©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
On the Mechanism of Hyperacidification in Lemon
COMPARISON OF THE VACUOLAR H-ATPase ACTIVITIES OF FRUITS AND EPICOTYLS (*)

(Received for publication, June 28, 1995; and in revised form, November 14, 1995)

Mathias L. Müller (§) Ursula Irkens-Kiesecker Bernard Rubinstein (¶) Lincoln Taiz (**)

From the Biology Department, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Lemon fruit vacuoles acidify their lumens to pH 2.5, 3 pH units lower than typical plant vacuoles. To study the mechanism of hyperacidification, the kinetics of ATP-driven proton pumping by tonoplast vesicles from lemon fruits and epicotyls were compared. Fruit vacuolar membranes were less permeable to protons than epicotyl membranes. H pumping by epicotyl membranes was chloride-dependent, stimulated by sulfate, and inhibited by the classical vacuolar ATPase (V-ATPase) inhibitors nitrate, bafilomycin, N-ethylmaleimide, and N,N`-dicyclohexylcarbodiimide. In addition, the epicotyl H pumping activity was inactivated by oxidation at room temperature, and oxidation was reversed by dithiothreitol. Cold inactivation of the epicotyl V-ATPase by nitrate (geq100 mM) was correlated with the release of V(1) complexes from the membrane. In contrast, H pumping by the fruit tonoplast-enriched membranes was chloride-independent, largely insensitive to the V-ATPase inhibitors, and resistant to oxidation. Unlike the epicotyl H-ATPase, the fruit H-ATPase activity was partially inhibited by 200 µM vanadate. Cold inactivation treatment failed to inhibit H pumping activity of the fruit membranes, even though immunoblots showed that V(1) complexes were released from the membrane. However, cold inactivation doubled the percent inhibition by 200 µM vanadate from 30% to 60%. These results suggest the presence of two H-ATPases in the fruit preparation: a V-ATPase and an unidentified vanadate-sensitive H-ATPase. Attempts to separate the two activities in their native membranes on linear sucrose density gradients were unsuccessful. However, following detergent-solubilization and centrifugation on a glycerol density gradient, the two ATPase activities were resolved: a nitrate-sensitive V-type ATPase that is also partially inhibited by 200 µM vanadate, and an apparently novel vanadate-sensitive ATPase that is also partially inhibited by nitrate.


INTRODUCTION

Eukaryotic cells contain a variety of acidic intracellular compartments, including coated vesicles, secretory vesicles, Golgi bodies, endosomes, lysosomes, and vacuoles, which have in common that their membrane transport processes are energized by the vacuolar H-ATPase (V-ATPase)(^1)(1, 2, 3) . In addition to the V-ATPase, plant vacuolar membranes contain an H-pyrophosphatase (H-PPase), although in most tissues the V-ATPase is the dominant pump(4, 5) .

In animal cells, different compartments of the endocytotic pathway have characteristic lumenal pHs, ranging from pH 6.5 in the coated vesicles to pH 5.0 in the lysosomes, suggesting that the lumenal pH of each organelle is tightly regulated(6) . A number of observations suggest that the pH of plant vacuoles is also regulated. In plants with crassulacean acid metabolism for example, the vacuolar pH of the leaves varies diurnally, from pH 3 at night to pH 6 in the day(7) . In stomatal guard cells, the vacuolar pH is 4.5 in the dark when the stomata are closed, and 6 in the light when the stomata are open(8) . During fruit development the vacuolar pH often changes, becoming either more or less acidic as ripening progresses. Such fluctuations indicate that the vacuolar pH is under metabolic and developmental control. However, even in the case of vacuoles with a constant pH the V-ATPase may be continually regulated, inasmuch as the typical steady state DeltapH across the tonoplast appears to be considerably less than the theoretical maximum. From the H/ATP stoichiometry of the pump (n), the membrane potential (Delta), the Faraday constant (F), and the DeltaG, the maximum DeltapH at equilibrium can be calculated according to the equation:

Bennett and Spanswick (9) determined an H/ATP stoichiometry of 2 for the plant V-ATPase, which was confirmed by Guern et al.(10) . Schmidt and Briskin (11) extended these studies to the H-PPase and included estimates of internal buffering capacity. They confirmed an n value of 2 for the V-ATPase and calculated a maximum possible DeltapH across the tonoplast of 5.0-5.4 units when the membrane potential is +20 mV. The authors concluded that the V-ATPase normally functions far from equilibrium and is regulated by factors other than energy supply. Mechanisms that have been proposed to regulate the V-ATPase include ``slip''(12) , cytosolic activators or inhibitors(13, 14, 15) , chloride(12, 16) , cytosolic pH(17) , and oxidation/reduction(18, 19) . As yet, none of these mechanisms has been shown to correlate with in vivo proton gradients.

In lemon fruit juice sacs, the vacuolar pH declines from 6.5 to as low as 2.2 during maturation(20) . Assuming a cytosolic pH of 7.2, the final pH corresponds to a DeltapH of 5 units. Thus, the fruit V-ATPase either operates near thermodynamic equilibrium, or, alternatively, the pump H/ATP stoichiometry is <2. If the V-ATPase operates near thermodynamic equilibrium, it follows that the regulatory mechanisms that normally prevent the V-ATPase from reaching equilibrium are absent or deficient in the fruit juice sac cells. On the other hand, if the proton pump responsible for hyperacidification has an n < 2, it could be either a V-ATPase with variable H/ATP stoichiometry (17) or a novel type of tonoplast H-ATPase with an n = 1. Here we report that the H pumping activity of juice sac tonoplast-enriched membrane vesicles is relatively insensitive to a variety of V-ATPase inhibitors and is inhibited 30% by 200 µM vanadate. Centrifugation of the detergent-solubilized fruit membranes on linear glycerol gradients resulted in the separation of two peaks of ATPase activities: a nitrate-sensitive V-ATPase that is partially inhibited by 200 µM vanadate, and an apparently novel vanadate-sensitive ATPase that is partially inhibited by high nitrate concentrations. The possibility that these two proton pumps may interact with each other during vacuolar hyperacidification is discussed.


