©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Plant Inorganic Pyrophosphatase Does Not Transport K in Vacuole Membrane Vesicles Multilabeled with Fluorescent Probes for H, K, and Membrane Potential (*)

(Received for publication, June 17, 1994; and in revised form, October 18, 1994)

Roc Ros (1)(§) Charles Romieu (2) Rémy Gibrat (3)(¶) Claude Grignon (3)

From the  (1)Departament de Biologia Vegetal, Facultat de Ciències Biològiques, Universitat de València, E-46100 Burjassot (València) Spain and the (2)Institut des Produits de la Vigne and (3)Biochimie et Physiologie Végétales, ENSA-M/Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique (URA 573), 34060 Montpellier Cedex 1, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

It has been claimed that the inorganic pyrophosphatase (PPase) of the plant vacuolar membrane transports K in addition to H in intact vacuoles (Davies, J. M., Poole, R. J., Rea, P. A., and Sanders, D.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11701-11705). Since this was not confirmed using the purified and reconstituted PPase consisting of a 75-kDa polypeptide (Sato, M. H., Kasahara, M., Ishii, N., Homareda, H., Matsui, H., and Yoshida, M. (1994) J. Biol. Chem. 269, 6725-6728), these authors proposed that K transport by the PPase is dependent on its association with other membrane components lost during purification. We have examined the hypothesis of K translocation by the PPase using native vacuolar membrane vesicles from Vitis vinifera suspension cells, multilabeled with fluorescent probes for K, H, and membrane potential. This material contained a high proportion of right-side-out, tightly sealed vesicles, exhibiting high PPase activity which was strongly stimulated by uncouplers and K. Proton pumping occurred in response to pyrophosphate addition in the absence of K. No K incorporation into the vesicles could be observed after PPase energization in the presence of K, although H transport was highly stimulated. The hydrolytic activity was stimulated by a protonophore and by a H/K exchanger but not by the K ionophore valinomycin. No evidence could be obtained supporting the operation of an endogenous K/H exchanger capable to dissipate the putative active K flux generated by the PPase. We conclude that PPase in native vacuolar membrane vesicles does not transport K.


INTRODUCTION

Since the K concentration in plant vacuoles can reach 40-200 mM (Lüttge and Higinbotham, 1979; MacRobbie, 1970), the thermodynamic equilibrium value predicted from cytosolic K concentration and vacuolar membrane potential may be exceeded. Thus, the possibility of an active K translocate in the vacuole has to be considered (Rea et al., 1992b and references therein). The plant vacuolar inorganic pyrophosphatase (EC 3.6.1.1, PPase) (^1)is known to function as an electrogenic H pump, translocating H from the cytosol to the vacuole lumen (Hedrich and Shroeder, 1989; Rea and Sanders, 1987; Sze, 1985). Considerable interest has resulted from recent electrophysiological data suggesting that the PPase of beet root vacuolar membrane transports K together with H. Patch clamping intact vacuoles (Davies et al., 1992) revealed an outwardly directed current dependent on vacuolar K after PPase activation in the reverse mode (i.e. PP(i) synthesis). Since the reversal potential of this current was determined by both H and K Nernst potentials, it was ascribed to simultaneous H and K transport out of the vacuole. The PPase would thus be the first primary active K transport system identified in plants.

However, recent work with highly purified pumpkin PPase reconstituted into proteoliposomes showed that K transport was independent on the activation of H translocation by the PPase and could be ascribed to passive diffusion. These results led to the conclusion that this enzyme does not translocate K (Sato et al., 1994). A possible explanation for the discrepancy between these results and those of Davies et al. (1992) is that another membrane protein is necessary to confer K translocation ability on the PPase. Alternatively, isolation and purification of the PPase may have altered its function.

We took advantage of several characteristics of the vacuolar membrane vesicles from Vitis vinifera cells to examine this question, using three independent criteria, viz. (i) direct, simultaneous determination of intravesicular K, pH, and membrane potential, (ii) ionophore effects on hydrolysis activity, and (iii) K effects on transport and hydrolytic properties of the PPase. The results show that H transport by the PPase is not directly linked to K transport in native vacuolar membranes from V. vinifera.


EXPERIMENTAL PROCEDURES

Chemicals

PBFI (potassium binding benzofuran isophthalate), pyranine (8-hydroxy-1,3,6-pyrene-trisulfonic acid), ACMA (9-amino-6-chloro-2-methoxy-acridine), quinacrine and oxonol were purchased from Molecular Probes, Inc. (Eugene, OR). All other chemicals used were of analytical grade.

