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

Abstract

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-(rn- [ '251]iodophenyl)-diazirine ([ '251]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, 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 ['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-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 CaZ+ pump. A labeled peptide of M, 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [1251]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 a-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N-terminus of the Ca2+ pump.Keywords: calcium pump; detection of the outside face of the helices; hydrophobic photolabeling; membrane proteins; secondary structure prediction for transmembrane segments; transmembrane topologyThe erythrocyte CaZ+ pump is a monomeric integral membrane protein that actively transports Ca2+ from the cytoplasm to the external milieu. The main portion of the pump faces the cytoplasm.

Identification of Bordetella pertussis virulence-associated outer membrane proteins

“…The BPTohama I virulence-associated bands were cut out and eluted by di¡usion as described previously [16]. The same regions of the gel corresponding to BP347 samples were processed as negative controls.…”

“…Preparative SDS-PAGE was performed as described above (circa 200 Wg protein gel 3I ), then, the gels were dyed by a reverse staining method [15] except that the toning step was avoided to prevent protein aggregation. The BPTohama I virulence-associated bands were cut out and eluted by di¡usion as described previously [16]. The same regions of the gel corresponding to BP347 samples were processed as negative controls.…”

Alpha-periodicity analysis of small multidrug resistance (SMR) efflux transporters

“…Such studies have been performed on a variety of proteins including the F 0 proteins of the E. coli F 1 F 0 ATPase (Vik and Dao 1992), the antenna complexes from photosynthetic bacteria (Donnelly and Cogdell 1993), the succinate:quinone oxidoreductases (Hägerhäll and Hederstedt 1996), the erythrocyte Ca 2+ pump (Castello et al 1997), and the rhodopsin family of G-protein-coupled receptors (Baldwin et al 1997). …”

Identification ofBordetella pertussisvirulence-associated outer membrane proteins

“…Preparative SDS‐PAGE was performed as described above (circa 200 μg protein gel −1 ), then, the gels were dyed by a reverse staining method [15] except that the toning step was avoided to prevent protein aggregation. The BPTohama I virulence‐associated bands were cut out and eluted by diffusion as described previously [16]. The same regions of the gel corresponding to BP347 samples were processed as negative controls.…”