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...