Materials

Plant Material

A colorless callus mutant established from green grape berries (V. vinifera cv. Ganay freau) was cultivated in liquid medium according to Ambid et al. (1983). Eighty g (fresh weight) of cells were inoculated in 250 ml of medium and shaken at 25 °C for 7-10 days, until they represented 50-70% (w/v) of the medium volume.

Preparation of Tonoplast Vesicles

All procedures described below were carried out at 4 °C. Cells were recovered by filtration through a nylon mesh under vacuum and resuspended in 0.3 M mannitol, 0.5% polyvinylpolypyrolidone (25,000, w/v), 0.1% (w/v) bovine serum albumin, 5 mM EDTA, 20 mM mercaptoethanol, 5 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride, and 20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) adjusted to pH 7.8 with ascorbic acid, in a 1:3 (w/v) cell/medium ratio. The material was homogenized with a Potter homogenizer and the slurry filtered through glasswool under vacuum to remove most of the cell walls. An aliquot of the crude homogenate was centrifuged at 100,000 times g for 25 min. The pellet was washed by resuspension in 0.3 M mannitol, 0.1 M KCl, 5 mM DTT, 20 mM BTP adjusted to pH 7.5 with MES, centrifuged at 100,000 times g for 25 min, and resuspended in 0.3 M mannitol, 20% (v/v) glycerol, 5 mM DTT, and 5 mM BTP-MES (pH 7.5) to give the total membrane fraction. The remaining homogenate was purified as follows. High density organelles were removed by centrifugation at 8,000 times g for 20 min, and the resulting supernatant was sedimented at 100,000 times g for 25 min. The pellet was washed by resuspension in 0.3 M mannitol, 0.1 M KCl, 5 mM DTT, 20 mM BTP-Mes, and centrifugation at 100,000 times g for 25 min. Tonoplast vesicles were purified by flotating the resuspended pellet on a 15%/28% (w/w) discontinuous sucrose gradient containing 5 mM DTT and 5 mM BTP-MES (pH 7.5) for 90 min at 100,000 times g. The 15%/28% (w/w) sucrose interface was collected and washed by centrifugation at 100,000 times g for 25 min in 0.3 M mannitol, 5 mM DTT, and 5 mM BTP-MES (pH 7.5). The pellet was resuspended (2-5 mg protein/ml) in the same medium plus 20% (v/v) glycerol and stored in liquid nitrogen until use.

Methods

Measurement of the ATPase and PPase Hydrolytic Activities

Hydrolytic activities were measured in a medium (500 µl of final volume) containing 0.25 M sucrose, 2 mM Mg-MES, 10 mM BTP-MES (pH 7.5 or 6.5), and 2-4 µg of proteins. Additions and modifications to this reaction mixture included various potassium salts, 0.1 mM vanadate, 1 mM azide, 0.02% (w/v) Triton X-100, and ionophores as detailed in the table legends. The reaction was started by addition of either 0.3 mM PP(i)-MES (pH 7.5) or 2.5 mM Mg-ATP and proceeded at 30 °C for 30 min. The P(i) liberated was determined according to Ames(1966). All pyrophosphatase activities are reported on the basis of nanomoles of PP(i) hydrolyzed per minute and per milligram of proteins.

Tonoplast Vesicle Multilabeling and Fluorescence Measurements

Tonoplast vesicles were multilabeled with two non-permeant probes for K (PBFI) and H (pyranine) and a permeant probe for membrane potential (oxonol VI).

Unless otherwise stated, aliquots of PBFI and pyranine (1 mM and 0.5 mM final, respectively) were added to a vacuolar membrane vesicle suspension (150 µg of proteins) containing 9% (v/v) glycerol, 2 mM Mg-MES, 10 mM potassium iminodiacetate (KIDA), and 50 mM BTP-MES (pH 7.5). This mixture was frozen in liquid nitrogen, thawed at room temperature, and sonicated twice for 1 min. Non-encapsulated probes were removed by gel filtration through a Sephadex G-50 column. The volume recovered was within 90-100% of the applied volume. A 10-µl aliquot (12 µg of proteins) of this volume was added to the assay cuvette (2 ml final) containing 9% glycerol, 2 mM Mg-MES, 10 mM KNO(3) or KIDA, 100 µM EGTA, 50 nM of the permeant dye oxonol VI, and 50 mM BTP-MES (pH 7.5). The cuvette was stirred and maintained at 30 °C.

