FIGURE 1: Effect of Bcl-xL on the localization and function of a Bax phosphomimetic mutant.

Mitochondria (mit.) and post-mitochondrial supernatant (S30) were isolated from strains co-expressing Bax-S184D and wild-type Bcl-xL (WT), Bax interaction-deficient mutant carrying a triple mutation G138E/R139L/I140N (GRI/ELN), or a cytosolic mutant carrying a single mutation A221R (see Fig. 2).

(A) Western-blot analysis of Bax and Bcl-xL localization in both fractions.

(B) Quantification of the ratio between mitochondrial and non-mitochondrial Bax. All the experiments were done in parallel to a control without Bcl-xL, for which the ratio was adjusted to 1, as a reference.

(C) Mitochondrial cytochrome c content, measured as the ratio cytochrome c/cytochrome b (unreleased cytochrome b serves as an internal control to the experiment). Each point represents an individual experiment. Data with wild-type Bax are given for a matter of comparison, and had been published previously [22,24,34], except for the results with wild-type Bax and Bcl-xL-A221R. Red bars indicates the averages. p values were calculated with an unpaired Student’s test.

22. Simonyan L, Renault TT, da Costa Novais MJ, Sousa MJ, Côrte-Real M, Camougrand N, Gonzalez C, and Manon S (2016). Regulation of Bax/mitochondria interaction by AKT. FEBS Lett 590(1):13-21. https://doi.org/10.1002/1873-3468.12030

24. Renault TT, Teijido O, Missire F, Ganesan YT, Velours G, Arokium H, Beaumatin F, Llanos R, Athané A, Camougrand N, Priault M, Antonsson B, Dejean LM, and Manon S (2015). Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737. Int J Biochem Cell Biol 64:136-146. https://doi.org/10.1016/j.biocel.2015.03.020

34. Arokium H, Camougrand N, Vallette FM, Manon S (2004). Studies of the interaction of substituted mutants of BAX with yeast mitochondria reveal that the C-terminal hydrophobic alpha-helix is a second ART sequence and plays a role in the interaction with anti-apoptotic BCL-xL. J Biol Chem 279(50):52566-73. https://doi.org/10.1074/jbc.M408373200