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 phagocytic clearance of injured or apoptotic cells. We recently identified a plasma membrane protein that induces rapid transbilayer movement of PL at elevated Ca2+, and we presented evidence that this PL scramblase mediates the transbilayer movement of plasma membrane PL in a variety of cells and tissues exposed to elevated intracellular Ca2+ [Zhou, Q. et al. (1997) J. Biol. Chem. 272, 18240-18244]. Activation of PL scramblase entails coordination of Ca2+ by a 12 residue segment resembling an EF hand loop motif that is adjacent to the single transmembrane helix of the polypeptide. On the assumption that correct orientation of the Ca2+-binding loop segment required a distal segment of the polypeptide to orient back toward the membrane, we considered the possibility 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 [3H]palmitate, and fusion protein immunoprecipitated with antibody against GFP-PL scramblase was found to covalently incorporate 3H, whereas no radioactivity was covalently associated with GFP. The identity of the covalently bound 3H in PL scramblase as a thioester-linked [3H]palmitate was confirmed by hydroxylamine cleavage and by thin-layer chromatography of the liberated fatty acid. Consistent with the assumption that activation by Ca2+ might require accessory site(s) of polypeptide attachment to the membrane, hydrolysis of thioester bonds in purified erythrocyte PL scramblase markedly reduced the Ca2+-dependent activity of the membrane-incorporated protein.
Zhao
J
J
Blood Research Institute of The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53201, USA.
Zhou
Q
Q
Wiedmer
T
T
Sims
P J
PJ
D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D019308:Palmitic Acid / Q000737:chemistry / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary
D002352:Carrier Proteins; D008051:Lipid Bilayers; D008563:Membrane Lipids; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D019308:Palmitic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium