Inhibition of the glucose-6-phosphate transporter in oilseed rape (Brassica napus L.) plastids by acyl-CoA thioesters reduces fatty acid synthesis

Addition of oleoyl-CoA (1 muM), or other acyl-CoA thioesters with a chain l ength of C-16 or greater, to oilseed rape plastids (Brassica napus L.) inhi bited the rate of D-glucose 6-phosphate (Glc6P) uptake by 70 % after 2 min. The IC50 value for oleoyl-CoA inhibition of the transporter was approx. 0. 2-0.3 muM. Inhibition was alleviated by the addition of acyl-CoA binding pr otein (ACBP) or BSA at slightly higher concentrations. Oleic acid (5-25 muM ), Tween 40 (10 muM), Triton-X 100 (10 muM) and palmitoylcarnitine (5 muM) had no effect on Glc6P uptake. The uptake of [1-C-14]Glc6P occurred in exch ange for P-i, 3-phosphoglycerate or Glc6P at a typical rate of 30 nmol Glc6 P/min per unit of glyceraldehyde-3-phosphate dehydrogenase (NADP(+)). The K -m(app) of the Glc6P transporter for Glc6P was 100 muM. Neither CoA. (0.3 m M) nor ATP (3 mM) inhibited Glc6P uptake, but the transporter was inhibited by 72 % when ATP and CoA were added together. This inhibition was attribut able to the synthesis of acyl-CoA thioesters, predominantly oleoyl-CoA and palmitoyl-CoA, by long-chain fatty acid-CoA ligase (EC 6.2.1.3) from endoge nous fatty acids in the plastid preparations. Acyl-CoA thioesters did not i nhibit the uptake of [2-C-14]pyruvate or D-[1-C-14]glucose into plastids. I n vivo quantities of oleoyl-CoA and other long-chain acyl-CoA thioesters we re lower than those for ACBP in early cotyledonary embryos, 0.7 +/- 0.2 pmo l/embryo and 2.2 +/- 0.2 pmol/embryo respectively, but in late cotyledonary embryos quantities of long-chain acyl-CoA thioesters were greater than ACB P, 3 +/- 0.4 pmol/embryo and 1.9 +/- 0.2 pmol/embryo respectively.