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. Author manuscript; available in PMC: 2021 Jul 2.
Published in final edited form as: Mol Cell. 2020 Jun 12;79(1):68–83.e7. doi: 10.1016/j.molcel.2020.05.029

Figure 6. BAXO is an execution-phase reagent that distinguishes between key steps of the BAX activation pathway and reveals dual roles of BAX α9.

Figure 6.

(A) I133A mutagenesis impairs BH3-triggered membrane translocation of BAXM, whereas BAXO I133A is essentially unaffected, as assessed at 1 h by liposomal translocation assay and BAX western analysis. BAXM proteins, 500 nM; BAXO proteins, 500 nM; BIM SAHBA2, 500 nM; tBID, 20 nM. Data shown are representative of two independent biological replicates. Fractions: liposomal, 4-6; supernatant, 7-14.

(B-C) tBID-triggered BAXM I133A exhibits decreased kinetics and 50% overall reduction of liposomal release compared to tBID-triggered BAXM (B), whereas I133A mutagenesis has no adverse effect in the context of BAXO (C). Error bars are mean ± s.e.m. for experiments performed in technical triplicate, with data representative of two independent experiments. BAXM proteins, 500 nM; BAXO proteins, 500 nM; tBID, 20 nM.

(D) Membrane translocation kinetics of BAXOΔC vs. BAXO, as monitored by liposomal translocation assay and BAX western analysis. BAXO proteins, 500 nM. Data shown are representative of two independent biological replicates. Fractions: liposomal, 4-6; supernatant, 7-14.

(E-F) Decreased kinetics and extent of membrane permeabilization of tBID-triggered BAXMΔC as compared to tBID-triggered BAXM (E) and of BAXOΔC as compared to BAXO (F), 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. BAXM, BAXMΔC, 500 nM; BAXO, BAXOΔC, 500 nM; tBID, 20 nM.

(G) Negative stain EM of liposomes incubated with BAXOΔC for 15 min. Black-squared region enlarged below.

(H) Comparative dose-responsive cytochrome c release upon treatment of BAX/BAK-deficient MLM with BAXOΔC or BAXO. Error bars are mean ± s.e.m. for experiments performed in technical triplicate, with data representative of two independent experiments. BAXM and BAXMΔC, 200 nM; BAXO and BAXOΔC, 6.25-200 nM.

See also Figure S6.