EXPERIMENTAL PROCEDURES

Materials

Lemon seeds (Citrus limon var. Schaub Rough Lemon) were generously supplied by Willits & Newcomb, Inc., Arvin, CA. Lemon fruits (var. Eureka) were harvested from trees on the campus of the University of California, Santa Cruz. Bafilomycin A(1) was from Sigma, BCA protein assay reagent was obtained from Pierce and n-dodecyl-beta-D-maltoside from Calbiochem. All bulk chemicals were purchased from Sigma and Fisher.

Juice Sac Membrane Preparation

Tonoplast-enriched membrane vesicles were prepared from lemon fruit juice sacs as follows. Three lemons were peeled, and their segments were carefully separated. The surrounding endocarp tissue was slit longitudinally along the narrow edge of each segment using a single-edged razor blade, and the juice sacs were released into 100 ml of cold homogenization buffer (HB; 1.5 M MOPS-KOH, pH 8.5, 1.5% polyvinylpyrrolidone-40, 0.75% bovine serum albumin, 7.5 mM EDTA, 2 mM DTT, and 0.1 mM PMSF). All subsequent steps were carried out at 4 °C and the membranes were maintained on ice. The juice sacs were ground using a mortar and pestle and filtered through a 0.28-mm nylon mesh. Cellular debris, nuclei and plastids were removed by centrifugation at 12,000 times g for 15 min in a Sorvall RC2-B refrigerated centrifuge (SS-34 rotor). The supernatant was subjected to ultracentrifugation in a Beckman SW-28 rotor at 132,000 times g for 60 min, and the pellet was resuspended in 15 ml of resuspension buffer (RB; 10 mM BTP-Mes, pH 7.6, 20 mM KCl, 1 mM EDTA, 2 mM DTT, and 0.1 mM PMSF). The microsomal fraction was further purified on a 10%/35% sucrose step gradient made up in gradient buffer (10 mM BTP-Mes, pH 7.6, 10% glycerol, 20 mM KCl, 1 mM EDTA, 2 mM DTT, and 0.1 mM PMSF). After 60 min of centrifugation at 132,000 times g in a Beckman SW-28.1 rotor, the 10%/35% interface containing tonoplast-enriched membranes was recovered, diluted with two volumes of RB, and pelleted at 174,000 times g for 20 min in a Beckman TLA-100.3 rotor. The tonoplast-enriched membranes were resuspended in RB at a final concentration of 10 µg of membrane protein/µl. Unless otherwise stated, when membranes were prepared for experiments involving oxidation or inhibition by N-ethylmaleimide (NEM) or N-phenylmaleimide (NPM), they were resuspended in RB in the absence of DTT.

Epicotyl Membrane Preparation

Lemon seeds were germinated and grown for 4 weeks at 29 °C, in the dark, in flats containing moist vermiculite. Epicotyls (40 g fresh weight) were harvested with a razor blade and homogenized in 150 ml of cold HB using a mortar and pestle. All other steps were as described in the fruit membrane preparation.

Liposome Preparation

L-alpha-Phosphatidylcholine type IV-S (Sigma) was dissolved to 10 mg/ml in a total volume of 10 ml of diethyl ether, evaporated to dryness under a stream of nitrogen, lyophilized, resuspended in 10 ml of water, and sonicated to clarity with a Braun-Sonic U probe sonicator.

Membrane Solubilization

Membranes were made up to 6 mg of protein/ml with RB. One volume of 4% (w/w) n-dodecyl-beta-D-maltoside in solubilization buffer (10 mM BTP/Mes, pH 7.6, 10% glycerol, 1 mM EDTA, 8 mM MgSO(4), 50 mM DTT, 200 µg/ml sonicated liposomes, and 0.012% butylated hydroxytoluene) was added drop by drop to the membranes while stirring on ice. The mixture was further incubated 30 min on ice with gentle stirring before being used in ATP hydrolysis experiments or further purified by glycerol gradient centrifugation.

Glycerol Density Gradient Centrifugation

Solubilized membrane proteins (3.5 mg) were centrifuged for 15 min at 412,000 times g (4 °C) in a Beckman TLA-100.3 rotor. The supernatant was filtered through a 0.45-µm cellulose nitrate filter and loaded on a linear 15-30% glycerol gradient in 10 mM BTP-Mes, pH 7.0, 0.1% Triton X-100, 1 mM EDTA, 4 mM MgSO(4), 10 mM KCl, 50 µg/ml sonicated liposomes, and 20 mM DTT. After centrifugation for 16 h at 177,000 times g (4 °C) in a Beckman SW-41 rotor, 0.5-ml fractions were collected and analyzed for ATPase activity in the presence or absence of nitrate and vanadate.

Dissociation of V(1) Complex

Membranes were made up to 0.3 mg of protein/ml in 2.5 ml of RB containing 5 mM ATP, 7 mM MgSO(4), and 0, 20, 100, 200, or 500 mM KNO(3) or KI. They were incubated on ice for 1 h and centrifuged 15 min at 412,000 times g (Beckman TLA-100.3). The membrane pellet was resuspended in 750 µl of RB and used for proton pumping and immunoblotting. The proteins in 50 µl of the resuspended pellet, and 167 µl of the supernatant were used for immunoblotting after being precipitated with 10% trichloroacetic acid, washed with cold acetone, lyophilized, and separated by SDS-PAGE.

