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 me mbrane Ca2+ pump of erythrocytes has been performed by hydrophobic pho tolabeling. Purified Ca2+ pump was labeled with trifluoromethyl)-3-(m- [I-125]iodophenyl)-diazirine ([I-125]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [H-3]bis-phosphatidylethanolami ne (trifluoromethyl) phenyldiazirine. A significant light-dependent la beling of an M-r 135,000-140,000 peptide, corresponding to the full Ca 2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe rind isolation of fragments by SDS-PAGE, a com mon pattern of labeled peptides was observed. Similarly, labeling of t he Ca2+ pump with [ I-125]TID, either in isolated red blood cell membr anes or after the enzyme was purified, yields a similar pattern of lab eled peptides, Taken together, these results validate the use of eithe r probe to study the lipid interface of the membrane-embedded region o f this protein, and sustain the notion that the conformation of the pu mp 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 domai n of the Ca2+ pump. A labeled peptide of M-r 40,000 belonging to this region was purified and further digested with Vs protease, The specifi c incorporation of [I-125]TID to proteolytic fragments pertaining to t he amino-terminal region indicates the existence of two transmembrane stretches in this domain, A theoretical analysis based on the amino ac id sequence 1-322 predicts two segments with high probability of membr ane insertion, in agreement with the experimental data. Each segment s hows a periodicity pattern of hydrophobicity and variability compatibl e with alpha-helical structure, These results strongly suggest the exi stence of a transmembrane helical hairpin motif near the N-terminus of the Ca2+ pump.