FIGURE 7.

Transport or flippase activity of Atp8a2. Loss in NBD fluorescence upon the addition of 2 mm dithionite (+Dithionite) to Atp8a2 proteoliposomes containing C12 NBD-PS pretreated with ATP (solid line) or ATP + NEM (dotted line) (A) and empty liposomes containing C12 NBD-PS pretreated with ATP (solid line) or ATP + NEM (dotted line) (B). The difference in the fluorescence between samples containing ATP and samples containing ATP + NEM (or no ATP) reflects the transport activity. Triton X-100 (1%) was added at the end of the trace to make all C12 NBD-PS accessible to bleaching by dithionite. C, transport activity of C6 NBD-PC, -PE, and -PS. D, transport activity of C12 NBD-PS in the absence or the presence of an equimolar amount of DOPS (2.5%). E, effect of pretreating Atp8a2 proteoliposomes with various nucleotides (AMP-PNP, GDP, GTP, ADP, and ATP) and inhibitors (NEM and vanadate) on the transport of C12 NBD-PS. NBD-labeled PS is transported by Atp8a2 in an ATP-dependent manner from the inside of the vesicle (corresponding to the exocytoplasmic side) to the outside of vesicles (corresponding to the cytoplasmic side of cell membranes). The transport of NBD-labeled PS is inhibited by PS, the physiological substrate of Atp8a2.