Proton Pumping Assays

Proton pumping by tonoplast vesicles was monitored by the quenching of quinacrine fluorescence as described previously(21) . The reaction mix contained 10 mM BTP-Mes, pH 7.0, 250 mM sorbitol, 100 mM KCl, 50 µM vanadate, 1 mM azide, 250 nM valinomycin, 2.5 mM ATP, and 10 µM quinacrine unless otherwise stated. 100 µg of membrane proteins were typically used and the reaction was usually started with 3.5 mM MgSO(4). In kinetic experiments, ionic interactions were taken into account by means of the SOLCON computer program (generously provided by D. C. S. White, University of York, York, United Kingdom, and Y. E. Goldman, University of Pennsylvania, Philadelphia, PA) using logarithmic association constants of all metal-ligand complexes present in the assay mix(22) . Proton pumping was initiated with MgSO(4) at the concentration indicated to provide the required concentration of MgATP. Typically, the concentration of MgSO(4) had to be present at approximately 2 mM in excess of the sum of the concentrations of ATP and ADP present in the assay mix. Fluorescence quenching (423 nm excitation, 502 nm emission) was measured in a Perkin-Elmer LS-5 fluorescence spectrophotometer. Proton pumping activity was expressed as % quenchbulletmgbulletmin.

ATPase Assays

Native or solubilized membranes were added to a reaction mix containing 2.5 mM ATP, 2.5 mM MgSO(4), 100 mM KCl, 1 mM azide, 1 mM molybdate, 2 µM gramicidin D, and 1 mg/ml sonicated liposomes in 25 mM BTP/Mes buffer, pH 7.0. The total reaction volume was 300 µl. After 30 min at 37 °C, the reaction was stopped by adding one volume of 10% trichloroacetic acid, 4% perchloric acid (ice-cold) to the reaction mix. The mixture was maintained on ice for 5 min, centrifuged 4 min at 14,000 times g (4 °C) in a Tomy MTX-150 microcentrifuge, and the supernatant assayed for released inorganic phosphate by the Fiske and Subbarow reaction(23) . Boiled membranes were used for background estimates. Where nitrate-sensitive activity is reported, 200 µM vanadate were present in the reaction mix and the results are expressed as the difference in activity in the presence or absence of 200 mM KNO(3). Where vanadate sensitive activity is reported, 200 mM KNO(3) were present in the mix and the results show the difference in activity in the presence or absence of 200 µM vanadate.

Immunoblotting

One-dimensional SDS-PAGE of tonoplast enriched membranes was performed as described by Laemmli (24) on 12% polyacrylamide gels. The proteins from SDS gels were electrotransferred to 0.45-µm nitrocellulose filters (35 V, 200-300 mA, overnight; or 100 V, 250-350 mA for 1 h) in a Transphor TE 50 transfer cell (Hoefer Scientific Instruments, San Francisco, CA) or in a Mini Trans-Blot® electrophoretic transfer cell (Bio-Rad). The blots were blocked with 3% nonfat instant milk in TPBS (10 mM Na(2)HPO(4), 0.05% Tween® 20, and 0.9% NaCl, pH 7.5, with NaH(2)PO(4)) for 1 h before being incubated for 7 h in TPBS containing 0.5% bovine serum albumin and the primary antibody to the 70-kDa subunit of the corn V-ATPase(25) . The blots were then washed in TPBS and visualized by a peroxidase-coupled secondary antibody reaction (Vectastain ABC kit, Vector Laboratories, Burlingame, CA).

Antiserum Purification

Preimmune serum and antiserum raised against the 70-kDa subunit of the corn V-ATPase were purified on a Protein A-Sepharose column according to Sambrook et al.(26) . The sera were made up to 2.25 µg of protein/µl in 100 mM TrisbulletHCl, pH 8.0, before use in proton pumping and ATP hydrolysis experiments.

Protein Concentration

Estimates of protein concentrations were done routinely by a modified BCA protein assay after trichloroacetic acid precipitation(27) .

Statistical Treatment of the Results

All experiments were performed at least three times in duplicates. Proton pumping results are given as the means (± range) of the duplicates of one representative experiment. For ATP hydrolysis experiments, the mean (± S.D.) of two independent experiments are presented. K(m) and V(max) values were calculated from Hanes-Woolf plots.


RESULTS

DeltapH and Membrane Permeability

The proton pumping activities of the tonoplast-enriched membrane fraction from epicotyls and mature fruits were compared by quinacrine fluorescence quenching. When the fruit membranes were diluted to give the same initial rate of H pumping as the epicotyl vesicles, they developed a greater pH gradient (total quench) than the epicotyl membranes (Fig. 1). When the gradient had stabilized, 10 mM EDTA was added to stop the reaction by chelating Mg. In the absence of MgATP, the fruit membranes maintained 80% of the pH gradient for up to 2 h after an initial leakage, whereas the pH gradient across the epicotyl tonoplast collapsed within 20 min. Addition of gramicidin at the end of the reaction confirmed that a pH gradient was still present across the fruit vesicle membranes. We conclude that the juice sac tonoplast is less permeable to protons than the epicotyl tonoplast.


Figure 1: ATP-dependent proton pumping and membrane permeability of tonoplast enriched vesicles isolated from lemon epicotyls and juice sacs. Membrane protein concentrations were adjusted to give the same initial rates of H pumping (70 µg of fruit protein and 100 µg of epicotyl protein were used in this representative experiment). The assay mix was as described under ``Experimental Procedures,'' and the reaction was started with MgSO(4). After equilibrium was reached, 10 mM EDTAbulletBTP (pH 7.0) was added to chelate the Mg. After a new equilibrium was reached, the residual pH gradient was collapsed with 4 µM gramicidin.



Chloride Dependence of H Pumping Versus ATPase Activity

As shown in Fig. 2A, the H pumping activity of the epicotyl V-ATPase was almost completely dependent on chloride. Because the reaction was started by adding membranes loaded with 20 mM KCl, and valinomycin was present in the assay mix, chloride stimulation of H pumping is probably due to direct activation of the enzyme rather than to the ability of chloride to collapse the membrane potential. Similar results were obtained in the presence of valinomycin and 50 mM potassium iminodiacetate in the assay mix (data not shown). In this latter case, the membrane were allowed to equilibrate for 20-30 min in the reaction mix containing 50 mM potassium iminodiacetate and the proton-pumping reaction was started with Mg. Because K and valinomycin were present in the mix, the buildup of a positive Delta should again have been prevented. In contrast to epicotyl membranes, juice sac vesicles did not require chloride for proton transport (Fig. 2A).


