Phospholipid distribution around the plasma membrane calcium pump. A hydrophobic photolabeling study

VILLAMIL GIRALDO AM; CASTELLO PR; GONZALEZ FLECHA FL; DELFINO JM; ROSSI JPFC

CELL BIOCHEMISTRY AND BIOPHYSICS

Humana Press

Año: 2006 vol. 44 p. 431 - 437

1085-9195

The functions of membrane proteins are highly dependent on their phospholipid environment. In this article, we have used a hydrophobic photolabeling method to study the noncovalent interactions between plasma membrane calcium pump (PMCA) and surrounding phospholipids. With this approach, we determined (1) the number of lipid molecules in close contact with the transmembrane surface, i.e., the lipid¨Cprotein stoichiometry, and (2) the distribution of lipid molecules among different regions of the protein. PMCAwas photolabeled in mixed micelles containing detergent, the phosphatidylcholine photoactivatable analog 1-palmitoyl-2-[9-[2¡ä-[125I]iodo-4¡ä(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine, and different amounts of dimyristoyl phosphatidylcholine (DMPC). The stoichiometry was estimated after the extent of the labeling reaction had been independently assessed. We determined a maximum number of 17 ¡À1 molecules of DMPC in close contact with the transmembrane surface per PMCAmolecule. In addition, a semiquantitative description of the phospholipid environment around different regions of PMCAwas carried out after limited proteolysis of the photolabeled protein. The distribution of labels among the N-terminal (1-322), the central (323-660), and the C-terminal (661-1205) regions was 26, 36, and 38%, respectively.