The electrochemical proton gradient and its influence on citrate uptake in tonoplast vesicles of Hevea brasiliensis

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Abstract
The relationship between the electrochemical proton gradient, Δ μH+ − , and citrate transport has been studied in tonoplast vesicles from Hevea brasiliensis (the rubber tree). Vesicles were generated from lyophilized samples of fresh vacuoles obtained from the latex sap. Methylamine was used to measure intravesicular pH and lipophilic ions to determine the electrical potential difference (ΔΨ) across the tonoplast. When incubated at pH 7.5 in the absence of ATP, the tonoplast vesicles showed a ΔpH of 0.6 units (interior acid) and a ΔΨ of about-100 mV (interior negative). This potential is thought to be made up of contributions from an H+ diffusion potential, diffusion potentials from other cations and a Donnan potential arising from the presence of internal citrate. In the presence of 5 mol m-3 MgATP the ΔpH was increased to about 1.0 unit and the ΔΨ to about-10 mV. Under these conditions the proton-motive force (Δ p Δ μH+ − /F) became positive and reached +50 mV. These effects were specific to MgATP (ADP and Mg2+ having no significant effect) and were prevented by the protonophore p-trifluoromethoxycarbonylcyanidephenylhydrazone (FCCP). Citrate uptake by the vesicles was markedly stimulated by MgATP; ADP and Mg2+ again had no effect. Nigericin greatly increased ΔpH and this was associated with a large increase in citrate accumulation. The results indicate that the vesicle membrane possesses a functional H+-translocating ATPase. The Δ μH+ − generated by this ATPase can be used to drive citrate uptake into the vesicles. The properties of the tonoplast vesicles are compared with those of the fresh latex vacuoles.