Figure 2: Effect of chloride on H pumping and ATP hydrolysis by tonoplast-enriched membranes from lemon epicotyls and juice sacs. For proton pumping (A), the reaction mix contained 2.5 mM ATP, 3.5 mM MgSO(4), 0.25 µM valinomycin, 50 µM vanadate, 1 mM azide, sorbitol, 10 µM quinacrine, and cholinebulletCl at the concentrations indicated. Constant osmolarity was achieved by balancing cholinebulletCl and sorbitol in the mix. Each proton pumping reaction was started by adding 10 µl of membranes (100 µg of protein), preloaded with 20 mM KCl by freeze-thawing to collapse the membrane potential (bullet, epicotyl; circle, juice sac). Experimental conditions for ATP hydrolysis (B) were similar to those for H pumping, except that 1 mM molybdate was present in the mix, and quinacrine was omitted.



When ATPase activity was measured under similar conditions, the epicotyl membranes exhibited only a 15% chloride stimulation (Fig. 2B). If only the nitrate-sensitive activity is considered, the percent chloride stimulation increases to 30%. There remains a marked discrepancy between the chloride dependence of proton pumping and ATPase activities in the epicotyl. In contrast, both the proton pumping and ATP hydrolytic activities of juice sacs were equally insensitive to chloride. Thus, the apparent uncoupling of H-transport from ATP hydrolysis observed in the epicotyl V-ATPase in the absence of chloride, defined as ``slip,'' does not occur in the juice sac V-ATPase.

pH Optima, K(m), and V(max)

As shown in Fig. 3, the pH optima for the fruit and the epicotyl V-ATPases were both 7.0. Table 1gives the K(m) and V(max) values for the fruit and epicotyl membranes based on proton pumping activity. Whereas the K(m) values for MgATP of the fruit and epicotyl membranes were similar, the average V(max) of the fruit enzyme was about double that of the epicotyl.


Figure 3: Determination of the pH optima for H pumping by the epicotyl and juice sac V-ATPases. 10 µl of tonoplast enriched membranes (100 µg of protein) were assayed for ATP-dependent proton pumping in a reaction mix containing 2.5 mM ATP, 100 mM KCl, 0.25 µM valinomycin, 50 µM vanadate, 1 mM azide, and 10 µM quinacrine. The mix was buffered with 10 mM BTP-Mes to the indicated pH. The reaction was started with 3.5 mM MgSO(4), and the initial rate of quinacrine quenching is reported (bullet, epicotyl; circle, juice sac).





Immunoblotting

The higher V(max) of the fruit V-ATPase could reflect a higher density of V-ATPases on the membrane. Immunoblots probed with antibodies to the 70-kDa subunit of the corn V-ATPase (25) suggest that the fruit tonoplast vesicles actually contain less V-ATPase per milligram of protein than the epicotyl membranes (Fig. 4). This implies either a higher proportion of active enzyme on the juice sac tonoplast or a higher K. Alternatively, the antibody may have a lower affinity for the fruit V-ATPase than for the epicotyl enzyme, which would imply that the fruit V-ATPase is a different isozyme.


Figure 4: Immunoblot of membrane proteins from lemon epicotyls and juice sacs. Tonoplast-enriched membranes were separated by SDS-PAGE on a 15% polyacrylamide gel, transferred onto nitrocellulose, probed with a polyclonal antibody raised against the 70-kDa subunit of the corn V-ATPase, and stained using horseradish peroxidase. 10 and 20 µg of membrane proteins of juice sacs and epicotyls were loaded on the gel. Standard proteins and their molecular mass in kDa are given on the right of the sample lanes.



Inhibitors

Both the fruit and the epicotyl V-ATPases were inhibited by ADP. Inhibition was largely competitive, although the sigmoidal nature of the curves at low [ATP]/[ADP] ratios suggested allosteric inhibition as well (data not shown). The average K(i) values for ADP calculated from replots of the slopes of double-reciprocal plots were about 30 µM for both the fruit and epicotyl membranes.

Whereas the epicotyl tonoplast proton pumping activity showed a typical inhibition by nitrate and bafilomycin A(1), the juice sac tonoplast vesicles were remarkably insensitive to these inhibitors (Fig. 5A and Fig. 6). Measurements of ATP hydrolytic activity also indicated that the fruit V-ATPase was less sensitive to nitrate than the epicotyl enzyme (Fig. 5B, dashed lines). Detergent solubilization increased the nitrate sensitivity of the fruit, and decreased that of the epicotyl, so that both activities were equally sensitive to nitrate at 200 mM (Fig. 5B, solid lines).


Figure 5: Nitrate inhibition of the V-ATPases of lemon epicotyls and juice sacs. A, the effect of increasing concentrations of tetramethylammonium nitrate on the initial rate of proton pumping by tonoplast-enriched membranes (100 µg of protein) as measured by quinacrine quenching. B, the effect of KNO(3) on ATP hydrolysis in native and solubilized tonoplast vesicles (60 µg of membrane protein), as monitored by P(i) release. H pumping was initiated with 3.5 mM MgSO(4). ATP hydrolysis was initiated by adding the membranes to the mix (- -bullet- -, epicotyl membranes; - -circle- -, juice sac membranes; -bullet-, solubilized epicotyl membranes; -circle-, solubilized juice sac membranes).




Figure 6: Inhibition of ATP-dependent H pumping by bafilomycin A(1) in tonoplast-enriched vesicles from epicotyls and juice sacs. Bafilomycin A(1) at the concentrations indicated and 10 µl of tonoplast-enriched membranes (100 µg of protein) were added to the reaction mix (composition as under ``Experimental Procedures''). Each reaction was started with 3.5 mM MgSO(4) and the initial rates of quinacrine fluorescence quenching are reported (bullet, epicotyl; circle, juice sac).