Fluorescence intensities of the probes pyranine, PBFI, and oxonol VI were simultaneously measured with a multichannel photon counting spectrofluorometer SLM-Aminco 8000 C interfaced with an IBM computer as described previously (Venema et al., 1993). The T-shaped configuration of this apparatus allows two simultaneous fluorescence measurements using two emission channels A and B (fitted with a monochromator and a 495-nm long-pass cut on filter, respectively). Furthermore, different excitation/emission wavelengths can be selected during brief macroprogrammed acquisition cycles. Briefly, channel B was used to acquire pyranine signals after excitation at 460 and 410 nm, corresponding to the non-protonated and protonated species, respectively. Channel B was also use to acquire alternatively the PBFI signal after excitation at 336 nm. Channel A was used to acquire the oxonol signal at 646 nm after excitation at 614 nm. Each individual fluorescence signal was first ratioed with the signal of a rhodamine reference cell (channel C) and corrected for the fluorescence of the buffer solution. The oxonol response was calibrated by imposing a H diffusion gradient through tonoplast membranes suspended in the same medium as that described for measurements plus 3 µM FCCP. A calibration curve was obtained: E(m) = -20.6 (F/F(o))^2 + 154.4 F/F(o) + 5.88, where E(m) is the Nernst membrane potential for H and F is the maximum fluorescence minus the final fluorescence (F(o)) obtained after clamping the potential to zero with 30 mM KSCN. In experiments where the PPase-generated potential was measured, F was corrected by the percentage of active vesicles (right-side-out vesicles). The ratio of the unprotonated to protonated forms of pyranine corrected by the percentage of active vesicles (sealed right-side-out) was used to monitor the internal pH changes in the vesicles. The signal was calibrated using the following equation: pH = 7.6 + 2.9 log (F). The calibration curve was obtained as for oxonol calibration by imposing pH gradients through tonoplast membranes in the presence of 100 nM valinomycin plus 3 µM FCCP. Potassium concentration in the vesicles was monitored using the PBFI signal ratioed by the pyranine signal at its 420-nm isosbest.

The quenching of the fluorescence of the permeant amines quinacrine (5 µM) and ACMA (1 µM) was assayed in the same standard medium described above. The excitation and emission wavelengths were 420 and 495 nm for quinacrine and 415 and 485 nm for ACMA, respectively.

Proteins

Protein concentrations were determined in the membrane fractions by the method of Schaffner and Weissman(1973) with bovine serum albumin as a standard.


RESULTS

PPase Activity in Relation to Sidedness and Tightness of Tonoplast Vesicles

The membrane fraction used in our experiments (15%/28% interface of the sucrose gradient) was highly enriched in vacuolar membrane enzyme markers (the nitrate-sensitive (H)ATPase (EC 3.6.1.3) and the PPase) as compared to the total membrane fraction (Table 1). The low hydrolytic activity of the vanadate-sensitive ATPase and azide-sensitive ATPase, considered as plasma membrane and mitochondrial markers, respectively, confirmed that the 15%/28% interface mainly consisted of vacuolar membranes. This fraction contained a 67-kDa polypeptide which reacted with a polyclonal antibody raised against the 66-kDa MgPP(i)-binding polypeptide of the PPase purified from Vigna radiata (Maeshima and Yoshida, 1989) (data not shown). This antibody has been used to clone a cDNA coding for a 81-kDa polypeptide of Arabidopsis, homologous to the 67-kDa MgPP(i)-binding polypeptide of the Beta vulgaris PPase (Sarafian et al., 1992).



The hydrolytic activities of the two vacuolar membrane markers in the absence and in the presence of the permeabilizing agent Triton X-100 (0.02%) gave similar latencies (about 30%) (Table 1), suggesting that 70% of vacuolar membrane vesicles were oriented right-side-out. The basal activity of the PPase (i.e. the one of right-side-out vesicles in the absence of permeabilizing agent) assayed in the presence of KIDA was 2-fold stimulated by the protonophore FCCP (Table 2), proving that native vesicles were tightly sealed to H and small ions. These data together indicate that cell suspensions of V. vinifera are a suitable source of native vacuolar membrane vesicles for studying the transport properties of the PPase in the direct mode (PP(i) hydrolysis and H translocation into the vacuole).



