ISOLATION OF AN ERYTHROCYTE-MEMBRANE PROTEIN THAT MEDIATES CA2-DEPENDENT TRANSBILAYER MOVEMENT OF PHOSPHOLIPID()

Elevation of intracellular Ca2+ in erythrocytes, platelets, and other cells initiates rapid redistribution of plasma membrane phospholipids (PL) between inner and outer leaflets, collapsing the normal asymmetri c distribution. Consequently, phosphatidylserine and other lipids norm ally sequestered to the inner leaflet become exposed at the cell surfa ce. This Ca2+-induced mobilization of phosphatidylserine to the surfac e of activated, injured, or apoptotic cells confers a procoagulant pro perty to the plasma membrane, which promotes fibrin clotting and provi des a signal for cell removal by the reticuloendothelial system, To id entify the constituent of the membrane that mediates this Ca2+-depende nt ''PL scramblase'' activity, we undertook purification and reconstit ution of membrane component(s) with this activity from detergent extra cts of erythrocyte ghosts depleted of cytoskeleton. Active fractions w ere identified by their capacity to mediate the Ca2+-dependent redistr ibution of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled PL between leafle ts of reconstituted proteoliposomes. This PL scramblase activity co-el uted through multiple chromatographic steps with a single polypeptide of similar to 37 kDa, which was purified to apparent homogeneity as re solved by silver staining. The activity associated with this protein b and was inactivated by trypsin, The isolated protein reconstituted in proteoliposomes mediated nonselective, bidirectional transport of 7-ni trobenz-2-oxa-1,3-diazol-4-yl-PL between membrane leaflets, with half- maximal activation between 20 and 60 mu M Ca2+ (saturation >100 mu M), mimicking the Ca2+-dependent transbilayer lipid movement intrinsic to the erythrocyte membrane.