Transport mechanism of the cloned potato H+/sucrose cotransporter StSUT1

The transport mechanism of the potato StSUT1 H+/sucrose cotransporter expressed in Xenopus oocytes was investigated using the 2-electrode voltage clamp and radiotracer flux methods. Sucrose induced inward currents through the transporter that were dependent on the extracellular sucrose and H+ concentrations and the membrane voltage. The activation of StSUT1 by H+ and sucrose displayed Michaelis-Menten-type kinetics suggestive of a 1:1 H+:sucrose stoichiometry. This was confirmed by simultaneously measuring inward currents and sucrose flux in voltage-clamped oocytes. The apparent affinities K-0.5 for H+ and sucrose were voltage-dependent, At -150 mV K-0.5(suc) was 0.5 +/- 0.07 mM at 10 mu M H-o(+) and K-0.5(H) was 0.1 +/- 0.05 mu M at 20 mM sucrose(o). StSUT1 exhibited presteady-state transient currents, which relaxed with time constants between <1 and 4 ms and fitted to the Boltzmann equation: maximum charge transfer Q(max) approximate to 1.8 nanocoulombs; apparent valence z approximate to 1; potential for 50% charge transfer V-0.5 approximate to -15 mV at 0.032 mu M H-o(+) and -45 mV at 10 mu M H-o(+). The steady state data were used to formulate a kinetic model for sucrose transport, and computer simulations were performed to obtain rate constants for the partial reaction steps. Our model is consistent with protons binding to StSUT1 before sucrose with both ligands transported simultaneously across the membrane.