IDENTITY OF A CONSERVED MOTIF IN PHOSPHOLIPID SCRAMBLASE THAT IS REQUIRED FOR CA2-ACCELERATED TRANSBILAYER MOVEMENT OF MEMBRANE PHOSPHOLIPIDS()

Accelerated transbilayer movement of plasma membrane phospholipids (PL ) upon elevation of Ca2+ in the cytosol plays a central role in the In itiation of plasma clotting and in phagocytic clearance of injured or apoptotic cells, We recently identified a human erythrocyte membrane p rotein that induces rapid transbilayer movement of PL at elevated Ca2, We also presented evidence that this PL scramblase is expressed in a variety of other cells and tissues where transbilayer movement of pla sma membrane PL is promoted by intracellular Ca2+ [Zhou, Q., et al, (1 993) J. Biol. Chem. 272, 18240-18244]. We have now cloned murine PL sc ramblase for comparison with the human polypeptide. Both human and mur ine PL scramblase are acidic proteins (pI = 4.9) with a predicted insi de-outside (type 2) transmembrane segment at the carboxyl-terminus, Wh ereas human PL scramblase (318 AA) terminates in a short exoplasmic ta il, murine PL scramblase (307 AA) terminates in the predicted membrane -inserted segment. The aligned polypeptide sequences reveal 65% overal l identity, including near identity through 12 residues of an apparent Ca2+ binding motif (D[A/S]DNFGIQFPLD) spanning codons 273-284 (human) and 271-282 (murine), respectively, This conserved sequence in the cy toplasmic domain of PL scramblase shows similarity to Ca2+-binding loo p motifs previously identified in known EF hand structures. Recombinan t murine and human PL scramblase were each expressed in Escherichia co li and incorporated into proteoliposomes. Measurement of transbilayer movement of NBD-labeled PL confirmed that both proteins catalyzed Ca2-dependent PL flip-flop similar to that observed for the action of Ca2 + at the cytoplasmic face of plasma membranes, Mutation of residues wi thin the putative EF hand loop of human PL scramblase resulted in loss of its PL mobilizing function, suggesting that these residues directl y participate in the CA(2+)-induced active conformation of the polypep tide.