Does a proton-pumping ATPase exist in the tonoplast?

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Biochimie, 68 (1986) 1293-1298 © Soci6t6de Chimiebiologique/Elsevier,Paris

Does a proton-pumping ATPase exist in the tonoplast? Alain DUPAIX, Max HILL, Pierre VOLFIN and Bernard ARRIO Bio~nergdtique Membranaire, CNRS E.R. 308, lnstitut de Biochimie, B~t. 432, Universitd de Paris-Sud, Centre d'Orsay, 91405 Orsay, France (Received 1-8-1986, accepted3-9-1986)

S u m m a r y - In order to account for the accumulation of metabolites in plant vacuoles, the existence of a proton-pumping ATPase has been widely suggested in the literature. The demonstration of such a tonoplast-bound ATPase was merely based on the characterization of a nitrate-sensitive microsomal fraction. In some examples, this ATPase activity has been evidenced on vacuole preparations Obtained under conditions which were criticized by Boiler. The application of the reverse phase high-performance liquid chromatography method (RP-HPLC) to the simultaneous separation of adenine nucleotides, in the presence of tonoplast vesicles isolated from Catharanthus roseus, showed results not necessarily correlated with the ATPase hypothesis. Moreover, in light of the H +-quenching of quinacrine fluorescence observed during ATP hydrolysis by vacuoles or tonoplast vesicles, the existence of a proton-pumping ATPase may be questioned. Catharanthus roseus I tonoplast / ATPase / RP-HPLC R~sum~ - R~alit~ de l ' A T P a s e pompe4t-proton dans le tonoplaste ? L'existence d'une A TPase, pompe-?t-protons, a dtd largement invoqude clans la littdrature pour rendre compte des ph~nom~nes d'aecumulations de m~tabolites clans les vacuoles des plantes supdrieures. La ddmonstration de l'existence d'une telle ATPase, li~e au tonoplaste, repose essentiellement sur la caractdrisation d'une fraction microsomale sensible aux ions nitrates. Dans quelques cas, cette activit~ A TPasique a dtd raise en dvidence directement sur des vacuoles dont les conditions d'isolement ont ~td critiqu~es par Boller. L'application de la chromatographie liquide haute performance en phase inverse (RP-HPLC) ~ la sdparation simultande des nucldotides, d#rivds de i'addnosine, en prdsence de vdsicules isoldes du tonoplaste de Catharanthus roseus conduit ~ des rdsultats dont les conclusions n'impliquent pas ndcessairement l'existence d'une A TPase. De plus, l'analyse des rdsultats obtenus lors de l'extinction de fluorescence de la quinaerine, en presence de vacuoles ou de v~sicules tonoplastiques, pose le problbme de la rdalitd d'une A TPase, pompe-~-protons. Catharanthus roseus I tonoplaste I ATPase I RP-HPLC

Introduction A large number of organic as well as inorganic components are contained in the vacuoles of superior plants. Some of them reach such high concentrations that it seems necessary to account for these concentrations, to invoke accumulation processes, and therefore the capacity of specific transports in tonoplasts.

Quite naturally, and as suggested by the chemiosmotic concept [1], the results found in the literature tend to demonstrate the existence of an energy transducing proton-pumping ATPase involved in these accumulation processes. In a complex system like the vacuoles, ATP hydrolysis can be catalyzed both by ATPases and by other phosphorolytic enzymes not necessarily involved in an 'energization' process of the tonoplast.

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A. Dupaix et al. half using the following buffer: 1 mM Hepes-NaOH, 2.5 mM DDT, pH 7.3. The vacuoles were manually broken into vesicles in a refrigerated Thomas homogenizer, and finally centrifuged at 4°C for 3 h at 100000 ×g. The pellet was suspended in 25 mM Hepes-NaOH buffer, 2.5 mM DTT, 0.25 M sorbitol, centrifuged again at 100000×g overnight, and resuspended in a minimum volume of the same buffer (0.5-1 ml). This suspension was then used either for the RP-HPLC studies or for the studies in the presence of quinacrine.