Energetic Coupling between H Transport, Membrane Potential, and PP(i) Hydrolysis

The basal hydrolytic activity of the PPase was highly dependent on the electrochemical gradient created by the pump. The lowest PPase hydrolytic activity was observed in the presence of IDA (Table 2), an anion reputed to be poorly permeant. A high membrane potential associated with a low acidification rate was observed under these conditions (Fig. 1A). A 40% increase in the basal hydrolytic activity was observed in the presence of the permeant anion NO(3) (Table 2), together with a reduction by 50% in the membrane potential and a high acidification rate (Fig. 1B).


Figure 1: Effect of NO(3) and IDA on the pH gradient and membrane potential generated by the PPase. Vacuolar membrane vesicles (12 µg of protein) in 9% glycerol, 2 mM Mg-MES, 10 mM KIDA, 50 mM BTP-MES (pH 7.5), containing 0.5 mM pyranine were added to 2 ml of reaction medium containing 9% glycerol, 2 mM Mg-MES, 100 µM EGTA, 50 nM oxonol VI, 50 mM BTP-MES (pH 7.5), and 10 mM KIDA (A) or 10 mM KNO(3) (B). At the indicated times (dashed lines) aliquots of PP(i) (0.15 mM), valinomycin (20 nM), or FCCP (5 µM) were added to the reaction medium. Internal pH and membrane potential were determined from the variations of the pyranine and oxonol signals, respectively, as described under ``Experimental Procedures.''



The anionic selectivity disappeared and a maximum basal activity was reached in the presence of the protonophore FCCP (Table 2), which totally dissipated both the pH gradient and the membrane potential gradient generated by the PPase (Fig. 6A). This confirms that the stimulation of PPase hydrolytic activity by anions depends on their capacity to dissipate the membrane potential and not on some specific, direct interaction with the PPase. We conclude that the PPase from V. vinifera is a highly electrogenic H proton pump, and its activity is sensitive to both electrical and osmotic components of the H electrochemical gradient.


Figure 6: Simultaneous determinations of membrane potential, H, and K concentrations in vacuolar membrane vesicles after PPase activation in the presence of FCCP and 10 mM K. Measurements were made in a medium containing 9% glycerol, 2 mM Mg-MES, 100 µM EGTA, 10 mM KIDA, 5 µM FCCP, and 50 mM BTP-MES (pH 7.5) in the absence (A) or the presence (B) of 0.08% (w/v) DOC. At the indicated times (dashed lines), aliquots of PP(i) (0.15 mM), valinomycin (20 nM), and KIDA were added to the reaction medium. Internal pH and membrane potential were, respectively, determined from the variations of the pyranine and oxonol signals as described under ``Experimental Procedures.'' Internal K was monitored using the PBFI fluorescence ratio after excitation at 336 and 420 nm.



Potassium Stimulation of the PPase

The hydrolysis of PP(i) was stimulated by 50 mM cations in the following order: NH(4) =K > Cs > Na > Li > BTP = Tris (relative activities: 100, 91, 66, 32, 20, 5, and 4). Using the permeant amines ACMA (Fig. 2) or quinacrine (Fig. 3), H pumping activity of the PPase from V. vinifera vacuolar membrane vesicles could not be observed in the absence of added K. Addition of 10 mM potassium triggered a high quenching rate of the ACMA (Fig. 2) and quinacrine probes (Fig. 3). Conversely, a significant acidification rate was observed in the absence of K using the non-permeant pH probe pyranine, but addition of K poorly increased the response of the dye (Fig. 2). The reason for these discrepancies is that the pyranine response is more sensitive, but restricted to a narrower pH range than that of ACMA: the pyranine response is completely saturated below pH 6.0 (data not shown).


Figure 2: Response of the pH probes ACMA (A) and pyranine (B) to PPase energization in the absence or the presence of K. Vacuolar membrane vesicles (12 µg of protein) containing 9% glycerol, 2 mM Mg-MES, 5 mM BTP-MES (pH 7.5), and 0.5 mM pyranine encapsulated as described under ``Experimental Procedures'' were added to 2 ml of reaction medium containing 9% glycerol, 2 mM Mg-MES, 100 µM EGTA, 1 µM ACMA, 10 mM BTP-NO(3), and 5 mM BTP-MES (pH 7.5). At the indicated times (arrows), aliquots of PP(i) (0.15 mM), FCCP (5 µM), or (NH(4)) (10 µM) were added to the reaction medium. Fluorescence signals are given in arbitrary units.




