Induction of cytochrome c release by BH3-only proteins without affecting Δψ. (a and b) Induction of cytochrome c release by BH3-only proteins in isolated mitochondria. Mitochondria (1 mg/ml) were incubated with rBid, rBax, rtBid, or rBik at the indicated concentrations. At the indicated times (a) and at 10 min (b), 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. (c and d) No effect of BH3-only proteins on Δψ in isolated mitochondria. Mitochondria (1 mg/ml) were incubated with 50 μg/ml (c) or the indicated concentration (d) of rBax (open bar in d), rBid (shaded bar in d), rtBid (hatched bar in d), rBik (solid bar in d), or mock protein, and Δψ was measured by using rhodamine uptake over 15 min (c) or at 15 min (d).

Prevention of Bax- and Ca²⁺-induced, but not Bid-induced, cytochrome c release by inhibition of respiration. Mitochondria (1 mg/ml) were incubated with rBax (20 μg/ml), Ca²⁺ (50 μM), or rBid (20 μg/ml) in the presence or absence of 5 mM succinate (a) or were incubated with or without 10 μM oligomycin, 10 μM antimycin A (antA), 1 mM KCN, or 1 mM CCCP (b). At the indicated times (a) and at 10 min (b), samples were centrifuged and aliquots (20 μl) of the supernatants were analyzed by Western blotting by using anticytochrome c antibody.

No direct effect of Bid on VDAC activity. (a and b) Inability of Bid to directly modulate VDAC activity. Plain liposomes and VDAC liposomes were incubated with 50 mM sucrose together with rBcl-x_L, rBax, or rBid at 20 μg/ml (a) or at indicated concentrations (b) and with an equivalent amount of mock protein. In a, liposome swelling was assessed continuously for 10 min by the decrease of light scatter by using a spectrophotometer at a wavelength of 520 nm. In b, the changes of light scatter at 10 min from the initial value (time 0) are shown. (c) Inability of BH3-only proteins and BH3 peptides to modulate VDAC activity. VDAC liposomes were incubated with 50 mM sucrose with or without rBax, rBcl-x_L, rBid, rtBid, or rBik at 50 μg/ml or with or without Bax BH3 peptide and Bid BH3 peptide at 10 μM, and the differences of light scatter at 10 min from the initial value (time 0) are shown. (d) No direct interaction of Bid and Bik with VDAC. Purified rat liver VDAC (0.2 μg) was incubated with GST-Bid, GST-Bik, or GST-Bcl-x_L fusion protein (0.2 μg each), followed by incubation with GSH-Sepharose as described in Materials and Methods. After washing three times, the bound VDAC was analyzed by Western blotting. “total” represents the amount of VDAC used for the experiment. (e) Effect of tBid on Bcl-x_L activity to inhibit VDAC activity. VDAC liposomes were incubated with 50 mM sucrose together with mock protein or Bcl-x_L (20 μg/ml) in the presence or absence of tBid at the indicated concentrations, and the changes of light scatter were monitored continuously. (f) Effect of tBid on interaction between Bcl-x_L and VDAC in VDAC liposomes. VDAC liposomes (20 μl) were incubated with Bcl-x_L (20 μg/ml) in the presence of tBid at the indicated concentrations. These samples were subjected to immunoprecipitation with an anti-VDAC antibody, and the immune complexes were analyzed by Western blotting by using anti-VDAC and anti-Bcl-x_L antibodies as described in Materials and Methods.

BH3-only proteins induce cytochrome c release without the PT. (a and b) Prevention of rBax-induced, but not Bid- and Bik-induced, cytochrome c release by Ca²⁺ chelation, CsA, and BK. Mitochondria (1 mg/ml) were incubated for 10 min with rBax (at the indicated concentrations in a and at 20 μg/ml in b) or with rBid (20 μg/ml) and rBik (20 μg/ml) in the presence or absence of 0.2 mM EGTA (a), 1 μM CsA (b), and 1 μM BK (b). 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.

Role of the BH3 domain in Bid-induced cytochrome c release. (a and b) Induction of Δψ loss and cytochrome c release by BH3 peptides. Mitochondria (1 mg/ml) were incubated in the presence of the following peptides (10 μM), and Δψ (a) and cytochrome c release (b) were measured at 10 min: Bax (amino acid residues 55–74), Bak (amino acid residues 73–87), Bid (amino acid residues 82–101), Bik (amino acid residues 53–72), BaxΔGD (Bax peptide lacking G⁶⁷D⁶⁸), and BakΔGD (Bak peptide lacking G⁸²D⁸³). Peptides derived from hexokinase (HK; amino acid residues 2–21) and Bcl-2 BH4 (amino acid residues 10–30) also were added as controls. Complete loss of Δψ was demonstrated by incubation of mitochondria with 1 mM CCCP for 10 min. (c) Prevention of BH3 peptide-induced cytochrome c release by CsA, Ca²⁺ chelation, and antimycin A. Mitochondria (1 mg/ml) were incubated for 10 min with Bid BH3 peptide (10 μM) in the presence of 1 μM CsA, 0.2 mM EGTA, and 10 μM antimycin A (antA). (d) Requirement of the BH3 domain for Bid-induced cytochrome c release. Mitochondria (1 mg/ml) were incubated with rBid and mutant Bid GD/EE (G⁹⁴D⁹⁵ to E⁹⁴E⁹⁵) at the indicated concentrations for 10 min, and cytochrome c release was measured as described in Materials and Methods. “total” in b–d represents an equivalent aliquot of mitochondria.

Decrease of mitochondrial Δψ by transfection of bax but not bid. (a) Effect of transient expression of Bax and Bid on mitochondrial Δψ and apoptosis. 293T cells were transfected with an expression construct (0.3 μg) for Bax (open symbols) and Bid (solid symbols), together with 0.1 μg of the GFP expression construct (for detection of DNA-transfected cells). At the indicated times, transfected cells were stained with MTR for measuring Δψ and with biotin-conjugated annexin V and streptavidin-red 617 for detecting apoptosis. Then, apoptosis (circles) and Δψ (squares) were measured by using a flow cytometer. The fractions of apoptotic cells and low Δψ cells were determined as the percentage of annexin V-positive, GFP-positive cells and MTR-negative, GFP-positive cells, respectively, among all GFP-positive cells. Data are shown as the mean ± SD for three independent experiments. (b) Representative flow cytometric profile of Bid- and Bax-overexpressing cells. The same experiments as in a were performed. Empty vector (0.3 μg) together with 0.1 μg of the GFP expression construct also was transfected as a control. After 24 hr, GFP-positive cells were gated and apoptosis (annexin V) and Δψ were analyzed by using a flow cytometer. (c) Representative morphology of bax- and bid-transfected cells. 293T cells were transfected with an expression construct (0.3 μg) for Bax, Bid, or empty vector, together with 0.1 μg of the GFP expression construct. After 24 hr, cells were stained with MTR to monitor Δψ and with Hoechst 33342 to assess nuclear morphology and were observed by using a fluorescent microscope. GFP-positive cells were stained green, and cells maintaining their Δψ were strongly stained orange (MTR).