Bcl-XL protects BimEL-induced Bax conformational change and cytochrome C release independent of interacting with Bax or BimEL.
The Bcl-2 homology (BH) 3-only pro-apoptotic Bcl-2 family protein Bim plays an essential role in the mitochondrial pathway of apoptosis through activation of the BH1-3 multidomain protein Bax or Bak. To further understand how the BH3-only protein activates Bax, we provide evidence here that BimEL induces Bax conformational change and apoptosis through a Bcl-XL-suppressible but heterodimerization-independent mechanism. Substitution of the conserved leucine residue in the BH3 domain of BimEL for alanine (M1) inhibits the interaction of BimEL with Bcl-XL but does not abolish the ability of BimEL to induce Bax conformational change and apoptosis. However, removal of the C-terminal hydrophobic region from the M1 mutant (M1DeltaC) abolishes its ability to activate Bax and to induce apoptosis, although deletion of the C-terminal domain (DeltaC) alone has little if any effect on the pro-apoptotic activity of BimEL. Subcellular fractionation experiments show that the Bim mutant M1DeltaC is localized in the cytosol, indicating that both the C-terminal hydrophobic region and the BH3 domain are required for the mitochondrial targeting and pro-apoptotic activity of BimEL. Moreover, the Bcl-XL mutant (mt1), which is unable to interact with Bax and BimEL, blocks Bax conformational change and cytochrome c release induced by BimEL in intact cells and isolated mitochondria. BimEL or Bak-BH3 peptide induces Bax conformational change in vitro only under the presence of mitochondria, and the outer mitochondrial membrane fraction is sufficient for induction of Bax conformational change. Interestingly, native Bax is attached loosely on the surface of isolated mitochondria, which undergoes conformational change and insertion into mitochondrial membrane upon stimulation by BimEL, Bak-BH3 peptide, or freeze/thaw damage. Taken together, these findings indicate that BimEL may activate Bax by damaging the mitochondrial membrane structure directly, in addition to its binding and antagonizing Bcl-2/Bcl-XL function.
Yamaguchi
Hirohito
H
Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa 33612, USA.
Wang
Hong-Gang
HG
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D003574:Cytochrome c Group / Q000378:metabolism*; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins; D008928:Mitochondria / Q000502:physiology; D011487:Protein Conformation; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C494811:BCL2L1 protein, human; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D002352:Carrier Proteins; D003574:Cytochrome c Group; D008565:Membrane Proteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein