Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization

Abstract

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.

Keywords: Bax; anticancer drug; apoptosis; liposome; mitochondrial apoptosis; mitochondrial localization; mitochondrial outer membrane permeabilization (MOMP); molecular cell biology; protein oligomers; translocation.

Figure 1.

Two helix 9 mutants of BAX, T182I and G179P, display divergent activities in liposomes. A, BAXT182I is less active than WT in liposomes, whereas BAXG179P is almost as active as WT. Left panel, a representative release kinetics of 70-kDa fluorescein-dextran from liposomes permeabilized with recombinant BAX proteins mixed with the activator, cBID(45 nm). Proteins and liposomes were incubated at 37 °C and dextran release was monitored as quenching by anti-fluorescein antibodies. Right panel, summary of activities of the mutants compared with the WT BAX. ns, not significant; numbers, p values. B, BAXT182I releases small molecules slowly. Left panel, release kinetics for liposomes loaded with quenching concentrations of ANTS and DPX (both ∼0.5 kDa). BAX and cBID were incubated with the liposomes and permeabilization was measured as dequenching of ANTS and DPX resulting from dilution of the dyes once released from the vesicles. Right panel, summary of activities of the mutants compared with the WT BAX. p < 0.0001. C, NBD-labeled BAX species maintain the relative activities of nonlabeled counterparts. Cascade blue (CB)-dextrans (10 kDa) were used to avoid interference with the spectrally overlapping NBD dye. Relative activity of the mutant is shown in the right panel compared with WT BAX. BAXG179P shows no significant difference from WT BAX. BAXT182I did not reach 50% release in time, therefore its activity was considered very close to 0. D, BAXT182I is inefficient in membrane insertion. Membrane insertion was monitored using NBD-labeled BAX (WT versus G179P in the left panel; WT versus T182I in the right panel) with and without cBID (45 nm) on nonloaded liposomes. E, BAXG179P permeabilizes OMVs more slowly. BAX WT, BAXT182, or BAXG179P were incubated with OMVs at 120 nm together with cBID (45 nm). Vesicle permeabilization was monitored as quenching of fluorescein-dextran (70 kDa). The one sample and Wilcoxon test shows borderline significance with BAXG179P (p = 0.054). BAXT182I did not reach 50% release; its activity was considered very close to 0. All the data in Fig. 1 were taken from three to four independent experiments and error bars are the standard deviations.

Figure 2.

BAX helix 9 mutants form size-restricted oligomers in the membrane. A, size-exclusion chromatography reveals that BAXT182I and BAXG179P form much smaller complexes than WT. BAX species (2 μm each) and cBID (1.3 μm) were incubated with liposomes for 2 h at 37 °C. After the incubation period, the membrane fraction was separated in a sucrose gradient centrifugation step to remove unbound BAX protein. Membranes were dissolved in 1.2% CHAPS and fractionated in a Superdex 200 Increase (10/300) column. Fractions were probed with anti-BAX antibody. The data shown are a representative of three independent experiments. B, an in situ TIRF-based assay for measuring the stoichiometry of BAX complexes in SLBs. Confirmation of monomeric status of BAX species. Schematic representation of the protocol used for confirmation of monomeric status of the BAX species used in the study (upper panel) and the data showing the monomeric status of BAX (lower panels). LUVs (gray) were used to prepare SLBs, which were then incubated with 2.5 nm labeled BAX (light green) for 30 min or 1 h at room temperature, washed carefully with buffer, and immediately imaged by TIRF microcopy. Percentage of occurrence of fluorophore units per particle for BAX (S4C,C62S,C126S)-488 (a), BAX G179P (S4C,C62S,C126S)-488 (b), and BAX T182I (S4C,C62S,C126S)-488 (c) mutants directly added on SLBs calculated as the average value from two different experiments. Due to unspecific interaction of BAX molecules with the SLB glass support, BAX molecules are unable to oligomerize, resulting mainly in monomers and some dimers and trimers. Data provided are the raw values, where no correction for partial labeling was applied. The error bars correspond to the standard deviations from two different experiments. Note that BAX Thr-182 (S4C,C62S,C126S)-488 was already oligomeric in solution. C, schematic representation of the stoichiometry distribution assay on BAX (S4C,C62S,C126S)-488 and BAXG179P (S4C,C62S,C126S)-488. LUVs (gray) were incubated with 2.5 nm BAX (light green) and 5 nm cBID (not shown) at room temperature for 1 h to form proteoliposomes (LUVs containing BAX oligomers). After incubation, these liposomes were used to prepare SLBs with labeled BAX oligomers associated with them. The right panel shows a representative TIRF image of a SLB containing BAX oligomers (bright spots). Scale bar is 10 μm. D and F, representative intensity distribution of BAX-488 (D) and BAX G179P-488 (F) mutant particles (minimum 1600 particles per experiment) bound to SLBs prepared from proteoliposomes after 1 h of incubation with the protein. The obtained brightness distribution was plotted as a probability density function (Pdf, black) or, alternatively, as a histogram and fitted with a linear combination of Gaussians to estimate, from the area under each curve, the percentage of occurrence of particles containing n-mer–labeled molecules (see color code in the graph). The cumulative fit is shown by a dashed black line. E and G, TIRF analysis reveals that BAXG179P fails to form high-order oligomers in SLBs. Percentage of occurrence of different oligomeric species for BAX-488 (E) and BAX G179P-488 (G) mutant particles were calculated as the average value from four (E) and two (G) different experiments. Data provided are the raw values, where no correction for partial labeling (80% for BAX-488 and 50% for BAX G179P-488) was applied (see “Materials and methods”). The error bars correspond to the standard deviations from the different experiments.

