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. 2000 Feb;20(3):929-35.
doi: 10.1128/MCB.20.3.929-935.2000.

Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane

R Eskes  1 , S Desagher, B Antonsson, J C Martinou
Affiliations

Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane

R Eskes et al. Mol Cell Biol. 2000 Feb.

Abstract

In many types of apoptosis, the proapoptotic protein Bax undergoes a change in conformation at the level of the mitochondria. This event always precedes the release of mitochondrial cytochrome c, which, in the cytosol, activates caspases through binding to Apaf-1. The mechanisms by which Bax triggers cytochrome c release are unknown. Here we show that following binding to the BH3-domain-only proapoptotic protein Bid, Bax oligomerizes and then integrates in the outer mitochondrial membrane, where it triggers cytochrome c release. Bax mitochondrial membrane insertion triggered by Bid may represent a key step in pathways leading to apoptosis.

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Figures

FIG. 1
FIG. 1
Bax integration into the mitochondrial membranes during apoptosis and after Bid treatment. (A) Mitochondria from HeLa cells cultured in the absence or presence of 1 μM staurosporine for increasing times were isolated and treated with 0.1 M Na2CO3 to produce alkali-sensitive (Att [attached]) and -resistant (Ins [inserted]) fractions. Both fractions were analyzed by Western blotting for the presence of Bax. Cox II was used as a gel-loading control. (B) Mitochondria from HeLa cells were incubated with 100 nM recombinant wild-type Bid and two Bid mutants (BidmIII-1 and BidmIII-3) for 15 min at 30°C, recovered by centrifugation, and treated with 0.1 M Na2CO3 as above. Various proteins were analyzed by Western blotting in alkali-sensitive (Att.) and -resistant fractions (Ins.). (C) Cytochrome c was analyzed by Western blotting in the mitochondrial suspension following incubation with wild-type and mutant Bid.
FIG. 2
FIG. 2
Bid induces the insertion of Bax into the outer mitochondrial membrane. Isolated mitochondria (100 μg) from HeLa cells were incubated with 100 nM Bid for 15 min at 30°C and treated with digitonin (1.2 mg/ml) for 25 min at 4°C. The digitonin-sensitive (Out. [outer mitochondrial membrane]) and resistant (In. [inner mitochondrial membrane]) fractions were analyzed by Western blotting for the presence of VDAC, Cox IV, Bax, and Bcl-xL.
FIG. 3
FIG. 3
Bid induces Bax oligomerization. Isolated mitochondria from HeLa cells were incubated with 1 μM Bid for 15 min at 30°C, and the mitochondrial pellet was treated with two different cross-linkers as described in Materials and Methods. After cross-linking, the mixture was immunoprecipitated with anti-Bax monoclonal antibody and analyzed by Western blotting with an anti-Bax polyclonal antibody.
FIG. 4
FIG. 4
Time course study of Bax oligomerization, Bax membrane insertion, and cytochrome c release after addition of Bid to isolated mitochondria. Isolated mitochondria from HeLa cells were incubated with recombinant Bid at 30°C for increasing times and analyzed for Bax oligomerization (A), Bax membrane insertion (B), and cytochrome c release (C). Att., attached (alkali sensitive); Ins., inserted (alkali resistant); Cyt.c, cytochrome c.
FIG. 5
FIG. 5
Bcl-xL and Bcl-2 inhibit Bid-induced oligomerization and insertion of Bax into the outer mitochondrial membrane. (A to C) Mitochondria isolated from HeLa cells were incubated with 100 nM Bid in the presence of 1 μM recombinant Bcl-xL or Bcl-xLm at 30°C for 15 min. Mitochondria were used to study Bax insertion into the outer mitochondrial membrane (A), cytochrome c release (B), and Bax dimerization (C). (D) Mitochondria from HeLa cells overexpressing Bcl-2 were isolated and incubated with 100 nM Bid at 30°C for 15 min and used to analyze Bax insertion into the outer mitochondrial membrane. Att., attached (alkali sensitive); Ins., inserted (alkali resistant); Ctl., Cont., control; Cyt.c, cytochrome c.
FIG. 6
FIG. 6
In contrast to cytochrome c release, Bid-induced insertion of Bax does not require the presence of Mg2+. (A) Mitochondria from HeLa cells were incubated with 100 nM Bid for 15 min at 30°C in the presence or absence of 2.5 mM MgCl2 or MnCl2 before analysis of Bax insertion into membranes. (B) Mitochondria were incubated in the presence or absence of 100 nM Bid and various salts including MgCl2 (2.5 mM), MnCl2 (2.5 mM), KCl (5 mM), NaCl (5 mM), and LiCl (5 mM). Both supernatants and mitochondrial pellets were analyzed for cytochrome c release. Ctl., control; Att., attached (alkali sensitive); Ins., inserted (alkali resistant); Cyt. c, cytochrome c.
FIG. 7
FIG. 7
Model for the activation of Bax by Bid during apoptosis. Following an apoptotic stimulus, Bid binds to Bax and triggers a change in the conformation of Bax. As a result, Bax dimerizes (or oligomerizes) and inserts into the outer mitochondrial membrane, which results in cytochrome c (Cyt. c) release from mitochondria.
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