Phospholipid distribution around the plasma membrane calcium pump: a hydrophobic photolabeling study

• Published in: Cell biochemistry and biophysics 2006-01, Vol.44 (3), p.431-437
• Main Authors: Villamil Giraldo, Ana María, Castello, Pablo Raúl, González Flecha, F Luis, Delfino, José María, Rossi, Juan Pablo F C
• Format: Article
• Language: English
• Subjects: Calcium; Calcium-Transporting ATPases - drug effects; Calcium-Transporting ATPases - isolation & purification; Calcium-Transporting ATPases - metabolism; Cation Transport Proteins - drug effects; Cation Transport Proteins - isolation & purification; Cation Transport Proteins - metabolism; Cell Membrane - metabolism; Erythrocyte Membrane - enzymology; Humans; Ion Channels; Membrane Lipids - metabolism; Micelles; Molecules; Peptide Fragments - metabolism; Phospholipids - chemistry; Phospholipids - metabolism; Plasma Membrane Calcium-Transporting ATPases; Proteins; Sodium-Potassium-Exchanging ATPase - metabolism; Staining and Labeling - methods
• ISSN: 1085-9195; 1085-9195; 1559-0283
• DOI: 10.1385/CBB:44:3:431

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-protein stoichiometry, and (2) the distribution of lipid molecules among different regions of the protein. PMCA was photolabeled in mixed micelles containing detergent, the phosphatidylcholine photoactivatable analog 1-palmitoyl-2-[9-[2'-[125I]iodo-4'- (trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine, and different amounts of dimyristoyl phosphatidylcholine (PC). The stoichiometry was estimated after the extent of the labeling reaction had been independently assessed. We determined a maximum number of 17 +/- 1 molecules of PC in close contact with the transmembrane surface per PMCA molecule. In addition, a semiquantitative description of the phospholipid environment around different regions of PMCA was 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 (661-1,205) regions was 26, 36, and 38%, respectively.