The substitution of Proline 168 favors Bax oligomerization and stimulates its interaction with LUVs and mitochondria.
Bax is a major player in the apoptotic process, being at the core of the mitochondria permeabilization events. In spite of the major recent advances in the knowledge of Bax organization within the membrane, the precise behavior of the C-terminal helix α9 remains elusive, since it was absent from the resolved structure of active Bax. The Proline 168 (P168) residue, located in the short loop between α8 and α9, has been the target of site-directed mutagenesis experiments, with conflicting results. We have produced and purified a recombinant mutant Bax-P168A, and we have compared its behavior with that of wild-type Bax in a series of tests on Large Unilamellar Vesicles (LUVs) and isolated mitochondria. We conclude that Bax-P168A had a greater ability to oligomerize and bind to membranes. Bax-P168A was not more efficient than wild-type Bax to permeabilize liposomes to small molecules but was more prone to release cytochrome c from mitochondria.
Institut de Biochimie et de Génétique Cellulaires, UMR5095, CNRS et Université de Bordeaux, CS61390, 1 Rue Camille Saint-Saëns, 33000 Bordeaux, France.
Lilit
L
Simonyan
Institut de Biochimie et de Génétique Cellulaires, UMR5095, CNRS et Université de Bordeaux, CS61390, 1 Rue Camille Saint-Saëns, 33000 Bordeaux, France.
Alexandre
A
Légiot
Institut de Biochimie et de Génétique Cellulaires, UMR5095, CNRS et Université de Bordeaux, CS61390, 1 Rue Camille Saint-Saëns, 33000 Bordeaux, France.
Ioan
I
Lascu
Institut des Biomolécules Max Mousseron, UMR 5247, CNRS, Université de Montpellier, ENSCM, et Université d'Avignon et des Pays de Vaucluse, BP 21239, 301 rue Baruch de Spinoza, 84916 Avignon cedex 9, France.
Grégory
G
Durand
Institut de Biochimie et de Génétique Cellulaires, UMR5095, CNRS et Université de Bordeaux, CS61390, 1 Rue Camille Saint-Saëns, 33000 Bordeaux, France.
Marie-France
MF
Giraud
Institut de Biochimie et de Génétique Cellulaires, UMR5095, CNRS et Université de Bordeaux, CS61390, 1 Rue Camille Saint-Saëns, 33000 Bordeaux, France.
Cécile
C
Gonzalez
Institut de Biochimie et de Génétique Cellulaires, UMR5095, CNRS et Université de Bordeaux, CS61390, 1 Rue Camille Saint-Saëns, 33000 Bordeaux, France. Electronic address: manon@ibgc.cnrs.fr.
Stéphen
S
Manon
D000409:Alanine / Q000737:chemistry* / Q000378:metabolism; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D045304:Cytochromes c / Q000378:metabolism; D015870:Gene Expression; D045325:HCT116 Cells; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D010539:Permeability; D011392:Proline / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C494826:BAX protein, human; D008081:Liposomes; D011994:Recombinant Proteins; D053835:Unilamellar Liposomes; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D011392:Proline; D000409:Alanine
Bax; Cell-free assays; Cell-free expression; LUV; Mitochondria