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

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 Westem) leaves. Symport mediated sucrose transport was electrogenic as demonstrated by the effect of membrane potential on ApH-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 on the kinetics of sucrose transport was on V,,, only with no apparent change in Km. Sucrose transport rates driven by membrane potential only, i.e. in the absence of ApH, were comparable to ApH-driven flux. Both membrane potential and ApHdriven 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 protoncoupled sucrose transport reported here provide further evidence in support of the chemiosmotic hypothesis of nutrient transport across the plant cell plasma membrane.

A Kinetic Model with Ordered Cytoplasmic Dissociation for SUC1, an Arabidopsis H + /Sucrose Cotransporter Expressed in Xenopus Oocytes

“…Furthermore, for SUC1 in yeast the value of K S m was 0.45 mM at an external pH of 5.5 (Sauer & Stolz, 1994), which is remarkably similar to the values obtained for the carrier expressed in oocytes. The stoichiometry of the H + / sucrose symport appears to be 1:1 and this agrees with measurements made using plasma membrane vesicles (e.g., Bush, 1990), but there are also reports of a variable stochiometry (e.g., Komor, 1997).…”

SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine‐tagged protein

“…Similar conclusions were drawn by Bush (1990) from experiments performed with plasma membrane vesicles from sugar beet. In this case changes of the membrane potential Au/influenced only the VMAX and not the KM.…”

“…All sucrose transporter activities studied so far seem to have several features in common: their KM-values are in the millimolar range, their pH optima are in the range of pH 5-6, they are sensitive to SH group inhibitors, and they are highly sensitive to uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (Buckhout, 1989;Bush, 1990;Delrot, 1989;Riesmeier et a/., 1992).…”

Kinetics Analysis of the Plasma Membrane Sucrose-H+ Symporter from Sugar Beet (Beta vulgaris L.) Leaves

“…The ability of the procedure to generate a negative Li# relative to the extemal medium and the ability of A$ to drive sugar uptake have been demonstrated ( Fig. 1A; Bush, 1990;Tubbe and Buckhout, 1992).…”

“…Transport is driven by the proton motive force established across the PM by the H+-ATPase in vivo and is electrogenic (Bush, 1990; Slone and Buckhout, 1991). The movement of H+ and Suc is tightly coupled with a stoichiometry of 1:l (Slone and Buckhout, 1991), and the carrier is specific for the Suc molecule, although phenylglucosides are also recognized by the carrier (Hecht et al, 1992).…”