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. 1998 Dec 8;95(25):14681-6.
doi: 10.1073/pnas.95.25.14681.

Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria

M Narita  1 , S Shimizu, T Ito, T Chittenden, R J Lutz, H Matsuda, Y Tsujimoto
Affiliations

Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria

M Narita et al. Proc Natl Acad Sci U S A. .

Abstract

Cytochrome c release and the mitochondrial permeability transition (PT), including loss of the transmembrane potential (Deltapsi), play an important role in apoptosis. Using isolated mitochondria, we found that recombinant Bax and Bak, proapoptotic members of the Bcl-2 family, induced mitochondrial Deltapsi loss, swelling, and cytochrome c release. All of these changes were dependent on Ca2+ and were prevented by cyclosporin A (CsA) and bongkrekic acid, both of which close the PT pores (megachannels), indicating that Bax- and Bak-induced mitochondrial changes were mediated through the opening of these pores. Bax-induced mitochondrial changes were inhibited by recombinant Bcl-xL and transgene-derived Bcl-2, antiapoptotic members of the Bcl-2 family, as well as by oligomycin, suggesting a possible regulatory effect of F0F1-ATPase on Bax-induced mitochondrial changes. Proapoptotic Bax- and Bak-BH3 (Bcl-2 homology) peptides, but not a mutant BH3 peptide nor a mutant Bak lacking BH3, induced the mitochondrial changes, indicating an essential role of the BH3 region. A coimmunoprecipitation study revealed that Bax and Bak interacted with the voltage-dependent anion channel, which is a component of PT pores. Taken together, these findings suggest that proapoptotic Bcl-2 family proteins, including Bax and Bak, induce the mitochondrial PT and cytochrome c release by interacting with the PT pores.