The methods used to measure A T P hydrolysis are more or less specific. The less specific and most widely used method is certainly the titration o f the appearing phosphate by colorimetric methods derived from that described by Fiske and Subbarow [2]. Some methods are a little more specific for A T P hydrolysis, such as those using the radioactivity of 32p, bioluminescence, titration of released A D P by coupled enzymatic reactions involving, f o r e x a m p l e , p y r u v a t e kinase a n d l a c t a t e dehydrogenase. Although the methods based upon the coupling of enzymatic reactions are convenient, because they are fast and allow the possibility o f performing a continuous assay, some precautions must be taken. In the sequence of the enzymatic reactions, one must make sure, in particular, that the limiting reaction is indeed that o f the transformation of the product to be titrated: A T P hydrolysis in the present case. Moreover, any modification o f any one of the parameters of the studied reaction, such as the use of inhibitors, modification o f the salt concentration, o f the pH, etc., requires an evaluation of the possible effects on the coupled enzymatic reactions. According to the specificity of the titration, the results obtained will describe different aspects o f the hydrolysis of nucleotides catalyzed by the tonoplast enzymes. Thus, we have focused our attention on developing a method enabling the simultaneous measurement of the evolution of adenylic nucleotides in the presence of tonoplasts. This method consists of separating and titrating the nucleotides by reversephase h i g h - p e r f o r m a n c e liquid c h r o m a t o g a p h y (RP-HPLC). However, demonstrating only A T P hydrolysis is not sufficient to characterize an ATPase. One should also be able to follow the proton gradient created during this hydrolysis. Monitoring the p H gradient was done by means o f a p H sensitive fluorescent probe, i.e., quinacrine.

pH gradient measurements Measurements of quinacrine fluorescence extinction were carried out with an Eppendorf fluorometer, the excitation and emission wavelengths being determined using interference filters at 405 and 530 nm, respectively. Kinetics were measured at 20°C in the presence of 10t~M quinacrine in 0.8 ml of 25 mM Hepes-NaOH buffer, 2.5 mM DTT, 10 mM MgCI2 or MgSO4, 50 mM KCI, pH 7.3, containing either 0.55 M sorbitol in the case of kinetic studies carried out in the presence of vacuoles, or 0.25 M sorbitol for tonoplasts. After 15 min of incubation, the reaction was triggered by the addition of 5 to 10/~1 of stock ATP solutions (final concentration 5 mM) previously adjusted to pH 7.3 by solid bis-tris propane.

Materials and methods

Protein titration Proteins were determined using the method of Bradford [5], modified according to Read and Northcote [6].

Isolation of vacuoles and protoplasts The method for preparing protoplasts and vacuoles from cells of Catharanthus roseus (L.) G. Don, strain C 20, is identical to that described by Gibrat et aL [3]. The only modification was the replacement of 7°70 Nycodenz by 10070 Ficoll (w/v). Tonoplast purification The vacuoles obtained were centrifuged for 10 min at 4500×g to eliminate most of the protoplasts contaminating the preparation (contamination < < 1070).The supernatant containing the vacuoles was then diluted to

Reverse-phase high-performance liquid chromatography Description of the equipment and techniques used is given by Hill et al. [4]. Kinetic studies Measurement of hydrolytic activities by RP-HPLC Generally, the reaction was triggered by mixing, under magnetic stirring at 20°C, 150 t~l of a stock solution of ATP, ADP or AMP 6 mM in 50 mM Tris-Mes buffer, pH 7.5, containing 5 mM MgCI2 and 50 mM KC1. At determined time intervals; 20f-I aliquots were collected and added to an equal volume of perchlorie acid to stop the reaction. The sample was then centrifuged at 15 000 × g for 2 min and 20/~I of the supernatant were adjusted to pH 5.5 by adding 20 ttl of 4 M ammonium acetate before being frozen until use in HPLC analyses.

Results Measurements o f 5'-nucleotidase activities by RP-HPLC The chromatograms shown in Fig. 1 represent, for three incubation times, a typical example of the nucleotide separation obtained during A T P hydrolysis in the presence o f tonoplast vesicles isolated f r o m Catharanthus roseus. Under our ex-

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perimental conditions, the retention times were 1.64, 2.49, and 3.57 min for A M P , A D P and ATP, respectively. The kinetic analysis o f these chromatograms is presented in Fig. 2. In addition to A T P hydrolysis and the concomitant appearance o f A D P , a slight release of A M P is also observed. It therefore seems, that even in the presence of 1 mM molybdate, described as a phosphatase inhibitor [7], not only A T P evolves but also A D P . The absence o f total inhibition o f nucleotide hydrolysis by molybdate is confirmed in Fig. 3, representing the evolution of A T P in the presence o f the 1 0 0 0 0 0 × g supernatant with or without 1 mM molybdate. The fate of A D P is presented in Fig. 4. A D P uptake is coupled with A M P appearance on the one hand, and A T P synthesis on the other hand. It is worth noting that the stoichiometry 2 A D P --~ A T P + A M P is rather well respected. This A T P synthesis from A D P seems relatively specific for tonoplasts, as shown by the comparison made in Fig. 5 between the effects of the supernatant and the 100 000 × g pellet.

