Inhibitors of the proton-sucrose symport.

Sucrose transporters are important components of the assimilate partitioning pathway in many plants. In the results reported here, we examined the effect of several inhibitors on proton-coupled sucrose transport into plasma membrane vesicles isolated from sugar beet leaf tissue. Three compounds that are reversible inhibitors of glucose transporters, phlorizin, cytochalasin B, and forskolin, also inhibited the proton-sucrose symport. Additionally, several reagents that covalently modify specific amino acid residues, including p-chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide (NEM), diethyl pyrocarbonate (DEPC), and Hg2+, were also examined. NEM was not an effective inhibitor of the symport under both energized (pH 6.0) and unenergized (pH 7.7) conditions. In contrast, PCMBS, DEPC, and Hg2+ blocked sucrose transport activity. However, in control experiments it was discovered that Hg2+, but not PCMBS or DEPC, dissipated the proton electrochemical potential difference (delta mu H) that drives sucrose accumulation. It was further demonstrated that Hg2+ dissipated an imposed delta mu+H in protein-free liposomes, thus obscuring its effect on the sucrose symport. In time- and concentration-dependent inactivation experiments, it was shown that DEPC binding was substrate protectable, thereby implicating binding at or near the active site of the carrier. In contrast, PCMBS activity was not linked to substrate binding. DEPC activity was partially reversed with hydroxylamine. This is consistent with specific modification of a histidine residue. Preloading purified vesicles with free histidine did not slow the DEPC-dependent inactivation kinetics. Since these membrane vesicles are predominantly right-side out, the last observation is consistent with a DEPC-sensitive site which is accessible from the outside face of the vesicle. The results with DEPC suggest that a histidine residue is at or near the active site of the sucrose symport and that this amino acid plays a critical role in the reaction mechanism.