Regulation of the lemon-fruit V-ATPase by variable stoichiometry and organic acids

The lemon-fruit V-ATPase can exist in two forms: nitrate-sensitive and nitrate-insensitive. Here we report the results of measurements of H+/ATP stoichiometries using two kinetic methods: one based on steady-state ΔpH and one based on initial rates of H+-pumping. Our findings indicate that the nitrate-insensitive fruit V-ATPase has an H+/ATP stoichiometry of ∼1, while both the nitrate-sensitive fruit V-ATPase and the epicotyl V-ATPase have stoichiometries of 2, under zero-load conditions. As ΔpH increases, the stoichiometry of the nitrate-sensitive fruit V-ATPase decreases to 1. Under similar conditions, the stoichiometry of the epicotyl enzyme remains 2. Thus, the pH-dependent variable stoichiometry of the lemon-fruit V-ATPase may represent a key factor in juice sac vacuolar hyperacidification. On the other hand, the H+/ATP stoichiometry of the epicotyl V-ATPase can decrease from 2 to 1 in the presence of a membrane potential. The low pH of the fruit vacuole is not due solely to the lower H+/ATP stoichiometry of its pump. We show that lumenal citrate and malate improve the coupling of both the epicotyl and fruit V-ATPases and enhance their ability to generate a pH gradient. Since citrate accumulation is restricted to fruit vacuoles, it may be another important determinant of vacuolar pH.