Measurements o f p r o t o n transport

Fig. I. Examples of chromatograms illustrating the evolution of ATP, at different incubation times, in the presence of 15,ug of tonoplast proteins (Catharanthusroseus). Chromatographic conditions: Supelcosil LC-18 column (5/~m, 4.6 mmx 15 cm, Supelco); eluants: 0.1 M KH:PO4, pH 7, 18°70methanol (v/v), 25 mM tetra-n-butylammoniumhydrogensulfate; injection of 5 yl of the incubation medium (ATP (Sigma) is contaminated by 2% ADP).

Addition of vacuoles to a quinacrine solution causes a fluorescence extinction o f the probe which indicates the existence o f a pH gradient between the intravacuolar and the outer medium (Fig. 6). Addition o f A T P only leads to a very slight acidification o f the inner medium (Fig. 7).

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Fig. 2. HPLC measurements of the variations of adenylic nucleotide concentration AC in tzM/mg of protein, as a function of time; initial substrate: ATP. Incubationconditions: 0.3 ml of 50 mM Tris-Mes buffer, pH 7.5, containing 5 mM MgC12, 50 mM KCI, 1 mM molybdate (ammonium heptamolybdate), and 15 yg of tonoplast (Catharanthusroseus); initial ATP concentration: 3 mM.

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Fig. 3. Evolutionof the ATP concentration, measuredby HPLC, in the presenceof proteins originating from the 100000x g supernatant: effect of molybdate. Incubation conditions: 0.3 ml of 50 mM Tris-Mes buffer, pH 7.5, containing 3 mM ATP, 5 mM MgCI2,50 mM KCI and 10 Fg of proteins from the 100000xg supernatant (Catharanthusroseus). _-U-Ca-_:1 mM molybdate (ammonium heptamolybdate); ~ : without ammonium heptamolybdate.

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Fig. 4. HPLC measurements of the variations of adenylic nucleotide concentration, AC in/zM/mg of protein, as a function of time; initial substrate: ADP. Incubation conditions: 0.3 ml of 50 mM Tris-Mes buffer, pH 7.5, containing 5 mM MgCI 2, 50 mM KCI, 1 mM molybdate (ammonium heptamolybdate) and 15/~g of tonoplasts (Catharanthus roseus); initial ADP concentration: 3 mM.

Fig. 5. Variations of the ADP concentration, measured by HPLC, as a function of time: comparison between the 100 000 x g supernatant and tonoplasts (Catharanthus roseus). Incubation conditions: 0.3 ml of 50 mM Tris-Mes buffer, pH 7.5, containing 3 mM ADP, 5 mM MgCI 2, 50 mM KCI, 1 mM molybdate (ammonium heptamolybdate), and 15 ~g or 10 vg of proteins originating from tonoplasts or the 100 000 x g supernatant, respectively.

The concept of a proton translocation coupled to the action of a tonoplast ATPase is not easy to reconcile with such a slight variation of the pH. Several hypotheses can be proposed: (1) The preexisting pH gradient does not allow any further acidification. (2) The buffer capacity of the intravacuolar medium masks the pH variation. (3) During the preparation of the vacuoles, some constituent, responsible for the creation of the pH gradient, has been lost, regardless of whether this constituent was specific for the vacuolar membrane or consisted of an interaction with the cytosoIic medium. Fig. 8 shows the creation of the proton gradient in tonoplast vesicles. Two remarks should be made: (1) the amplitude of the phenomenon is, here again, very weak; (2) the activating effect of KCI, described in the literature, is not clearly shown [8-11].

Published data on the ATPase activity of tonoplasts, mainly evaluated by the appearance of inorganic phosphate, usually for a unique reaction time, could not enable one t o reveal this. The very weak amplitudes of the fluorescence extinction kinetics of quinacrine, caused by ATP in the presence of vacuoles as well as tonoplasts, do not allow us to ascertain the existence of a tonoplast ATPase. In the literature, two experimental approaches have been developed to show the existence of such an ATPase. The first consists, starting from tissue homogenates, of separating and characterizing the various fractions obtained by density gradient centrifugations. The second leads to vacuole isolation either after mechanical disruption of the cells in a few favorable cases [12, 13] or from protoplats obtained by enzymatic digestion of tissues previously excised, or of cultured cells. Then the ATPase properties of the tonoplast membrane can be studied. In the first approach, one can, to begin with, note some puzzling term definitions. For Sze [14, 15] and some other authors, the microsomes are made out of all the membranes, excepting mitochondrial membranes separated by pre-centrifugations at 6 000-13 000 × g for 10 min. For still other authors, microsomes involve all of the membranes [16-18]. And finally, some authors adopt either definition [19, 20].

