Fig. 5.

Reconstitution of NBD-PS flippase activity with TAP-Drs2p proteoliposomes. (A) Dithionite quenching of NBD-PS (Left) or NBD-PC (Right) fluorescence in TAP-Drs2p proteoliposomes before and after 30 min of incubation with Mg²⁺-ATP or Na⁺-ATP. Dithionite was added at 30 s to quench outer leaflet NBD-phospholipid, and Triton X-100 was added at 150 s to quench remaining inner leaflet NBD-phospholipid. (B) Time course of NBD-PS translocation. The flippase assay was conducted as described in Materials and Methods, except that the incubation time varied from 0 to 60 min as indicated. Results were averaged from 4–6 independent experiments. (C) Flippase assay with liposomes containing TAP-Drs2p, Drs2p-TAP, or no Drs2p and the indicated NBD-phospholipid was performed as described in Materials and Methods. The increase in outer leaflet NBD-phospholipid after a 30-min incubation with Mg²⁺-ATP or Na⁺-ATP was plotted. Results were averaged from 6–9 independent experiments. ***, P < 0.001.