Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

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Abstract
The electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport were examined in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L. cv Great Western) leaves. Symport mediated sucrose transport was electrogenic as demonstrated by the effect of membrane potential on .DELTA.pH-dependent flux. In the absence of significant charge compensation, a low rate of sucrose transport was observed. When membrane potential was clamped at zero with symmetric potassium concentrations and valinomycin, the rate of sucrose flux was stimulated fourfold. In the presence of a negative membrane potential, transport increased six-fold. These results are consistent with electrogenic sucrose transport which results in a net flux of positive charge into the vesicles. The effect of membrane potential of the kinetics of sucrose transport was on Vmax only with no apparent change in Km. Sucrose transport rates driven by membrane potential only, i.e. in the absence of .DELTA.pH, were comparable to .DELTA.pH-driven flux. Both membrane potential and .DELTA.pH-driven sucrose transport were used to examine proton binding to the symport and the apparent Km for H+ was 0.7 micromolar. The kinetics of sucrose transport as a function of proton concentration exhibited a simple hyperbolic relationship. This observation is consistent with kinetic models of ion-cotransport systems when the stoichiometry of the system, ion:substrate, is 1:1. Quantitative measurements of proton and sucrose fluxes through the symport support a 1:1 stoichiometry. The biochemical details of proton-coupled sucrose transport reported here provide further evidence in support of the chemiosomotic hypothesis of nutrient transport across the plant cell plasma membrane.