Discussion Our results lead to several remarks. Using RPHPLC, ATP and ADP hydrolyses are observed, as well as an ATP synthesis from ADP and AMP. The first point, i.e., ATP hydrolysis, is not by itself proof of the existence of an ATPase, but at most that of a 5'-nucleotidase. The consumption of ADP suggests the presence of an enzyme whose mode of action would recall that of an adenylate kinase.

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Fig. 6. pH gradient in vacuoles isolated from Catharanthus roseus as measured by quinacrine fluorescence extinction. General conditions: 25 mM Hepes-NaOH buffer, 2.5 mM DTT, 0.55 M sorbitol, pH 7.3. The final concentrations in the measuring cuvette were as follows: 10/zM quinacrine, 25/~g of vacuole proteins, 10 mM MgCI 2, 50 mM KCI and 10 ~tM gramicidin.

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Fig. 7. Effect of ATP on intravacuolar pH as measured by quinacrine fluorescence extinction; vacuoles isolated from Catharanthus roseus. General conditions: 25 mM Hepes-NaOH buffer, 2.5 mM DTT, 0.55 M sorbitol, pH 7.3. The final concentrations in the measuring cuvette were: 10/~M quinacrine, IO3 #g of vacuole proteins, 10 mM MgCI 2, 50 mM KCI, 5 mM ATP.

Moreover, most of the time, the centrifugation conditions are insufficiently specified: either the type of rotor or the relative gravitational field is not indicated, or the duration of centrifugation is absent, so that it is not always easy to determine whether the centrifugation is of the isokinetic or isopyknic type.

Time (mlnl Fig. 8. Effect of ATP on intravesicular pH (tonoplasts isolated from Catharanthus roseus) as measured by quinacrine fluorescence extinction. General conditions: 25 mM Hepes-NaOH buffer, 2.5 mM DTT, 0.25 M sorbitol, 0. I o7oBSA (w/v), pH 7.3. Final concentrations in the measuring cuvette: 10tzM quinacrine, 100kd of tonoplast (about 10/~g of protein), 10 mM MgSO 4, 50 mM KCI, 5 mM, ATP, 10t~M gramicidin.

In the vacuoles, membrane proteins and soluble proteins represent, at the outmost, 10070of the protoplast proteins. Likewise, 10070, at the most, of the vacuolar proteins constitute the tonoplast proteins. The problem is therefore to characterize a membrane fraction representing at best 1°70 of the total proteins loaded onto the gradient, whose buoyant density is quite close to that of the Golgi apparatus and that of the endoplasmic reticulum, and for which a specific biochemical marker has yet to be discovered. According to some authors [10, 11] a nitratesensitive ATPase would be associated to the tonoplast and could be used as an enzymatic marker for tonoplast [21]. According to other authors, a similar activity would be found on membrane fractions originating from either the Golgi [22] or the endoplasmic reticulum or mitochondria [23-27]. A recent publication [28] even shows that, in the case of tomato cells, most of the proton-pumping activity (between 50 and 90%), as revealed by fluorescence extinction of orange acridine in the presence of ATP, would be located in the mitochondrial and not in the microsomal fraction. One wonders why this activity is claimed to be bound to the tonoplast ! ! Obviously, considering the contradictory results in the literature and taking into account the low percentage of the tonoplast membrane, the low resolving power and the possible artifacts of the method [29], the use of cell frac-

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tionation on density gradients does not seem to be, presently, the best a d a p t e d t e c h n i q u e for characterizing the tonoplast. Moreover, one should be most cautious as to the interpretations derived from the differential effects o f inhibitors, whose specificities are more or less well established and whose actions might vary according to the tissue origin, the inhibitor concentrations, or the ions and proteins present in the medium. Concerning the second approach, Boiler [30] has published a review emphasizing a number o f gaps in the experimental results. In particular, a clear distinction between a possible specific ATPase and nonspecific phosphatases was lacking, the only criterion used was the presence or absence o f magnesium during activity measurements [8, 31-33]. On the other hand, the state o f purity o f the vacuoles was not accurately established [11,34], and the levels o f contamination could let us suppose that they were responsible for the effects observed [9]. In our opinion, the existence of an ATPase that would be responsible for the accumulation processes in the vacuoles has not really been demonstrated. The demonstration based on density gradient separations is not really convincing. As regards the demonstration based upon tonoplasts originating from isolated vacuoles, the major ambiguity comes from the difficulty in obtaining well characterized tonoplast membranes with a good yield. Our results, based upon observations o f tonoplasts obtained by experimental methods described in the literature, although not strictly invalidating the reality o f a tonoplast ATPase, evidence, at any rate, the existence of other enzymes responsible for the evolution of the adenylic nucleotides brought into their environment.

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