisTris-propane. Since K⁺ stimulated ATPase activity more than Bistris-propane, K⁺ appeared to collapse formation of the pH gradient by an H⁺/K⁺ countertransport. The sensitivity to vanadate and K⁺ provides evidence that the plasma-membrane ATPase is an electrogenic H⁺ pump.     

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.     

Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization

Mg:ATP-dependent H⁺ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO₃⁻. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO₄²⁻, Cl⁻, Br⁻, NO₃⁻. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K⁺ (+ 80%) and to a lesser extent by Na⁺ and choline (+28% and +14%, respectively). The characteristics of H⁺ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.     

Fusicoccin Binding to its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase. II. Stimulation of the H+-ATPase in a Plasma Membrane Fraction Purified by Phase-partitioning

The effect of fusicoccin (FC) on the activity of the PM H⁺-ATPase was investigated in a plasma membrane (PM) fraction from radish seedlings purified by the phase-partitioning procedure. FC stimulated the PM H⁺-ATPase activity by up to 100 %; the effect was essentially on V_max with only a slight decrease of the apparent K_M of the enzyme for ATP. FC-induced stimulation of the PM H⁺-ATPase was evident within the first minute and maximal within five minutes of membrane treatment with the toxin indicating that transmission of the signal from the activated receptor to the PM H⁺-ATPase is very rapid. Both FC-induced stimulation of the PM H⁺-ATPase and FC binding to its receptor decreased dramatically upon incubation of the membranes in ATPase assay medium at 33 °C in the absence of FC, due to the lability of the free FC receptor. FC-induced stimulation of the PM H⁺-ATPase was strongly pH dependent: absolute increase of activity was maximal at pH 7, while percent stimulation increased with the increase of pH up to pH 7.5; FC binding was scarcely influenced by pH in the pH range investigated. Taken as a whole, these results indicate that FC binding is a condition necessary, but not sufficient, for FC-induced stimulation of the PM H⁺-ATPase.     

A mutant of Arabidopsis thaliana partially resistant to fusicoccin has reduced plasma membrane H+-ATPase

5-2 is a mutant of Arabidopsis thaliana which is partially resistant to fusicoccin in vivo. We have analysed fusicoccin binding and the activity and amount of H⁺-ATPase in plasma membrane isolated from mature leaves of the wild type and of mutant 5-2. Fusicoccin binding was similar in plasma membrane from the two genotypes, while H⁺-ATPase activity was markedly (c. 50%) lower in plasma membrane from mutant 5-2 than in that from the wild type. The H⁺-ATPase of mutant 5-2 was activated by fusicoccin as much as that of the wild type. In plasma membrane from mutant 5-2, the amount of immunodetectable H⁺-ATPase, quantified by densitometry of Western blots, was about half that in the wild type. These results indicate that the major defect of mutant 5-2 detectable at the plasma membrane level is a reduction in the amount of H⁺-ATPase.     

Characterization of native and reconstituted plasma membrane H+-ATPase from the plasma membrane ofBeta vulgaris

Summary Characteristics of the native and reconstituted H⁺-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H⁺-ATPase retained characteristics similar to those of the native plasma membrane H⁺-ATPase following reconstitution into proteoliposomes. ATPase activity and H⁺ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO ₃ ^– was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H⁺ transport showed a pH optimum of 6.5, required a divalent cation (Co²⁺>Mg²⁺>Mn²⁺>Zn²⁺>Ca²⁺), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aK _m of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H⁺-ATPases were stimulated by monovalent ions. Native plasma membrane H⁺-ATPase showed an order of cation preference of K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Cs⁺>Li⁺>choline⁺. This basic order was unchanged following reconstitution, with K⁺, NH ₄ ⁺ , Rb⁺ and Cs⁺ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H⁺ transport, both enzymes required a cation such as K⁺ for maximal transport activity, but were stimulated preferentially by Cl^– even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H⁺-ATPase.     

Cell physiological aspects of the plasma membrane electrogenic H<Superscript>+</Superscript> pump

This article deals with cell physiological aspects of the plasma membrane electrogenic proton (H⁺) pump and emphasizes the contribution of the giant algal cells of the Characeae in elucidating the mechanism of the pump. First, a history of the development of intracellular perfusion techniques in characean internodal cells is described, including preparation of tonoplast-free cells. Then, an outline of the hypothesis of the electrogenic H⁺ pump proposed by Kitasato is introduced, who prophesied the existence of an electric potential generated by an active H⁺ efflux. Subsequently, a history of finding ATP as the direct energy source of the electrogenic ion pump is presented. Quantitative agreement between the pump current and the ATP-dependent H⁺ efflux supports the notion that the ion carried by the electrogenic ion pump is H⁺. The role of the H⁺ pump in regulation of the cytosolic pH is discussed. Mechanisms of light-induced potential change through photosynthesis-controlled activation of the H⁺ pump are discussed in terms of changes in the levels of adenine nucleotides and in modulation of the K_m value for the ATP of H⁺-ATPase. Recent progress in the molecular mechanism of the blue-light-induced activation of the H⁺-ATPase in guard cells is presented. However, there are cases where H⁺-ATPase activity is inhibited by blue light, indicating the flexibility of the control mechanisms of H⁺-ATPase activity. Finally, modulation of H⁺-pumping or H⁺-ATPase activities in response to environmental factors, such as anoxia, membrane excitation, osmotic and salt stresses, nutrient deficiencies and aluminum toxicity are described. Discussions are presented on the regulation of the electrogenic H⁺ pump.     

