Studies of the interaction of substituted mutants of BAX with yeast mitochondria reveal that the C-terminal hydrophobic alpha-helix is a second ART sequence and plays a role in the interaction with anti-apoptotic BCL-xL.
The role of the two ends of the pro-apoptotic protein BAX in its interaction with mitochondria was challenged by assaying substituted mutants in yeast cells for the ability to bind and insert into the mitochondrial membrane and to promote the release of cytochrome c. Mutations at the N-terminal end confirmed the inhibitory function of this zone, known as apoptotic regulation of targeting (ART). On the other hand, mutations at the C-terminal end of the protein support the hypothesis that the hydrophobic helix alpha9 is not required for the insertion of BAX. In addition, three mutations (a T174D single substitution in the helix alpha9, a K189E/K190E double substitution at the end of the protein, and a P168A mutation in the loop before alpha9) exhibited a strong binding capacity, a strong insertion, as well as high ability to induce cytochrome c release. Considering the positions of these mutations and their potential effect on the movement of helix alpha9, we propose that the C-terminal end of the protein behaves like a second ART. Also, opposite to a mutation that changes the conformation of the N-terminal ART, the mutations in the C-terminal part of the protein impaired the inhibitory effect of anti-apoptotic BCL-xL over BAX insertion, suggesting that the conformation of the alpha9-helix plays a significant role in BAX/BCL-xL interaction.
Arokium
Hubert
H
UMR5095 CNRS/Université de Bordeaux 2, F-33077 Bordeaux cedex, France.
Camougrand
Nadine
N
Vallette
François M
FM
Manon
Stéphen
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017209:Apoptosis; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D066298:In Vitro Techniques; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494826:BAX protein, human; C494811:BCL2L1 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D004247:DNA