The proteolipid-binding inhibitor, N,N`-dicyclohexylcarbodiimide (DCCD), inhibited both the juice sac and the epicotyl H pumping, although the inhibition was less pronounced in juice sac membranes than in epicotyl membranes (Fig. 7). The sensitivity of both enzymes to low concentrations of the sulfhydryl reagent, NEM, is shown in Fig. 8. The epicotyl V-ATPase was highly sensitive to NEM, with 94% of its activity being inhibited by 50 µM NEM, whereas the fruit enzyme was almost unaffected at this concentration. The more hydrophobic sulfhydryl reagent, NPM, was more effective than NEM in inhibiting proton pumping by the fruit V-ATPase (Fig. 8).


Figure 7: Inhibition of ATP-dependent H pumping by DCCD in tonoplast-enriched vesicles from epicotyls and juice sacs. The reaction mix was as described under ``Experimental Procedures''. 100 µg membrane proteins and DCCD at the concentrations indicated were added. Each reaction was started with 3.5 mM MgSO(4) and the initial rates of quinacrine fluorescence quenching were determined (-bullet-, epicotyl; -circle-, juice sac).




Figure 8: Inhibition of ATP-dependent H pumping by NEM and NPM in tonoplast-enriched vesicles from epicotyls and juice sacs. 100 µg membrane proteins and NEM or NPM at the concentrations indicated were added to a reaction mix (see ``Experimental Procedures''). Each reaction was started with 3.5 mM MgSO(4), and the initial rates of fluorescence quenching were measured (-bullet-, epicotyl, NEM; -circle-, juice sac, NEM; - -circle- -, juice sac, NPM).



Azide (1 mM) and vanadate (50 µM) were routinely included in the reaction mix to suppress residual plasma membrane ATPase and mitochondrial ATPase activities, respectively. As shown by the dose-response curves in Fig. 9, these two contaminants would represent no more than 10% of the total activity in the fruit membranes under standard assay conditions. Moreover, the membrane-bound vanadate-sensitive activity could not be separated from the nitrate-sensitive activity by linear sucrose or dextran gradients (data not shown), suggesting that the nitrate- and vanadate-sensitive activities are on the same membrane.


Figure 9: Effects of vanadate and azide on ATP-dependent H pumping by tonoplast-enriched vesicles from epicotyls and juice sacs. A, the effect of vanadate on the initial rates of H pumping as measured by quinacrine quenching. B, the effect of azide on the initial rates of H pumping. The reaction mix contained 2.5 mM ATP, 100 mM KCl, 0.25 µM valinomycin, 10 µM quinacrine, and either sodium vanadate or sodium azide at the concentrations indicated. In the vanadate experiment,1 mM azide was included into the mix and in the azide experiment 50 µM vanadate was present. Each reaction was started with 3.5 mM MgSO(4) (bullet, epicotyl; circle, juice sac).



Inhibition by 70-kDa Antibody

When fruit and epicotyl membranes were treated with polyclonal antibody to the catalytic subunit of the corn V-ATPase, the proton pumping activity of the epicotyl was inhibited by up to 80%, whereas the fruit membranes were inhibited by only 25% (Fig. 10A). However, following solubilization, the nitrate-sensitive ATPase activities of the fruit and epicotyl were equally inhibited by the 70-kDa antibody (Fig. 10B).


Figure 10: Inhibition of H pumping and ATPase activity by antibody to the 70-kDa subunit of corn. A, the effect of polyclonal antibody to the corn 70-kDa subunit on the initial rate of proton pumping by lemon tonoplast-enriched membranes measured by quinacrine fluorescence quenching. B, the effect of the same antibody on ATP hydrolysis by the solubilized enzymes, as monitored by P(i) release. Preimmune serum was used in both cases as a control. For H pumping and ATP hydrolysis assay conditions, see ``Experimental Procedures.'' 100 µg of membrane proteins were added to the mix in proton pumping experiments and each reaction was started with 3.5 mM MgSO(4). In ATP hydrolysis experiments, each reaction was started by adding 20 µg of solubilized membrane proteins to the mix and the nitrate-sensitive activity was determined (-bullet-, inhibition of the epicotyl V-ATPase by the 70-kDa antibody; -circle-, inhibition of the juice sac V-ATPase by the 70-kDa antibody; - -bullet- -, effect of preimmune serum on the epicotyl V-ATPase; - -circle- -, effect of preimmune serum on the juice sac V-ATPase).



Oxidation/Reduction

The sensitivities of the fruit and epicotyl V-ATPases to DTT-reversible oxidation was examined by measuring the decay of proton pumping activity over time at 20 °C, and the restoration of activity by 50 mM DTT. The membranes were resuspended either in RB containing 1 mM DTT (Fig. 11A), or RB without DTT (Fig. 11, B and C). As shown in Fig. 11(A-C), the epicotyl V-ATPase was almost completely inhibited after a 4-h incubation in the presence or absence of 1 mM DTT. In contrast, the activity of the fruit V-ATPase was stable over the same time period in the presence of 1 mM DTT and was only slowly inactivated in the absence of DTT. Incubation of the epicotyl membranes in the presence of 50 mM DTT or 5 mM ATP over the same time period completely protected the epicotyl V-ATPase from inactivation (Fig. 11, A and C). Addition of 50 mM DTT to the oxidized epicotyl preparation restored proton transport activity to >60% of the activity at time 0. When epicotyl membranes were incubated on ice, inactivation was greatly reduced, and was completely reversible with DTT, demonstrating that the oxidation was temperature-dependent. In contrast, the slow loss of activity of the fruit V-ATPase that occurred in the absence of DTT was temperature-independent and could not be reversed by DTT (Fig. 11B). In addition, ATP did not protect against inactivation of the juice sac V-ATPase (Fig. 11C). To determine whether the greater oxygen sensitivity of the epicotyl membranes was due to the release of a soluble oxidant, a 1:1 mixture (µg protein) of fruit and epicotyl membranes was incubated at 20 °C and the inactivation kinetics measured. The oxidation curve of the mixture corresponded to the average curve of the two membrane fractions measured separately (data not shown). If a soluble oxidant were involved, the loss of activity would have been additive.


Figure 11: Oxidative inactivation of the V-ATPases of epicotyls and juice sacs. Tonoplast-enriched membranes of lemon epicotyls and juice sacs were incubated either at 4 °C or at 20 °C, and the initial rates of ATP-dependent proton pumping were assayed at different time intervals. A, the H pumping activities of membranes incubated in the presence of 1 mM DTT (-bullet-, epicotyl, 20 °C; -circle-, juice sac, 20 °C; --, epicotyl, 4 °C) or in the presence of 50 mM DTT (- -bullet- -, epicotyl, 20 °C). Where indicated, 50 mM DTT was added to the membranes. B, H pumping activities of membranes incubated in absence of DTT (-bullet-, epicotyl, 20 °C; -circle-, juice sac, 20 °C; - -bullet- -, epicotyl, 4 °C; - -circle- -, juice sac, 4 °C; --, 1:1 mixture (µg of protein) of epicotyl and juice sac membranes). At 240 min, 50 mM DTT was added to all membranes. C, H pumping activities of membranes incubated at 20 °C in the presence or absence of 5 mM ATP (-bullet-, epicotyl, -ATP; - -bullet- -, epicotyl, +ATP; -circle-, juice sac, -ATP; - -circle- -, juice sac, +ATP). At time 240 min, 50 mM DTT was added to all membranes. The composition of the reaction mix was as described under ``Experimental Procedures,'' 100 µg of membrane proteins were assayed in each case, and the reactions were started with 3.5 mM MgSO(4).



To further characterize the oxidative inactivation of the epicotyl V-ATPase, the ability of agents other than ATP to protect the enzyme was examined. As shown in Table 2, a variety of chelators failed to protect against oxidation, arguing against a role for metals as cofactors in an enzymatic oxidation process. Catalysis is not required for protection, since ADP was shown to be as effective as ATP in protecting the enzyme. Surprisingly, 100 mM sulfate not only protected the enzyme against inactivation, it also caused a doubling of the original activity. The effects of sulfate and nucleotides were not additive (Table 2). Other anions also protected against oxidation to varying degrees, including chloride and nitrate. The ability of nitrate to protect against oxidation is surprising inasmuch as nitrate is a potent inhibitor of catalysis. The combination of nitrate plus ATP or MgATP was less effective than nitrate alone in protecting against oxidation. Nevertheless, it clearly protected relative to the control.



Dissociation of the V(1) Complex with Chaotropic Agents

We hypothesized that the nitrate insensitivity of the fruit membranes might be due to a tighter attachment of the peripheral V(1) complex to the membrane-bound portion, V(0). To test this, dissociation of V(1) from V(0) in fruit and epicotyl membranes was induced by KNO(3), on ice, in the presence of 5 mM MgATP. As shown in Fig. 12(A and B), the proton pumping activity of the epicotyl membranes was strongly inhibited at nitrate concentrations > 20 mM, and the inhibition was correlated with the release of V(1) complexes from the membrane, as shown by immunoblotting. Surprisingly, the total proton pumping by the fruit membranes was insensitive to as high as 500 mM nitrate, even though the pattern of V(1) dissociation paralleled that of the epicotyl. The only detectable effect of nitrate on the fruit proton pumping activity was a progressive increase in the proportion of vanadate-sensitive activity and a concomitant decrease in the vanadate-insensitive activity (Fig. 12B). Similar results were obtained with KI (data not shown). The data suggest the presence of a second, vanadate-sensitive proton pump on the fruit tonoplast.


Figure 12: Dissociation of the V(1) complex with nitrate. A, the effect of increasing concentrations of nitrate on the cold release of V(1) complex, as probed on immunoblots with antibody to the catalytic subunit of the corn V-ATPase. Tonoplast-enriched vesicles were incubated for 1 h, on ice, in the presence of 5 mM MgATP and KNO(3) as indicated. After centrifugation, equal volume fractions of the pellets and the supernatants were analyzed by SDS-PAGE and blotted onto nitrocellulose. B, the effect of V(1) release on the initial rate of proton pumping by the pelleted and resuspended membranes (-bullet-, epicotyl membranes in the presence of 50 µM vanadate; bullet -circlebullet -, fruit juice sac membranes in the absence of vanadate; -circle-, fruit juice sac membranes in the presence of 500 µM vanadate; - -circle- -, resultant of the difference of the two precedent curves (vanadate-sensitive activity).



Solubilization and Glycerol Gradient Centrifugation

Following solubilization of the fruit tonoplast-enriched membranes with 2% dodecylmaltoside and centrifugation, the nitrate-sensitive ATPase activity released into the supernatant was double that of the initial membrane-bound activity (Fig. 13A). The total nitrate-sensitive activity after solubilization (pellet plus supernatant) was 2.5 times that of the native membrane (Fig. 13B). In contrast, the nitrate-insensitive activity remained constant over the range of the detergent concentrations used.


Figure 13: Solubilization of fruit juice sac membranes with dodecylmaltoside. Tonoplast-enriched membranes of lemon juice sacs were solubilized with dodecylmaltoside added to the concentrations indicated. After centrifugation, equal volume fractions of the pellets and the supernatants were assayed for ATPase activity in the presence or the absence of 200 mM KNO(3). A, the nitrate-sensitive ATPase activities remaining in the pellets (- -circle- -) and the nitrate-sensitive activities released into the supernatants (-circle-). B, the total ATPase activities after solubilization (pellet plus supernatant). bullet -circlebullet -, total activity; -circle-, nitrate-sensitive activity; - -circle- -, nitrate-insensitive activity.



Partial purification of the solubilized enzymes by density gradient centrifugation on a linear 15-30% glycerol gradient resulted in the separation of the nitrate-sensitive from the vanadate-sensitive ATPase activities. However, the nitrate-sensitive peak also appeared to be partially vanadate-sensitive, and the vanadate-sensitive peak was partly inhibited by nitrate (Fig. 14). The vanadate-sensitive peak was missing in glycerol gradients of solubilized epicotyl membranes and is thus specific for the fruit membranes (data not shown).