Figure 3: Stimulation of vacuolar membrane PPase hydrolytic and proton pumping activities by K. Vacuolar membrane vesicles suspended in 0.25 M sucrose, 2 mM Mg-MES, and 10 mM BTP-MES (pH 7.5) were diluted in a reaction medium of the same composition plus 100 µM EGTA and 0-140 mM KNO(3). PP(i) hydrolysis (A) was measured on the same samples as H transport (B). The later was monitored by the quenching of quinacrine. PanelC represents the relationship between the data of panelsA and B.



Therefore, the use of the non-permeant dye pyranine demonstrated that H translocation by the PPase does not actually depend on the presence of K, as expected from an obligatory H/K cotranslocation mechanism.

It has been proposed that each of the transport sites of the PPase might be occupied by K or H, resulting in a variable H/K stoichiometry (Davies et al., 1992). In such a system, a pure H transport (0 K/n H) would be possible and would explain the results of Fig. 2. We further examined the K stimulation of the PPase by measuring hydrolytic and H pumping activities in the same assay sample. A permeant amine dye (quinacrine) was used in this experiment despite a lower sensitivity than pyranine because such a dye was not saturated upon establishment of high pH gradients. Both H pumping and hydrolytic activities were strongly stimulated by K (K(M) = 10 mM) but remained linearly correlated (Fig. 3), which suggests that K did not compete with H ions for a common translocation site, as previously suggested (Davies et al., 1992).

The hypothesis of a K/H cotranslocation by the PPase was also examined by studying the effect of K and H ionophores on the PPase hydrolytic activity. A stimulation of the hydrolytic activity should be expected from the dissipation of such electrochemical gradients. As showed in Table 2, FCCP strongly stimulated the hydrolytic activity of the enzyme in agreement with the total dissipation of the large H electrochemical gradient generated by the pump ( Fig. 1and 6A). Addition of the K ionophore valinomycin to dissipate the putative K electrochemical gradient did not stimulate the activity, irrespective of the K concentration used in the assay medium ( Table 2and Fig. 4). Moreover, no additional stimulation was observed when valinomycin was used together with FCCP. On the other hand, high stimulations were observed in the presence of the H/K exchanger nigericin. These experiments indicate that the PPase responded to the dissipation of the electrochemical gradient of H but not to that of K.


Figure 4: Effect of H and K ionophores on the PPase hydrolytic activity at different K concentrations. Vacuolar membranes (1-2 µg) suspended in 0.25 M sucrose, 2 mM Mg-MES, and 10 mM BTP-MES (pH 7.5) were diluted in a reaction medium containing 0.25 M sucrose, 2 mM Mg-MES, 100 µM EGTA, 10 mM BTP-MES (pH 7.5), and 0-150 mM KNO(3). Where appropriate, valinomycin, FCCP or nigericin (5 µM) were added. The ionic strength was kept constant over all the range of K concentrations by adding BTP-NO(3) (BTP + K = 150 mM).



Effect of the PPase Activation on the Responses of the K, H, and Membrane Potential Probes in Multilabeling Experiments

The K and H electrochemical gradients following PPase energization were simultaneously monitored by multilabeling the vacuolar membrane vesicles with pyranine, PBFI, and oxonol VI dyes. Experiments were performed in the presence of 10 mM K both inside and outside the vesicle. This concentration was sufficient to stimulate the PPase, while PBFI remained approximately half-saturated with K.

In the first experiment (Fig. 5A), the PPase was activated in the presence of the non-permeant anion IDA. Energization of the PPase generated a membrane potential up to 270 mV, and a small active H influx associated with a passive K efflux. This could mean either that the PPase does not transport K in these conditions or that K is recirculated by, for example, a K channel. Addition of valinomycin caused a rapid additional K efflux associated with a transient depolarization of the vacuolar membrane. This suggests that the vacuolar membrane did not contain an efficient K uniport system, capable of short-circuiting the PPase at low internal K concentration (10 mM). When the PPase was energized after addition of valinomycin (Fig. 5B), the initial observed membrane potential was lower (200 mV). This was associated with a higher K efflux, as shown by the 4-fold increase in the rate of PBFI fluorescence variation. However, after a few minutes, the membrane potential was similar to that observed in the absence of the K ionophore. Thus, valinomycin failed to permanently short-circuit the PPase, owing to the small intravesicular volume which was rapidly depleted in K. This result is in agreement with the absence of a stimulation of the hydrolytic activity by valinomycin ( Table 2and Fig. 4), since hydrolysis was measured over a 30-min period.


