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 ATPdriven 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 ( 100 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 VATPase inhibitors, and resistant to oxidation. Unlike the epicotyl H ؉ -ATPase, the fruit H 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 ⌬pH across the tonoplast appears to be considerably less than the theoretical maximum. From the H ϩ /ATP stoichiometry of the pump (n), the membrane potential (⌬), the Faraday constant (F), and the ⌬G ATP , the maximum ⌬pH at equilibrium can be calculated according to the equation:Bennett and Spanswick (9) determined an H ϩ /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 ϩ -PPase and included estimates of internal buffering capacity. They confirmed an n value of 2 for the V-ATPase and calculated a maximum possible ⌬pH across the tonoplast of 5.0 -5.4 units when the membrane potential is ϩ20 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 (13-15), chloride (12, 16), cytosolic pH (17), and oxidation/reduction (18,19). As yet, none of these mech...
The vacuolar H؉ -ATPases (V-ATPases) of lemon fruits and epicotyls were detergent-solubilized, purified by column chromatography, and reconstituted into artificial proteoliposomes. During purification, a vanadateand nitrate-sensitive ATPase activity, consisting of partially disassembled V-ATPase complexes, was resolved from the V-ATPase peak. ATPase and H ؉ -transport activities of the purified, reconstituted V-ATPases of both fruit and epicotyl exhibited similar inhibitor profiles, except that the fruit V-ATPase retained partial vanadate sensitivity. Since the V-ATPase activity of native fruit tonoplast vesicles is insensitive to inhibitors (Mü ller, M. L., Irkens-Kiesecker, U., Rubinstein, B., and Taiz The juice sacs of lemon fruits contain cells that can acidify their vacuoles to as low as pH 2.2 (1). In contrast, the vacuoles of the surrounding fruit tissues as well as those of vegetative tissues are maintained in the typical vacuolar pH range, 5.0 -6.0. The occurrence in lemon of two types of vacuoles with vastly different lumenal pH values provides a convenient experimental system to probe the mechanisms underlying the control of steady state vacuolar pH. One hypothesis to explain the extreme acidity of the juice sac vacuoles is that their H ϩ -ATPase (V-ATPase) 1 is a functionally specialized isoform capable of generating a greater pH gradient than vegetative VATPases. In an earlier report (2), we compared the ATP-driven H ϩ -pumping activities of tonoplast-enriched membrane vesicles isolated from juice sacs and seedling epicotyls. In native vesicles, the juice sac V-ATPase generated a steeper proton gradient than the V-ATPase of epicotyls. However, since the epicotyl tonoplast was more permeable to protons than the juice sac tonoplast, the steeper ⌬pH generated by the juice sac V-ATPase may have resulted from differences in membrane permeability rather than from intrinsic properties of the pumps. On the other hand, the two H ϩ -pumping activities differed with respect to several kinetic parameters. The epicotyl activity showed a typical V-ATPase profile with respect to ions and inhibitors (i.e. stimulation by chloride, inhibition by nitrate, bafilomycin A 1 , and N-ethylmaleimide (NEM), and insensitivity to vanadate). In contrast, the proton pumping activity of juice sac tonoplasts was insensitive to nitrate, bafilomycin, and NEM, and was partially inhibited by vanadate. Sensitivity of the juice sac ATPase activity to nitrate and NEM increased following detergent treatment, consistent with the juice sac proton pump's identity as a V-ATPase. However, evidence for the possible existence of a second H ϩ -ATPase on the juice sac tonoplast was also obtained. In nitrate-induced V 1 -dissociation experiments, the epicotyl vacuolar H ϩ -pumping activity became inactivated with the release of the catalytic subunit from the membrane. Despite the loss of a major portion of the catalytic subunit, the juice sac membranes retained 100% of their H ϩ -pumping activity following nitrate treatment, although vanadate sensi...
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