Lysophosphatidylcholine stimulates ATP dependent proton accumulation in isolated oat root plasma membrane vesicles

Lysophosphatidylcholine at concentrations of 30 micromolar stimulated the rate of MgATP-dependent H⁺-accumulation in oat (Avena sativa L. cv Rhiannon) root plasma membrane vesicles about 85% while the passive permeability of H⁺ was unchanged. Activation was dependent on chain length, degree of saturation, and head group of the lysophospholipid. A H⁺-ATPase assay was developed that allowed the simultaneous measurement of proton pumping and ATPase activity in the same sample. ATP hydrolysis was also stimulated by lysophospholipids and showed the same lipid specificity, but stimulation was only about 25% at 30 micromolar. At higher concentrations of lysophosphatidylcholine the ATPase activity in a latency-free system could be stimulated about 150%. The enzymic properties of proton pumping and ATP hydrolysis were otherwise identical with respect to vanadate sensitivity, K_m for ATP and pH optimum. The stimulatory effect of lysophospholipids suggests that these compounds could be part of the regulatory system for plant plasma membrane H⁺-ATPase activity in vivo.     

Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl

To determine whether the detergent-solubilized and purified vacuolar H⁺-ATPase from plants was active in H⁺ transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K⁺-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K⁺ and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H⁺ pumping was electrogenic. Both H⁺ pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A₁, a specific vacuolar type ATPase inhibitor. The reconstituted H⁺ pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO₃⁻ but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H⁺ pumping by the reconstituted ATPase in the presence of K⁺ and valinomycin. Hence, our results support the idea that the vacuolar H⁺-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl⁻ concentration. The purified and reconstituted H⁺-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H⁺ pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H⁺ translocation.     

Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied the effect of Cl⁻, NO₃⁻, and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H⁺-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (μh⁺). A permeant anion (e.g. Cl⁻), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([¹⁴C]thiocyanate distribution) and stimulated formation of a pH gradient ([¹⁴C]methylamine distribution). However, Cl⁻-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl⁻ independent of the proton electrochemical gradient. Unlike the plasma membrane H⁺-ATPase, the Cl⁻-stimulated ATPase was inhibited by NO₃⁻ (a permeant anion) and by DIDS. In the absence of Cl⁻, NO₃⁻ decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO₃⁻ of Cl⁻-stimulated pH gradient formation and Cl⁻-stimulated ATPase activity was noncompetitive. In the absence of Cl⁻, DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl⁻-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H⁺-ATPase by NO₃⁻ or DIDS suggest that the vanadate-insensitive H⁺-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.     

Subunit Composition and Organization of the Vacuolar H-ATPase from Oat Roots

The vacuolar H⁺-translocating ATPase (H⁺-ATPase), originally reported to consist of three major subunits, has been further purified from oat roots (Avena sativa var Lang) to determine the complete subunit composition. Triton-solubilized ATPase activity was purified by gel filtration on Sephacryl S400 and ion-exchange chromatography (Q-Sepharose). ATP hydrolysis activity of purified preparations was inhibited by 100 nanomolar bafilomycin A₁, a specific vacuolar-type ATPase inhibitor. The purified oat H⁺-ATPase (relative molecular weight = 650,000) was composed of polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. To analyze the organization of the H⁺-ATPase subunits, native vacuolar membranes were treated with KI and MgATP to dissociate peripheral proteins. Release of 70, 60, 44, 42, 36, and 29 kilodalton polypeptides from the membrane was accompanied by a loss of ATP hydrolysis and ATP-dependent H⁺-pumping activities. Five of the peripheral subunits were released from the membrane as a large complex of 540 kilodaltons. Vesicles that had lost the peripheral sector of the ATPase could hold a pH gradient generated by the proton-translocating pyrophosphatase, suggesting that the integral sector of the ATPase did not form a H⁺-conducting pathway. Negative staining of native vesicles revealed knob-like structures of 10 to 12 nanometers in dense patches on the surface of vacuolar membranes. These structures were removed by MgATP and KI, which suggested that they were the peripheral sectors of the H⁺-ATPase. These results demonstrate that the vacuolar H⁺-ATPase from oat roots has 10 different subunits. The oat vacuolar ATPase is organized as a large peripheral sector and an integral sector with a subunit composition similar, although not identical to, other eukaryotic vacuolar ATPases. Variations in subunit composition observed among several ATPases support the idea that distinct types of vacuolar H⁺-ATPases exist in plants.     