Figure 14: Glycerol density gradient centrifugation of solubilized fruit juice sac membrane proteins. Tonoplast-enriched membranes were solubilized with 2% dodecylmaltoside and layered on a 15-30% glycerol gradient. After centrifugation for 16 h at 177,000 times g, 0.5-ml fractions were collected and analyzed for vanadate-sensitive (- -circle- -) and nitrate-sensitive (-circle-) ATPase activities (activities in the presence or absence of 400 mM nitrate and 200 µM vanadate). Fraction 1 represents the top of the gradient, fraction 21, the bottom. The amount of activity recovered from the gradients was 50% of the initial activities.




DISCUSSION

During maturation, the vacuolar pH of the juice sac cells of lemon fruits declines gradually from pH 6.0 to pH 2.2, a process that may take over a year. This prolonged acidification is accompanied by citrate accumulation in the form of the free acid to a final concentration of about 300 mM(20) . Since citrate typically enters vacuoles as citrate(28) , it would tend to bind protons and raise the vacuolar pH rather than lower it. Thus, the dramatic decline in pH is brought about by active proton pumping, rather than by citrate uptake. The H-PPase can be ruled out as the primary proton pump because: 1) H-PPase activity is negligible in juice sacs, (^2)and 2) calculations indicate that under typical cellular conditions the H-PPase is incapable of generating such a large pH gradient(11, 17) .

Membrane Permeability

The equilibrium DeltapH across the tonoplast is governed by two main factors: proton pumping and proton leakage across the membrane(29) . We have shown that juice sac tonoplast vesicles are less permeable to protons than epicotyl vesicles. The slower leak rate of the juice sac tonoplast allows the buildup of a steeper pH gradient in the in vitro assays and undoubtedly plays an important role in vivo. However, it is unlikely that the vacuolar pH in epicotyls is regulated by proton leakage alone since futile cycling of protons would be costly energetically. Thus, V-ATPases must be kinetically regulated in vivo.

The Juice Sac Tonoplast Contains a Vanadate-sensitive V-ATPase

The H-ATPase activity of the native tonoplast-enriched membrane vesicles from juice sacs was insensitive to a wide array of V-ATPase inhibitors, including nitrate, bafilomycin A(1), NEM, and a polyclonal antibody to the 70-kDa subunit of corn. Nevertheless, a V-type H-ATPase appears to be present based on the following criteria. First, both the fruit and epicotyl H pumping activities have similar pH optima and K(m) values for MgATP. Second, although the fruit-derived vesicles were insensitive to NEM, the hydrophobic sulfhydryl inhibitor, NPM, was a more effective inhibitor. Third, detergent solubilization increased the sensitivity of the fruit ATPase to inhibition by nitrate by 2.5-fold, indicating that the lack of sensitivity to nitrate is caused by molecular interactions in the native membrane. Fourth, the solubilized ATPase activity was inhibited by antibody to the 70-kDa antibody and by NEM. (^3)Fifth, cold treatment in the presence of MgATP and nitrate caused the release of immunodetectable V(1) complexes from the fruit membrane. Interestingly, the solubilized, partially purified nitrate-sensitive V-ATPase from the fruit is also partly inhibited by 200 µM vanadate. Since the epicotyl V-ATPase, when treated in similar fashion, is not inhibited, (^4)the fruit V-ATPase appears to be a different isozyme. Vanadate-sensitive V-ATPases have previously been shown to be present on the plasma membranes of animal osteoclast cells, although they are more sensitive to vanadate than the fruit enzyme(30) . More recently, a vanadate- and nitrate-sensitive V-ATPase has been demonstrated on the tonoplast of vacuoles isolated from Acer pseudoplatanus cell cultures(31) . Whether or not vanadate-sensitive V-ATPases are specifically adapted for hyperacidification remains to be determined.

Regulation by ADP

ADP has been suggested to be involved in the kinetic regulation of the V-ATPase(32) . In lemon, ADP inhibition of the V-ATPases appears to have both a competitive and an allosteric component. The K(i) values determined for the competitive ADP inhibition of proton pumping for both the fruit and the epicotyl V-ATPases were surprisingly low (20-40 µM) compared to previously published values for plant V-ATPases (140-180 µM(32) , 120 µM(33) ). Since the K(m) for MgATP is about 0.8-0.9 mM, the low K(i) value for ADP suggests that the V-ATPase in epicotyls is partially inhibited by ADP in vivo, assuming that epicotyl cells contain typical millimolar concentrations of ATP and ADP(9) .

Mature juice sacs are hypoxic, which raises the question of whether or not there is sufficient ATP energy to maintain the observed pH gradients. Based on the published values of 16 µM ATP, 21 µM ADP(34) , and 3 mM PO(4)(20) , and using -8.8 kcal/mol for DeltaG`(35) , the calculated DeltaG in mature juice sacs is around -12 kcal/mol. Since this value is in the same range as in normal vegetative tissues(11) , there is sufficient free energy to drive proton transport in mature juice sacs, although the rate will be slower than in epicotyls because of the low substrate concentration.

Regulation by Slip

Moriyama and Nelson (12) proposed that the vacuolar H-ATPase of chromaffin granules is controlled by slip, defined as ATPase catalytic activity uncoupled from proton transport. In epicotyl tonoplast vesicles, the proton pumping activity was strongly stimulated by chloride in the presence of K and valinomycin to collapse Delta. Under the same conditions, ATPase activity was only marginally stimulated by chloride. Since the H pumping in the epicotyl was much more dependent on chloride than the ATPase activity, the epicotyl V-ATPase appears to undergo slip when chloride is limiting. In contrast, the H pumping and ATPase activities of the juice sac V-ATPase were both equally insensitive to chloride. One interpretation of the results is that the juice sac V-ATPase remains coupled under low chloride conditions and does not undergo slip. Alternatively, the chloride-independent activity could be due to the presence of a second proton pump on the membrane that does not require chloride.

