The membrane topology of the amino‐terminal domain of the red cell calcium pump

A systematic study of the membrane‐associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3‐(trifluorometnyl)‐3‐(m‐[ 125I]iodophenyl)‐diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane‐bound probe, [3H]bis‐phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light‐dependent labeling of an Mr 135,000‐140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS‐PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with [125I]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane‐embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N‐terminal domain of the Ca2+ pump. A labeled peptide of Mr 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [125I]TID to proteolytic fragments pertaining to the amino‐terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1‐322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with α‐helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N‐terminus of the Ca2+ pump.