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

The effects of NO₃⁻ and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H⁺-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO₃ when assayed at 24°C or above and was tentatively identified as the tonoplast H⁺-ATPase. A smaller peak of H⁺-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO₃ and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H⁺-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO₃ inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO₃. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO₃ caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO₃ was explained by the temperature-dependent delay of the inhibition by KNO₃. The plasma membrane H⁺-ATPase maintained a pH gradient in the presence of KNO₃ for up to 30 minutes at 24°C.     

Dissociation and Reassembly of the Vacuolar H-ATPase Complex from Oat Roots

Conditions for the dissociation and reassembly of the multi-subunit vacuolar proton-translocating ATPase (H⁺-ATPase) from oat roots (Avena sativa var Lang) were investigated. The peripheral sector of the vacuolar H⁺-ATPase is dissociated from the membrane integral sector by chaotropic anions. Membranes treated with 0.5 molar KI lost 90% of membrane-bound ATP hydrolytic activity; however, in the presence of Mg²⁺ and ATP, only 0.1 molar KI was required for complete inactivation of ATPase and H⁺-pumping activities. A high-affinity binding site for MgATP (dissociation constant = 34 micromolar) was involved in this destabilization. The relative loss of ATPase activity induced by KI, KNO₃, or KCl was accompanied by a corresponding increase in the peripheral subunits in the supernatant, including the nucleotide-binding polypeptides of 70 and 60 kilodaltons. The order of effectiveness of the various ions in reducing ATPase activity was: KSCN > KI > KNO₃ > KBr > K-acetate > K₂SO₄ > KCl. The specificity of nucleotides (ATP > GTP > ITP) in dissociating the ATPase is consistent with the participation of a catalytic site in destabilizing the enzyme complex. Following KI-induced dissociation of the H⁺-ATPase, the removal of KI and MgATP by dialysis resulted in restoration of activity. During dialysis for 24 hours, ATP hydrolysis activity increased to about 50% of the control. Hydrolysis of ATP was coupled to H⁺ pumping as seen from the recovery of H⁺ transport following 6 hours of dialysis. Loss of the 70 and 60 kilodalton subunits from the supernatant as probed by monoclonal antibodies further confirmed that the H⁺-ATPase complex had reassembled during dialysis. These data demonstrate that removal of KI and MgATP resulted in reassociation of the peripheral sector with the membrane integral sector of the vacuolar H⁺-ATPase to form a functional H⁺ pump. The ability to dissociate and reassociate in vitro may have implications for the regulation, biosynthesis, and assembly of the vacuolar H⁺-ATPase in vivo.     

Characteristics and Regulatory Properties of the H+-ATPase in a Plasma Membrane Fraction Purified from Arabidopsis thaliana

The aqueous two-phase partitioning technique was utilized to isolate a plasma membrane (PM) fraction from etiolated seedlings of Arabidopsis thaliana. The purification procedure adopted yielded a fraction highly enriched in PM as compared to inner membranes, with a recovery of about 30%, as judged from the activities of PM markers such as vanadate-sensitive ATPase, FC binding and UDP-glucose sterol glucosyltransferase. The purified PM fraction displayed vanadate-sensitive H⁺ pumping activity. Its purity was confirmed by the biochemical characteristics of its ATPase activity assayed in the absence of Ca²⁺: sensitivity to vanadate (IC₅₀ ca. 1 μM), Mg²⁺-dependence, insensitivity to molybdate, oligomycin and nitrate, pH optimum at 6.6. The PM H⁺-ATPase activity was stimulated by fusicoccin and by a controlled treatment of the PM with trypsin. In both cases stimulation was much stronger on the activity assayed at pH 7.5 than on the activity at pH 6.6. Moreover, neither fusicoccin nor the treatment with trypsin stimulated the portion of activity (30 to 40% at pH 7.5) which decayed upon preincubation of the PM in assay medium without ATP.     

Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte

The plasma membrane H⁺-ATPase provides the driving force for solute transport via an electrochemical gradient of H⁺ across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H⁺-ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate Thr-containing H⁺-ATPase (pT H⁺-ATPase) and non-pT H⁺-ATPase as in the green algae, and that pT H⁺-ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H⁺-ATPase genes, designated PpHA (Physcomitrella patens H⁺-ATPase). Six isoforms are the pT H⁺-ATPase; a remaining isoform is non-pT H⁺-ATPase. An apparent 95-kD protein was recognized by anti-H⁺-ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H⁺-ATPase. Furthermore, we could not detect the pT H⁺-ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H⁺-ATPase most likely appeared for the first time in bryophyte.