Bcl-XL Protects BimEL-induced Bax Conformational Change and Cytochrome c Release Independent of Interacting with Bax or BimEL

Hirohito Yamaguchi,Hong-Gang Wang

doi:10.1074/jbc.m207516200

Abstract

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.

Highlights

Mitochondrion is the crucial regulatory organelle for the signaling pathway of apoptosis [1,2,3,4]
These results indicate that the pro-apoptotic activity of the BH3-mutated BimEL protein requires its C-terminal hydrophobic region, deletion of this hydrophobic domain alone does not much affect BimEL to induce apoptosis and Bax conformational change
The intracellular localization of M1⌬C was only found in the cytosol, indicating that both the C terminus hydrophobic region and the BH3 domain are required for the mitochondrial localization of BimEL, which may be critical for BimEL-mediated Bax activation and apoptosis

Summary

Introduction

Mitochondrion is the crucial regulatory organelle for the signaling pathway of apoptosis [1,2,3,4]. We show that BimEL triggers Bax conformational change and cytochrome c release, which can be suppressed by Bcl-XL but is independent from its ability to heterodimerize with this anti-apoptotic Bcl-2 family protein. The effects of these Bim mutants on cell viability was determined by luciferase assay (Fig. 1B), Bax conformational change was examined by immunoprecipitation with anti-Bax 6A7 antibody (Fig. 1C), and Bax translocation to mitochondria was detected by subcellular fractionation/immunoblot analysis (Fig. 1D).

Results

Conclusion