“The Membrane Topology of the Amino-Terminal Domain of the red cell Ca2+-pump”.

CASTELLO PR; CARIDE, A.J.; FERNÁNDEZ, H; DELFINO, J.M.; ROSSI, J.P.F.C.

PROTEIN SCIENCE

Cold Spring Harbor Laboratory Press,

Lugar: New York; Año: 1997 vol. 6 p. 1708 - 1717

0961-8368

AbstractA systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has beenperformed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(rn-[ '251]iodophenyl)-diazirine ([ '251]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzymelabeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine(t rifluoromethyl) phenyldiazirine. Asignificant light-dependent labeling of an M, 135,000-140,000 peptide, corresponding to the full Ca2+ pump, wasobserved with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments bySDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with ['251]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-embeddedregion of this protein, and sustain the notion that the conformation of the pump is maintained throughout the proceduresof solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis ona detailed analysis of the N-terminal domain of the CaZ+ pump. A labeled peptide of M, 40,000 belonging to this regionwas purified and further digested with V8 protease. The specific incorporation of [1251]TID to proteolytic fragmentspertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. Atheoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membraneinsertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity andvariability compatible with a-helical structure. These results strongly suggest the existence of a transmembrane helicalhairpin motif near the N-terminus of the Ca2+ pump.