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

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.

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

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-x_L.

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

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

Bcl-x_L 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-x_L or Bcl-x_Lm 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

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

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.