Figure 5: Simultaneous determinations of membrane potential, H, and K concentrations in vacuolar membrane vesicles after PPase activation in the absence or the presence of valinomycin. Vacuolar membranes were incubated during 10 min in the absence (A) or the presence (B) of 20 nM valinomycin. The reaction medium contained 9% glycerol, 2 mM Mg-MES, 100 µM EGTA, 10 mM KIDA, and 50 mM BTP-MES (pH 7.5) with or without valinomycin. At the indicated times (dashed lines) PP(i) (0.15 mM), valinomycin (20 nM), KIDA (12 mM), or FCCP (3 µM) were added to the reaction medium. Internal pH and membrane potential were, respectively, determined from the variations of the pyranine and oxonol signals as described under ``Experimental Procedures.'' Internal K was monitored using the PBFI fluorescence ratio after excitation at 336 and 420 nm.



In a second experiment (Fig. 6A), the H electrochemical gradient was clamped to zero using FCCP, which led to a 2-fold increase in the hydrolytic activity (Table 2). According to the H/K cotranslocation model, a K influx should be observed after energization. Data in Fig. 6A indicate that the internal K concentration did not vary after energization of the pump. However, a K influx could have been detected in such an experiment. After valinomycin addition, K supply to the outside induced a significant increase in the PBFI signal, associated with an alkalinization of the interior of the vesicles, as expected from the catalysis of H and K fluxes by the two ionophores. When the vesicles were permeabilized with 0.08% (w/v) deoxycholate (DOC), only the PBFI response was observed, confirming that the H/K exchange previously observed was due to a true coupled process (Fig. 6B).

A H/K antiporter has been shown at the vacuolar membrane of Brassica napus (Cooper et al., 1991). It could therefore be hypothesized that the putative K electrochemical gradient generated by the PPase would be partly dissipated by such a system and transformed into an additional H influx. However, imposition of inward K gradient to non-energized membranes did not create a detectable H efflux (Fig. 7), in spite of the high sensitivity of the pyranine probe. Addition of FCCP and valinomycin was necessary to observe a H/K exchange. Finally, energization of the PPase in a pH clamping experiment using FCCP was repeated in the presence of 50 mM K (Fig. 8), a condition thought to inhibit the oilseed rape H/K antiporter (Cooper et al., 1991). In the absence of a permeant anion, active K translocation by the PPase should produce an alkalinization of the interior of the vesicle associated with the establishment of a H diffusion potential (positive inside). Neither pyranine nor oxonol responded to pump energization under these conditions (PBFI fluorescence was not measured since it is saturated at 50 mM K). Thus, the hypothesis of a H/K antiport was ruled out.


Figure 7: Alkalinization of non-energized vacuolar membrane vesicles in response to an imposition of a K gradient. The reaction medium contained 9% glycerol, 2 mM Mg-MES, 100 µM EGTA, 10 mM KIDA, 50 mM BTP-MES (pH 7.5) (A) plus 5 µM FCCP and 50 nM valinomycin (B), or 5 µM FCCP, 50 nM valinomycin, and 0.08% (w/v) DOC (C). At the indicated times (dashed lines) aliquots of KIDA (10 mM) were added to the reaction medium. Internal pH was determined from the variations of the pyranine signals as described under ``Experimental Procedures.''




Figure 8: Simultaneous determinations of membrane potential and H concentration in vacuolar membrane vesicles after PPase activation in the presence of FCCP and 50 mM K. Vacuolar membranes vesicles were suspended in a medium containing 9% glycerol, 2 mM Mg-MES, 50 mM KIDA, and 5 mM BTP-MES (pH 7.5). Measurements were made in a medium containing 9% glycerol, 2 mM Mg-MES, 100 µM EGTA, 50 mM KIDA, 6 µM FCCP, and 5 mM BTP-MES (pH 7.5). At the indicated times (dashed lines) aliquots of PP(i) (0.15 mM), valinomycin (20 nM), and KIDA (200 mM) were added to the reaction medium. Internal pH and membrane potential were respectively determined from the variations of the pyranine and oxonol signals as described under ``Experimental Procedures.''




