FIGURE 4.
Transport of Fl-PL by ABCA1, ABCA7, and ABCA4. The dithionite Fl-PL bleaching assay was used to determine the phospholipid specificity and effect of nucleotides and inhibitors on phospholipid transport by ABCA proteins. A, phospholipid transport (flipping) by WT ABCA1 and ABCA7 and MM mutants reconstituted into PG liposomes containing 0.6% (w/w) Fl-PL. B, phospholipid transport by ABCA1 reconstituted into PC or PG liposomes. Unlabeled PC effectively competes with Fl-PC in PC liposomes reducing the transport of Fl-PS and Fl-SM (sphingomyelin) compared with liposomes composed of PG base lipid. C, phospholipid transport by ABCA4 reconstituted into PC and PG liposomes. D, effect of increasing Fl-PL concentration on transport by ABCA1 reconstituted into PG liposomes. E and F, effect of nucleotides, phosphate analogs, and inhibitors on the Fl-PC transport or flipping by ABCA1 (E) and Fl-PE by ABCA4 (F). Proteoliposomes were incubated at 37 °C for 60 min in the presence of AMP-PNP or ATP with or without the addition of 200 μm Vi, 200 μm AlFx, or 200 μm BeFx, 1 mm ouabain, and 1 mm azide. In the case of N-ethylmaleimide, the ABCA proteins were incubated with 10 mm N-ethylmaleimide for 25 min prior to Fl-PL transport. Results are the mean of three independent experiments. Error bars show S.E.