Regulation by Oxidation/Reduction

The epicotyl V-ATPase was strongly inhibited by the sulfhydryl reagent, NEM. It is now well established that the highly conserved cysteine residue in the catalytic site of V-ATPases (Cys in the bovine coated vesicle H-ATPase and Cys in yeast) is the site of NEM binding and inhibition(18, 36) . Moreover, Krauss et al.(37) and Feng and Forgac (18, 19) have provided evidence that the V-ATPase can undergo DTT-reversible oxidative inactivation. Our results show that DTT-reversible inactivation of the lemon epicotyl V-ATPase occurred in native tonoplast vesicles upon incubation at 20 °C. Moreover, the reduction in the V(max) of the epicotyl V-ATPase compared to the fruit enzyme appeared to be proportional to the amount of oxidized enzyme. When 20 mM DTT was included in the vesicle isolation buffers, instead of the usual 2 mM, the V(max) of the epicotyl enzyme doubled, making it equal to that of the fruit.^4 This is similar to the results of Feng and Forgac (19) for bovine coated vesicles and it suggests that as much as 50% of the epicotyl V-ATPase may be in the oxidized, inactive state in vivo.

Oxidation of the lemon epicotyl V-ATPase was temperature-dependent and was blocked by ATP and ADP. Feng and Forgac (19) showed that the oxidation of the coated vesicle V-ATPase was also blocked by nucleotides and was partly prevented by 100 mM KCl, which they attributed to an effect of ionic strength. Our results indicate that the protective effect of salts is anion-specific. Sulfate, which has been shown to inhibit the V-ATPase of chromaffin granules (38) not only protects the lemon epicotyl V-ATPase against oxidation, it strongly activates it. However, neither sulfate nor other anions protected against inhibition by NEM.^4 This suggests that the site of protection against oxidation is distinct from the NEM-binding site. The ability of anions to block oxidation could be due to a conformational change.

We also tested the ability of sulfate to substitute for chloride in the proton pumping assay. As previously reported for chromaffin granules (38) , chloride was required by the epicotyl V-ATPase even in the presence of sulfate, suggesting that there are two distinct anion binding sites.

In contrast to the epicotyl, the juice sac V-ATPase was insensitive to NEM, and exhibited no DTT-reversible oxidation. However, NPM was more inhibitory than NEM, suggesting that the critical cysteine is in a hydrophobic pocket. In accord with this view, detergent solubilization of the juice sac V-ATPase increased the sensitivity of the juice sac V-ATPase to NEM. The insensitivity of the juice sac V-ATPase to oxidation may be another factor which contributes to the unusually low pH of juice sac vacuoles. Inasmuch as maturation in lemon fruits occurs over a year, and during much of that time the tissue is hypoxic, a highly stable V-ATPase is needed to maintain the steep DeltapH across the tonoplast over the entire life of the fruit.

A Second Vacuolar H-ATPase?

Under conditions known to cause the physical dissociation of the catalytic V(1) complex from the membrane sector, V-ATPases from juice sacs and epicotyls released comparable amounts of V(1) complexes into the incubation medium. Whereas V(1) release from epicotyl membranes was correlated with a decrease in the total H pumping activity, the total H pumping activity of the fruit membranes was unaffected. The net increase in vanadate-sensitive H pumping activity accompanying the dissociation of the catalytic complex of the juice sac V-ATPase suggests that a second, vanadate-sensitive H-ATPase is activated under the conditions promoting V(1) dissociation. This interpretation is supported by the identification of a vanadate-sensitive ATPase peak that separates from the nitrate-sensitive V-ATPase peak after centrifugation of the detergent-solubilized fruit enzymes on linear glycerol gradients. This activity is not likely to be due to a contaminating plasma membrane H-ATPase because: (a) the concentration of vanadate needed to inhibit it is 10 times greater than that needed to inhibit a normal P-type ATPase, and (b) the activity is also nitrate-sensitive. Moreover, if the two proton pumps were to reside on different membranes, one would expect the two activities to be additive. The cold inactivation experiment shows, however, that this is not the case. Rather, the putative second proton pump appears to take over the function of the V-ATPase as the V(1) complex becomes dissociated, so that the initial rate of proton pumping by the vesicles remains the same. A corollary of the two proton pump model is therefore that the two H-ATPases interact in some way on the membrane. Reconstitution studies are currently under way to determine whether the two pumps behave differently when reconstituted into artificial liposomes either individually or as a pair.


FOOTNOTES

*
This research was supported in part by Grant DE-FG03-84ER13245 (to L. T.) from the United States Department of Energy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Swiss National Foundation for Scientific Research and from the Ciba-Geigy Jubiläumsstiftung.

Permanent address: Biology Dept., University of Massachusetts, Amherst, MA 01003-5810.

**
To whom correspondence and reprint requests should be addressed. Tel.: 408-459-2036; Fax: 408-459-3139; :taiz{at}biology.ucsc.edu.

(^1)
The abbreviations used are: V-ATPase, vacuolar H-ATPase; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; DCCD, N,N`-dicyclohexylcarbodiimide; DTT, dithiothreitol; HB, homogenization buffer; H-PPase, H-pyrophosphatase; Mes, 2-(N-morpholino)ethanesulfonic acid; MOPS; 3-(N-morpholino)propanesulfonic acid; n, H/ATP stoichiometry; NEM, N-ethylmaleimide; NPM, N-phenylmaleimide; PMSF, phenylmethylsulfonyl fluoride; RB, resuspension buffer; Delta, membrane potential; PAGE, polyacrylamide gel electrophoresis.

(^2)
U. Irkens-Kiesecker and L. Taiz, unpublished data.

(^3)
M. L. Müller and L. Taiz, unpublished data.

(^4)
M. L. Müller, unpublished data.


ACKNOWLEDGEMENTS

We thank Megan Jensen for technical assistance in the preparation of membranes. We also thank Ronald Poole, Roger Spanswick, Richard Cross, Don Briskin, Dale Sanders, and Roberto Bogomolni for valuable discussions.


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