Analysis of carbohydrate transport across the envelope of isolated cauliflower-bud amyloplasts

Using isolated amyloplasts from cauliflower buds, we have characterized the interaction and transport of various carbohydrates across the envelope membrane of a heterotrophic plastid. According to our results, glucose 6-phosphate (Glc6P) and glucose 1-phosphate (Glc1P) do not share the same transport protein for uptake into cauliflower-bud amyloplasts. Glc6P-dependent starch synthesis is strongly inhibited in the presence of dihydroxyacetone phosphate (DHAP) or 4,4'-di-isothiocyano-2,2'- stilbenedisulphonic acid (DIDS), whereas Glc1P-dependent starch synthesis is hardly affected by these compounds. Analysis of the Glc6P uptake into proteoliposomes reconstituted from the envelope proteins of cauliflower-bud amyloplasts indicate that Glc6P is taken up in a counter-exchange mode with Pi, DHAP or Glc6P, whereas Glc1P does not act as a counter-exchange substrate. Pi is a strong competitive inhibitor of Glc6P uptake (Ki 0.8 mM) into proteoliposomes, whereas Glc1P does not significantly inhibit Glc6P transport. Beside a hexose-phosphate translocator, these amyloplasts possess an envelope protein mediating the transport of glucose across the membrane. This translocator exhibits an apparent Km for glucose of 2.2 mM and is inhibited by low concentrations of phloretin, known to be a specific inhibitor of glucose-transport proteins. Maltose inhibits the uptake of glucose (Ki 2.3 mM), indicating that both carbohydrates share the same translocator.