Fig. 2.

VDAC2 promotes the association of BAX and BAK with a VDAC complex. a Endogenous BAX and BAK associate with independent complexes in mitochondria. Mitochondria-enriched fractions from HeLa or HCT116 cells were solubilized in 1% digitonin prior to incubation with a control antibody or an antibody that binds inactive human BAK (7D10), prior to BN-PAGE and immunoblotting for BAK or BAX. * likely cross-reactivity of anti-rat secondary antibody with rat IgG used for gel-shift. Importantly, whilst all of the BAK:VDAC2 complex was gel-shifted by the BAK antibody, the BAX:VDAC2 complex was unaffected. b Mass spectrometry analysis of the native BAX complex. Mitochondria from MEFs expressing FLAG-BAX^S184L or untagged BAX^S184L were solubilized in 1% digitonin prior to anti-FLAG affinity purification and proteins identified by quantitative mass spectrometry analysis. Volcano plot illustrating the log₂ protein ratios of proteins enriched in the native complex following quantitative pipeline analysis. Proteins were deemed differentially regulated if the log₂ fold change in protein expression was greater than two-fold (red) or four-fold (green) and a –log₁₀ p value ≥ 1.3, equivalent to a p value ≤ 0.05. c Mass spectrometry of the native BAK complex. Mitochondria from MEFs expressing FLAG-BAK or untagged BAK harvested and analyzed as in (b). d Deletion of VDAC2 impacts mitochondrial localization of BAX and BAK. Clonal populations of Bax^−/− and Bak^−/− MEFs with deleted Vdac1, Vdac2 or Vdac3 (denoted ∆) were fractionated into cytosol and membrane and immunoblotted for BAX, BAK or TIM44 as a mitochondrial control. e VDAC2 plays the major role in BAX and BAK complex stability. Mitochondria isolated from clonal populations of Bax^−/− and Bak^−/− MEFs with deleted Vdac1, Vdac2 or Vdac3 were analyzed on BN-PAGE. Data are representative of two independent clones (see Supplementary Fig. 2e). Intermediate complexes indicated (arrows). f BAX-mediated apoptosis is impaired in the absence of VDAC2 and to a lesser extent by VDAC3. Polyclonal populations were treated with etoposide (10 μM) and cell death was assessed by PI uptake. Data are mean+/− SEM of three independent experiments. ***p < 0.001; **p < 0.01; n.s, not significant; based on unpaired Student’s t-test