Figure 2. The biochemical activity of BAX_O emulates BH3-triggered BAX.

(A) BAX_O and tBID-triggered BAX_M translocate to liposomes, whereas vehicle-treated BAX_M remains in the soluble fraction, as assessed at 1 h by liposomal translocation assay and BAX western analysis. BAX_O and BAX_M, 500 nM; tBid, 20 nM. Data shown are representative of two independent biological replicates. Fractions: liposomal, 4-6; supernatant, 7-14.

(B) BAX_O and tBID-triggered BAX_M induce liposomal poration with similar kinetics and potency, as assessed by liposomal release assay. Error bars are mean ± s.e.m. for experiments performed in technical triplicate, with data representative of two independent experiments. BAX_M, 500 nM; BAX_O, 500 nM (equivalent total protein amounts); tBID, 20 nM.

(C-D) Anti-apoptotic proteins effectively blocked liposomal poration induced by BAX_M upon tBID triggering (C), but had no inhibitory effect on BAX_O (D). Error bars are mean ± s.e.m. for experiments performed in technical triplicate, with data representative of two independent experiments. BAX_M and BAX_O 500 nM; tBID, 20 nM; anti-apoptotic proteins, 10 μM.

(E-G) Negative stain EM of liposomes incubated with BAX_M (E), tBID-triggered BAX_M (F), or BAX_O (G), showing similar morphology of membrane disruption and pore size induced by BAX_O and BH3-triggered BAX_M after 15 min. Mean pore diameter (nm) ± s.e.m.: tBID-triggered BAX, 48.9 ± 2.3; BAXo, 56.2 ± 2.6; p value = 0.195.

(H) BAX_O induces dose-responsive cytochrome c release from BAX/BAK-deficient mouse liver mitochondria (MLM). tBID-triggered BAX release is shown as a positive control. Error bars are mean ± s.e.m. for experiments performed in technical triplicate, with data representative of two independent experiments. BAX_M, 200 nM; BAX_O, 6.25-200 nM; tBID, 40 nM.

See also Figures S3 and S4.