Reconstitution of the hexose phosphate translocator from the envelope membranes of wheat endosperm amyloplasts

Amyloplasts were isolated and purified from wheat endosperm and the envelope membranes reconstituted into liposomes. Envelope membranes were solubilized in n-octyl β-D-glucopyranoside and mixed with liposomes supplemented with 5.6 mol% cholesterol to produce proteoliposomes of defined size, which showed negligible leakage of internal substrates. Transport experiments with proteoliposomes revealed a counter-exchange of glucose 1-phosphate (Glc1P), glucose 6-phosphate (Glc6P), inorganic phosphate (P_i), 3-phosphoglycerate and dihydroxyacetone phosphate. The Glc1P/P_i counter-exchange reaction exhibited an apparent K_m for Glc1P of 0.4 mM. Glc6P was a competitive inhibitor of Glc1P transport (K_i 0.8 mM), and the two hexose phosphates could exchange with each other, indicating the operation of a single carrier protein. Glc1P/P_i antiport in proteoliposomes showed an exchange stoichiometry at pH 8.0 of 1 mol of phosphate transported per mol of sugar phosphate.