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Figures

Figure 1
Figure 1
Δψ loss induced by rBakΔC and rBax in isolated mitochondria. (A and B) Δψ loss induced by rBakΔC. Isolated mitochondria (1 mg/ml) were incubated with 100 μg/ml (A) or the indicated concentration (B) of rBakΔC, and Δψ was measured by using Rh123 uptake over 20 min (A) or at 20 min (B). (C) Uptake of rBakΔC into mitochondria. After the same experiments as in B were performed, mitochondria (30 μg) were centrifuged, washed, and subjected to Western blot analysis using anti-Bak antibody. The total amount of rBakΔC protein in 100 μg/ml is indicated as total. (D) Δψ loss induced by rBax. Isolated mitochondria (1 mg/ml) were incubated with 100 μg/ml rBax, and Δψ was measured by using Rh123 uptake over 20 min.
Figure 2
Figure 2
Induction of the PT by rBax and rBakΔC. (A) Prevention of rBax-induced mitochondrial swelling by CsA. Isolated mitochondria (0.1 mg/ml) were incubated with rBax (10 μg/ml) or an equivalent concentration of mock protein in the presence or absence of CsA at 100 nM, and light scatter was monitored as described in Materials and Methods. (B and C) Prevention of rBax-induced release of mAST by CsA or Ca2+ chelation. Mitochondria (1 mg/ml) were incubated with rBax (100 μg/ml) or an equivalent concentration of mock protein in the presence or absence of CsA at 100 nM (B) or at the indicated concentrations (C). Mitochondria were also incubated with rBax (100 μg/ml) after Ca2+ chelation in B. mAST activity was measured over 20 min (B) or at 20 min (C) as described in Materials and Methods. •, rBax; ○, mock protein; ■; rBax + CsA (100 nM); □, rBax with Ca2+ chelation. (D) Prevention of rBax-induced Δψ loss by CsA or Ca2+ chelation. Mitochondria (1 mg/ml) were incubated with rBax (100 μg/ml) or an equivalent concentration of mock protein in the presence or absence of CsA at the indicated concentrations. Mitochondria also were incubated with rBax (100 μg/ml) after Ca2+ chelation. Δψ was measured by using Rh123 at 20 min. (E) Inhibition of rBakΔC-induced Δψ loss by BK and l-carnitine. Mitochondria (1 mg/ml) were incubated with rBakΔC (100 μg/ml) in the presence or absence of 10 mM BK or 5 mM l-carnitine. Δψ was measured by using Rh123 at 20 min.
Figure 3
Figure 3
Requirement for the BH3 domain in rBakΔC-induced PT. (A) Lack of Δψ loss induction by rBakΔGDΔC. Mitochondria (1 mg/ml) were incubated with 100 μg/ml of rBakΔC, rBakΔGDΔC, or mock protein and Δψ was measured using Rh123 at 20 min. (B) Δψ loss induction by BH3 peptides. Mitochondria (1 mg/ml) were incubated in the presence of the following peptides (10 μM), and Δψ was measured at 20 min using Rh123: Bak (amino acid residues 73–87), mutant BakL78A (amino acid residues 73–87), and Bax (amino acid residues 55–74).
Figure 4
Figure 4
Interaction of Bax and Bak with VDAC. (A and B) Interaction of rBax with VDAC in mitochondria. Mitochondria (1 mg/ml) were incubated with rBax (100 μg/ml). After induction of the PT, the mitochondria were centrifuged and washed twice. The mitochondrial lysates were subjected to immunoprecipitation (IP) with anti-human Bax antibody (αBax) or normal rabbit IgG (NRI) in A and with anti-human VDAC antibody (αVDAC) or normal mouse IgG (NMI) in B. The immune complexes were analyzed by Western blotting using anti-human VDAC antibody (A) and anti-Xpress antibody (B). An aliquot of mitochondria (mit) in A and rBax in B were used as the controls. (C) Interaction of Bax or Bak with VDAC in mammalian cells. Cos7 cells were cotransfected with pUC-CAGGS-mouse bax or pUC-CAGGS-human bak and pUC-CAGGS-human vdac1, and were treated with DTBP as described in Materials and Methods. Cell lysates were immunoprecipitated with anti-Bax (αBax) or anti-Bak (αBak) polyclonal antibody and normal rabbit IgG (NRI). Then the immune complexes were analyzed by Western blotting using anti-human VDAC antibody.
Figure 5
Figure 5
Induction of cytochrome c release by rBax and rBakΔC. Mitochondria (1 mg/ml) were incubated with 100 μg/ml of rBax (A), 100 μg/ml of rBakΔC (B), or 10 μM BH3 peptides (C). Mock proteins were used as controls in A and B. At 20 min, samples were centrifuged and aliquots (20 μl) of the supernatants were subjected to Western blot analysis for cytochrome c (cyt.c). “Total” represents an equivalent aliquot of mitochondria.
Figure 6
Figure 6
Inhibition of rBax and rBakΔC-induced cytochrome c release by inhibiting the PT. (A) Prevention of rBax-induced cytochrome c release by CsA. Mitochondria (1 mg/ml) were incubated with rBax (100 μg/ml) in the presence of CsA at the indicated concentrations. At 20 min, samples were centrifuged and aliquots (20 μl) of the supernatants were analyzed by Western blotting using anti-cytochrome c antibody. (B and C) Prevention of rBax- and BH3-peptide-induced cytochrome c release by depletion of Ca2+. Mitochondria were incubated with 100 μg/mg of rBax, mock proteins, or BH3 peptides derived from Bak or Bax (10 μM) under Ca2+-depleted conditions. At 20 min, samples were obtained as described in A and the amount of cytochrome c (cyt.c) released was analyzed by Western blotting.
Figure 7
Figure 7
No requirement of cytochrome c release for rBax-induced Δψ loss. (A) Cytochrome c depletion from mitochondria. Cytochrome c was depleted by washing mitochondria with a hypotonic buffer as described in Materials and Methods. Intact mitochondria (intact) and cytochrome c-depleted mitochondria (cyt.c-depleted) (30 μg each) were analyzed for cytochrome c (cyt.c) by Western blotting. (B) rBax-induced Δψ loss in cytochrome c-depleted mitochondria with exogenous cytochrome c. Cytochrome c-depleted mitochondria (1 mg/ml) were incubated with rBax (50 μg/ml) or mock protein in the presence of exogenous cytochrome c (1 mM), and Δψ was measured using Rh123.
Figure 8
Figure 8
Prevention of rBax-induced Δψ loss and cytochrome c release by rBcl-xL, transgene-derived Bcl-2, and oligomycin. (A) Prevention of rBax-induced Δψ loss by rBcl-xL and oligomycin. Mitochondria (1 mg/ml) were incubated with mock protein or rBax at 50 μg/ml in the presence or absence of rBcl-xL (wt) and its mutants (mt1 and mt7) (20 μg/ml) or oligomycin (10 μM), and Δψ was measured using Rh123 at 20 min. (B) Prevention of rBakΔC-induced Δψ loss by rBcl-xL. Mitochondria (1 mg/ml) were incubated with rBakΔC (100 μg/ml) in the presence or absence of rBcl-xL (20 μg/ml), and Δψ was measured using Rh123 at 30 min. (C) Prevention of rBax-induced cytochrome c release by rBcl-xL and oligomycin. Mitochondria (1 mg/ml) were incubated with rBax (50 μg/ml) in the presence or absence of Bcl-xL (wt) and its mutants (mt 1 and mt 7) (20 μg/ml), CsA (100 nM) or oligomycin (10 μM). After 12 min, samples were centrifuged and aliquots of the supernatants (20 μl each) were subjected to Western blot analysis for cytochrome c (cyt.c). (D) Prevention of rBax-induced Δψ loss by transgene-derived Bcl-2. Mitochondria were isolated from the livers of hepatic bcl-2 transgenic mice (Bcl-2) and nontransgenic littermates (non-Tg). Mitochondria (1 mg/ml) were incubated with mock protein or rBax at 50 μg/ml, and Δψ was measured at 30 min.
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