DISCUSSION

Characteristics of the Vacuolar Membrane Fraction

The present study is the first report on PPase transport properties using multilabeling with non-permeant H and K dyes. Such sequestered probes have high sensitivity, in contrast to permeant H probes, which must be accumulated at high concentration in the vesicles by a large pH gradient to display significant quenching. The magnitude of the response of encapsulated dyes to PPase energization depends on the proportion of right-side-out vesicles with external facing catalytic sites and not on the absolute concentration of active vesicles, as is the case of permeant probes. Furthermore, only the fraction of these right-side-out vesicles which are also tightly sealed to H is involved in the response of encapsulated dyes. This proportion is usually low, although dependent on the tissue source and on the isolation method used (Brauer et al., 1988). The vacuolar membrane vesicles from V. vinifera culture cells were tightly sealed to small ions, as indicated by the 2-fold stimulation by FCCP of the activity assayed in medium containing poorly permeant anions (Table 2). To our knowledge, this is the highest stimulation by FCCP reported. Furthermore, most of the vacuolar membrane vesicles from V. vinifera culture cells were right-side-out (70%), making the PPase catalytic site directly accessible to the substrates (Table 1). Finally, the specific PPase hydrolytic activity from V. vinifera is the highest reported in the literature, being about 3-6-fold greater than that measured in B. vulgaris (Rea et al., 1992b), Avena sativa (Pope and Leigh, 1987; Wang et al., 1986), Kalanchoe daigremontiana (White et al., 1990), or Chara corallina (Takeshige and Hager, 1988).

Energetic Coupling of the PPase

FCCP led to a stimulation of hydrolytic activity in the absence of permeant anion, demonstrating the stalling of PP(i) hydrolysis by the H electrochemical gradient generated by the PPase. The selectivity of the stimulation of the H pump by anions has been attributed to their capacity to remove electrical constraint on transport (Gianini and Briskin, 1987; Kaestner and Sze, 1987; Marquardt-Jarcyk and Lütte, 1990; Pope and Leigh, 1987). In the present case, this conclusion is supported by the relation between anion selectivity and anion relative ability to short-circuit the membrane potential generated by the PPase (Fig. 1) and by the disappearance of anion selectivity in the presence of FCCP (Table 2). The energetic coupling between the hydrolytic activity of the PPase and its electrochemical gradient offers the possibility to examine simply the hypothesis of a K translocation by the PPase by studying the stimulation of its hydrolytic activity by K ionophores, as discussed below.

Interaction of K and the PPase

The strict dependence of H translocation on K (Wang et al., 1986; White et al., 1990) was the first indication supporting the hypothesis that K activated the PPase by being involved in the ion translocation cycle of this enzyme (Rea et al., 1992b). Electrophysiological analysis on isolated vacuoles indicated that the PPase responded to K at its cytoplasmic face (Davies et al., 1991). More recently, use of this approach led to the conclusion that the PPase could generate both H and K currents, i.e. that it could function as a H/K symporter (Davies et al., 1992).

PP(i) hydrolysis by the vacuolar membrane PPase from V. vinifera suspension cells was strongly stimulated by K (Fig. 3). However, we showed that a significant H translocation occurred in the absence of K (Fig. 2). This indicates that the putative K translocation site of the PPase does not display a strict requirement for K. The fractional K/H stoichiometry of transport (1.7 K/1.3 H) deduced from the electrophysiological measurements in vacuoles from red beet roots suggests that K and H could compete for three transport sites (Davies et al., 1992). However, the relationship between H translocation and PP(i) hydrolysis remained linear when using increasing concentrations of K (Fig. 3), indicating that K and H probably did not compete for the same translocation sites. Despite the tight energetic coupling between PP(i) hydrolysis and the H electrochemical gradient, demonstrated by the large stimulation of hydrolysis by FCCP, the K ionophore valinomycin had no effect on PPase hydrolytic activity at any K concentration used ( Table 2and Fig. 4). The strong stimulation of the PPase by K does not seem to be associated with the creation of a K electrochemical gradient, as would be expected from an electrogenic cotranslocation of K and H.

Finally, the hypothesis of a K translocation by the PPase has been examined directly using the PBFI probe under conditions (10 mM K) where PBFI could detect K net influx as well as net efflux ( Fig. 5and Fig. 6). Clamping the membrane potential at zero using FCCP, we have shown that the energization of the PPase does not affect the K equilibrium (Fig. 6).

