title: A CRAC-like motif in BAX sequence: relationship with protein insertion and pore activity in liposomes.
abstract: Several proteins that interact with cholesterol have a highly conserved sequence, corresponding to the cholesterol recognition/interaction amino acid consensus. Since cholesterol has been proposed to modulate both oligomerization and insertion of the pro-apoptotic protein BAX, we investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix, LFYFASK, correspond with the sequence motif described for the consensus pattern, -L/V-(X)(1-5)-Y-(X)(1-5)-R/K. Functional characterization of the point mutations, K119A, Y115F, and L113A in BAX, was performed in liposomes supplemented with cholesterol, comparing binding, integration, and pore forming activities. Our results show that the mutations Y115F and L113A changed the cholesterol-dependent insertion observed in the wild type protein. In addition, substitutions in the BAX sequence modified the concentration dependency of carboxyfluorescein release in liposomes, although neither pore activity of the wild type or of any of the mutants significantly increased in cholesterol-enriched liposomes. Thus, while it is likely that the putative CRAC motif in BAX accounts for its enhanced insertion in cholesterol-enriched liposomes; the pore forming properties of BAX did not depend on cholesterol content in the membranes, albeit those mutations changed the pore channeling activity of the protein.
authors: Departamento de Bioquímica, Instituto Nacional de Cardiología, Mexico.
authors: Eduardo
authors: E
authors: Martínez-Abundis
authors: Francisco
authors: F
authors: Correa
authors: Emma
authors: E
authors: Rodríguez
authors: Elizabeth
authors: E
authors: Soria-Castro
authors: José S
authors: JS
authors: Rodríguez-Zavala
authors: Diana
authors: D
authors: Pacheco-Alvarez
authors: Cecilia
authors: C
authors: Zazueta
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001704:Biopolymers / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D008081:Liposomes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051028:bcl-2-Associated X Protein / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001704:Biopolymers; D005452:Fluoresceins; D008081:Liposomes; D051028:bcl-2-Associated X Protein; C024098:6-carboxyfluorescein; D002784:Cholesterol