INVESTIGADORES
GONZALEZ FLECHA Francisco Luis
artículos
Título:
The membrane topology of the amino-terminal domain of the red cell calcium pump
Autor/es:
PABLO R. CASTELLO; F. LUIS GONZÁLEZ-FLECHA; ARIEL J CARIDE; HORACIO N FERNANDEZ; JOSÉ MARÍA DELFINO; JUAN PABLO F.C. ROSSI
Revista:
PROTEIN SCIENCE
Referencias:
Año: 1997 vol. 6 p. 1708 - 1717
ISSN:
0961-8368
Resumen:
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-(trifluoromethyl)-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 M(r) 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 M(r) 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 alpha-helical structure. These results strongly suggest
the existence of a transmembrane helical hairpin motif near the
N-terminus of the Ca2+ pump.