PALMITOYLATION OF PHOSPHOLIPID SCRAMBLASE IS REQUIRED FOR NORMAL FUNCTION IN PROMOTING CA2-ACTIVATED TRANSBILAYER MOVEMENT OF MEMBRANE PHOSPHOLIPIDS()

Accelerated transbilayer movement of plasma membrane phospholipids (PL ) plays a central role in the initiation of plasma clotting and in pha gocytic clearance of injured or apoptotic cells. We recently identifie d a plasma membrane protein that induces rapid transbilayer movement o f PL at elevated Ca2+, and we presented evidence that this PL scrambla se mediates the transbilayer movement of plasma membrane PL in a varie ty of cells and tissues exposed to elevated intracellular Ca2+ [Zhou, Q. et al. (1997) J. Biol. Chem. 272, 18240-18244], Activation of PL sc ramblase entails coordination of Ca2+ by a 12 residue segment resembli ng an EF hand loop motif that is adjacent to the single transmembrane helix of the polypeptide. On the assumption that correct orientation o f the Ca2+-binding loop segment required a distal segment of the polyp eptide to orient back toward the membrane, we considered the possibili ty of membrane anchoring through covalent fatty acid, Human Raji cells transformed with PL scramblase cDNA in the expression vector pEGFP-C2 were metabolically labeled with [H-3]palmitate, and fusion protein im munoprecipitated with antibody against GFP-PL scramblase was found to covalently incorporate H-3, whereas no radioactivity was covalently as sociated with GFP. The identity of the covalently bound H-3 in PL scra mblase as a thioester-linked [H-3]palmitate was confirmed by hydroxyla mine cleavage and by thin-layer chromatography of the liberated fatty acid. Consistent with the assumption that activation by Ca2+ might req uire accessory site(s) of polypeptide attachment to the membrane, hydr olysis of thioester bonds in purified erythrocyte PL scramblase marked ly reduced the Ca2+-dependent activity of the membrane-incorporated pr otein.