Interactions between plasma membrana calcium pump and phospholipids studied with a photoactiva-table probe

VILLAMIL GIRALDO, AM; CASTELLO, P.R.; GONZÁLEZ FLECHA, F.L.; DELFINO, J. M.; ROSSI, J.P.F.C.

Angra dos Reis, RJ

Congreso; 1st Latin American Protein Society Meeting; 2004

Protein Society

INTERACTIONS BETWEEN PLASMA MEMBRANE CALCIUM PUMP AND PHOSPHOLIPIDS STUDIED WITH A PHOTOACTIVATABLE PROBE Villamil Giraldo, A.M., Castello, P.R., Gonz´alez Flecha, F.L., Delfino, J.M. and Rossi, J.P.F.C1, ∗  1 IQUIFIB, Facultad de Farmacia y Bioqu´ýmica, U.B.A., Jun´ýn 956, Buenos Aires, Argentina. The functions of membrane proteins are highly dependent on their phospholipid environment. To study the inter­action between plasma membrane calcium pump (PMCA) and surrounding phospholipids we used the phosphatidyl­choline photoactivatable analogue ([125I] TID-PC/16) on a pure soluble preparation of PMCA. With this approach, we analysed the lipid annulus around the transmembrane domain. Specifically, we determined (i) the number of lipid molecules in close contact with the transmembrane surface, and (ii) the distribution of lipid molecules among different regions of the protein. PMCA was photolabeled in the presence of different amounts of added phosphatidylcholine (PC). Lipid-protein stoichiometry was determined by estimating the extent of the labeling reaction. The number of PC molecules in direct contact with the protein follows a hyperbola when plotted as a function of PC concentration. The maximum number of PC molecules that result in close contact with the transmembrane surface is 17 +/-1 PC per protein molecule. With the aim of evaluating its general applicability, we assayed this analytical method on sarcoplasmic reticulum calcium pump (SERCA). Lipid stoichiometry was previously measured for this protein by spin-label electron paramagnetic resonance (EPR) and the values thus obtained ranged between 22 +/-2 and 24 +/­5 phospholipid molecules per protein monomer [1]; [2]. Accordingly, our results indicated a value of 19 +/-3 PC molecules surrounding the transmembrane surface of this protein. Our experimental approach could be advantageous when -as in the case of PMCA -protein concentration is too low to apply EPR techniques or, when more than one membrane protein is simultaneously present in the sample. In addition, a semi-quantitative analysis of the interac­tion of PMCA with its phospholipid environment was carried out after limited proteolysis of photolabeled PMCA and further analysis of the incorporation of the probe into each fragment. 26% of the total labeling was found to be associated to N-terminal (1-322) region while C-terminal region (661-1205) incorporated 38% of the probe. This relative distribution of phospholipid molecules around the transmembrane surface of the protein does not depend on PC concentration, pointing to the absence of particular regions with enhanced affinity for PC. ACKNOWLEDGEMENTS This work was supported by ANPCYT, CONICET and UBACYT ∗ Electronic address: anamaria@qb.ffyb.uba.ar [1] Silvius et al. Biochemistry 23:538-547 (1984) [2] Thomas et al. Biophys J. 37:217-25 (1982)