Surcose transport in isolated plasma-membrane vesicles from sugar beet (Beta vulgaris L.) Evidence for an electrogenic sucrose-proton symport

Abstract

An analysis of the molecular mechanism of sucrose transport across the plasmalemma was conducted with isolated plasma-membrane (PM) vesicles. Plasma membrane was isolated by aqueous two-phase partitioning from fully expanded sugar beet (Beta vulgaris L.) leaves. The isolated fraction was predominantly PM vesicles as determined by marker-enzyme analysis, and the vesicles were oriented right-side-out as determined by structurally linked latency of the PM enzyme, vanadate-sensitive Mg(2+)-ATPase. Sucrose uptake was investigated by equilibrating PM vesicles in pH 7.6 buffer and diluting them 20-fold into pH 6.0 buffer. Using this pH-jump technique, vesicles accumulated acetate in a pH-dependent, protonophore-sensitive manner, which demonstrated the presence of a pH gradient (ΔpH) across the vesicle membrane. Addition of sucrose to pH-jumped PM vesicles resulted in a pH-dependent, protonophoresensitive uptake of sucrose into the vesicles. Uptake was sucrose-specific in that a 10-fold excess of mannose, glucose, fructose, mannitol, melibiose, lactose or maltose did not inhibit sucrose accumulation. The rate of pH-dependent uptake was saturable with respect of sucrose concentration and had an apparent K m, of 0.45 mM. Sucrose uptake was stimulated approximately twofold by the addition of valinomycin and K(+), which indicated an electrogenic sucrose-H(+) symport. Membrane potentials (ΔΨ) were imposed across the vesicle membrane using valinomycin and K(+). A membrane potential, negative inside, stimulated pH-dependent sucrose uptake while a ΔΨ, positive inside, inhibited uptake. Conditions that produce a negative ΔΨ in the absence of a pH gradient supported, although weakly, sucrose uptake. These data support an electrogenic sucrose-H(+) symport as the mechanism of sucrose transport across the PM in Beta leaves.