Bax-induced cytochrome C release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions

Abstract

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homologue of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). We now report that Bax is sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A-sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg2+ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A, the other Bax dependent, Mg2+ sensitive but cyclosporin insensitive.

Figure 1

Mitochondrial localization of Bax. HeLa cells were transiently transformed with cDNA encoding a His-tagged Bax and immunostained 15 h later for both the mitochondrial marker HSP-70 (A) and Bax (B). Note that the shape as well as the distribution of the mitochondria has change in the Bax-transfected cell (arrow).

Figure 2

Immunofluorescence studies of cytochrome C release from mitochondria after overexpression of Bax in HeLa cells. HeLa cells were transiently transfected with a cDNA encoding a His-tagged human Bax and cultured for 15 h in the presence (D–F) or absence (A–C) of z-VAD-fmk before immunostaining for Bax (A and D) and cytochrome C (B and E). C and F are nuclear Hoechst stainings. Arrows, transfected cells.

Figure 3

Bax-induced cytochrome C release from isolated mitochondria. (A) Mitochondria isolated from mouse liver were incubated for 1 h at 30°C with 5 μM COOH-terminal truncated Bax (BaxΔTM), 5 μM COOH-terminal truncated Bcl-2 or 170 nM full-length Bax in 200 μl KCl buffer. After 1 h, the reaction mixture was centrifuged at 13,000 g for 5 min. Supernatant and mitochondrial pellets corresponding to 5 μg mitochondrial proteins were subjected to 4–20% SDS-PAGE and analyzed by Western blot. Loading for the mitochondrial pellet was controlled with a Cox IV antibody. (B) Kinetics of Bax-induced cytochrome C release. Mitochondria were incubated with 170 nM Bax or buffer control at 30°C and the mitochondria pellet was analysed over time as in A. In addition to cytochrome C and Cox IV, Bax levels were measured in the mitochondrial pellets. (C) Mitochondria were incubated with different amounts of Bax for 1 h at 30°C and cytochrome C release was measured as in A. Data shown are representative of 3–12 independent experiments performed with at least three different preparations of Bax protein.

Figure 4

Bax effect is ion dependent. Mitochondria isolated from mouse liver cells were incubated with 170 nM Bax at 30°C in sucrose-mannose buffer with or without addition of salts as indicated in the figure. The salt concentrations were 4 mM MgCl₂ and 5 mM Na₂HPO₄. Cytochrome C release was measured after 1 h as described in Fig. 3.

Figure 5

Bax-induced release of cytochrome C from isolated mitochondria is not inhibited by PTP blockers. Several PTP blockers have been tested for their ability to inhibit the release of cytochrome C from isolated mitochondria induced by 170 nM Bax. (A) We compared Bax effect with that of calcium, a PTP opener, and their sensitivity to 10 μM CsA and 0.5 mM EGTA. Cytochrome C and Cox IV in the mitochondrial pellet were measured after a 1-h incubation in KCl buffer at 30°C by Western blotting. The PTP inhibitors BKA (100 μM) (B), a combination of CsA (10 μM) and ARA (50 μM) (C) and ADP (10 μM) (D) were also tested. Mitochondria were preincubated with these compounds for 5 min before addition of Bax. Note that although CsA and EGTA were capable of inhibiting the calcium effect, they failed to block Bax-induced cytochrome C release.

Figure 6

Both Cyclosporin A and BKA fail to inhibit Bax-induced release of cytochrome C in COS cells. COS cells were transfected with a cDNA encoding His-Bax and cultured for 15 h in the presence of 10 μM CsA and 100 μM z-VAD-fmk (A and B) or 100 μM BKA and 100 μM z-VAD-fmk (C and D) before immunostaining for Bax (A and C) and cytochrome C (B and D). Note that all cells that overexpress Bax display a diffuse cytosolic cytochrome C immunostaining. Arrows, transfected cells.