On the mechanism of hyperacidification in lemon: comparison of the vacuolar H+-ATPase activities of fruits and epicotyls

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 (greater than or equal to 100 mM) was correlated with the release of V1 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 micromolar vanadate. Cold inactivation treatment failed to inhibit H+ pumping activity of the fruit membranes, even though immunoblots showed that V1 complexes were released from the membrane. However, cold inactivation doubled the percent inhibition by 200 micromolar 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 micromolar vanadate, and an apparently novel vanadate-sensitive ATPase that is also partially inhibited by nitrate.