We also examined the existence of a K/H antiporter with a high affinity for K, as demonstrated in vacuolar membrane from oilseed rape hypocotyls (Cooper et al., 1991). Indeed, the cooperation between a H/K active symporter (i.e. the PPase) and such an antiporter would be expected to partly dissipate the K electrochemical gradient. Nevertheless, K/H exchange across non-energized vacuolar membrane from V. viniferacould not be observed in the absence of H and K ionophores (Fig. 7). Since the antiporter described in the vacuolar membrane of rape hypocotyls was inhibited at a K concentration greater than 50 mM, the response of the pH probe pyranine was studied after energization of the PPase at a high K concentration and in the presence of FCCP (Fig. 8). No net H efflux was detected as expected from the activation of a H/K cotransporting PPase in the presence of a protonophore.

In conclusion, the present study of the in vitro energization of the PPase in native vacuolar membrane vesicles from V. vinifera does not confirm the H/K cotranslocation indicated by the electrophysiological analysis performed on isolated vacuoles from B. vulgaris (Davies et al., 1992). Using K and a highly purified and reconstituted PPase, Sato and colleagues(1994) also could not find evidence of such an active K translocation. The use of native vacuolar membrane vesicles makes unlikely the possibility of a regulation of PPase transport properties by other polypeptides present in the native vacuolar membrane such as the 20-24-kDa polypeptides previously described in the literature (Britten et al., 1992). In some experiments, we used a vesicle sonication and freeze-thawing procedure to load the non-permeant fluorescent indicators. Only 2% of the membrane proteins were lost as a result of this procedure, but neither a change in the PPase specific activity nor in its stimulation by K and H ionophores was observed (data not shown). Furthermore, it has been found (Kim et al., 1994) that the heterologous expression of the gene encoding for the 66-kDa PPase unit in yeast is sufficient to fully restore all the known properties of this enzyme, including its K stimulation. It is important to emphasize that our study and that of Sato and colleagues(1994) were performed in the direct mode (PP(i) hydrolysis), whereas the electrophysiological one was performed in the reverse mode (PP(i) synthesis) and that these studies were made on different species. We conclude that K transport is not a general property of the vacuolar membrane PPase. Like this enzyme, the plant plasma membrane ATPase was thought to transport K in addition to H, as a result of the stimulatory effect of K on its activity. This hypothesis has been made unlikely because (i) there is a significant H transport in the absence of K, provided that permeant ions ensure electrical charge compensation for the H translocation, and (ii) the K site is on the wrong side of the ATPase (Gibrat et al., 1990). The stimulation of ATPase and PPase activities by K may reflect some structural requirement for this cation, which is the major ionic species in the natural environment of these enzymes.


FOOTNOTES

*
This work was supported by grants from the Spanish Ministry Ministerio de Educación y Ciencia, the French Ministry Ministère de l'enseignement supérieur et de la recherche, and from an European Economic Community grant (to R. R.). 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.

§
Present address: Biochimie et Physiologie Végétales; E.N.S.A.-M./Institut National de la Recherche Agronomique; Centre National de la Recherche Scientifique (U.R.A. 573), 34060 Montpellier Cedex 1, France.

To whom correspondence should be addressed: Biochimie et Physiologie Végétales, ENSA-M/Institut National de la Recherche Agronomique, 34060 Montpellier Cedex 1, France. Fax: 33-67525737.

(^1)
The abbreviations used are: PPase, inorganic pyrophosphatase; PBFI, potassium binding benzofuran isophthalate; pyranine, 8-hydroxy-1,3,6-pyrene-trisulfonic acid; ACMA, 9-amino-6-chloro-2-methoxy-acridine; DTT, dithiothreitol; BTP, 1,3-bis(tris(hydroxymethyl)-methylamino)propane; MES, 2-(N-morpholino)ethanesulfonic acid; IDA, iminodiacetic acid; FCCP, carbonyl cyanide p-trifluoromethyoxyphenylhydrazone; DOC, deoxycholate; KIDA, potassium iminodiacetate.


ACKNOWLEDGEMENTS

We are grateful to Dr. M. Maeshima for the provision of the antibody against the PPase, to Dr. J. Fallot, Dr. G. C. Pech, and Dr. A. Latché for the gift of the Vitis callus culture, and to Dr. D. C. Logan for correcting the manuscript.


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