Figure 3.

In cells, BAXT182I translocates strongly to mitochondria and, as a result, mediates MOMP and apoptosis as well as WT BAX; BAXG179P remains cytoplasmic and functions poorly. A, permeabilized cells incubated with recombinant BAX species. BAX/BAK DKO MEFs were permeabilized with digitonin and recombinant BAX proteins and cBID were added for 1 h at 30 °C. The supernatant and the pellet fractions were isolated and immunoblotted for cytochrome c, SMAC, or BAX. B, expression of BAX species in transduced cells. Mouse BAX WT, T182I, or G179P were transduced in BAX/BAK DKO MEFs using retroviral transduction with IRES-GFP. We sorted out cell populations with equivalent expression levels of GFP, and therefore of BAX species. Note that each mutant BAX migrated slightly differently from WT. Ponceau S stain was used to verify equivalent sample loading. C, transduced BAXT182I is recruited strongly to mitochondria in permeabilized cells incubated with cBID. BAXWT-, T182I-, or G179P-transduced DKO MEFs were permeabilized with digitonin and incubated with cBID for 1 h at 30 °C. The supernatant and the pellet fractions were immunoblotted for cytochrome c or anti-BAX antibodies. T182I-expressing cells had a higher level of BAX in the pellet than WT and released cytochrome c even more efficiently than WT. Basal BAXT182I levels in the pellet were higher than that of WT BAX and increased dramatically in the presence of cBID, showing that BAXT182I is recruited to mitochondria more efficiently than WT. D, BAXT182I mediates apoptosis effectively, but BAXG179P functions poorly. Staurosporine-induced cell death of transduced cells was measured by propidium iodide (PI) stain. Error bars are S.D. of triplicate samples. Representatives of 2–4 independent experiments are shown.

Figure 4.

BAXT182I constitutively localizes to mitochondria, whereas G179P is completely cytosolic. A, BAXT182I accumulates on mitochondria to a greater extent than WT, whereas BAXG179P fails to translocate to mitochondria. GFP-BAX was transiently expressed in BAX/BAX DKO MEFs and imaged by epifluorescence microscopy. GFP-BAX was stained with anti-GFP antibody. Mitochondria were stained with mitochondrial HSP70 (mHSP70) and the nuclei, with DAPI. Mitochondria were fragmented as previously reported in these cells (48). WT BAX distributed both at mitochondria and cytoplasm, whereas the localization of BAXT182I was more pronounced in mitochondria. BAXG179P was diffuse cytoplasmic. Scale bar: 10 μm. B, microscopic detection of activated BAX species in apoptotic cells. BAX/BAK DKO MEFs transduced with BAX-IRES-GFP (WT, T182I, or G179P) were treated with staurosporine in the presence of caspase inhibitor Q-VD, then fixed and stained with antibodies 6A7 (for the activated form of BAX) and anti-SMAC. The diffuse green GFP fluorescence served as a marker for BAX co-expression. WT and BAXT182I showed characteristic staining of punctate BAX in apoptotic cells, whereas BAXG179P-expressing cells were not stained with 6A7. Red SMAC staining was only seen in cells with no BAX puncta, i.e. nonapoptotic cells. Note the morphology of the cells regardless of the species of BAX expression was altered due to staurosporin treatment. Scale bar, 10 μm.

Figure 5.

BAXT182I retrotranslocates substantially more slowly than WT BAX. A, monitoring of photobleached cells over time. Cytoplasmic portions of cells expressing GFP-BAXWT, BAXS184V, and BAXT182I were photobleached in the yellow-lined area and fluorescence was monitored in the green region of interest. The red line indicates the outline of the imaged cell. BAXS184V is constitutively expressed in mitochondria and was used here as a control for slow retrotranslocation. Scale bars: 10 μm. B and C, BAXT182I retrotranslocates slowly. Fluorescence data from A was normalized to 100% fluorescence pre-bleach and the average decrease in fluorescence over time plotted (B). Data represents values from three independent experiments and error bars represent standard deviation. Data from B was fitted to a one-phase dissociation curve and the average fluorescence recovery calculated. Data were analyzed via ANOVA, *, p < 0.05. Error bars represent standard deviation (C). D, total fluorescence does not decrease in unbleached cells. The total cell fluorescence of unbleached cells within the same region of interest as cells analyzed in A was measured and the average total fluorescence plotted over time.

Figure 6.

Permeabilized cell system shows BAX retrotranslocation. BAX/BAK DKO MEFs transduced with WT-BAX or BAXT182I were digitonin-permeabilized and incubated at room temperature over time. Supernatant and pellet were separated at each time point and BAX and other proteins were probed by immunoblot. VDAC1 was shown as loading controls for the pellet. A portion of a cytosolic protein, HSP70, still remained in the permeabilized cells, but the level of HSP70 in the soluble fraction was unchanged over time unlike WT BAX. This controls for the genuine dissociation of BAX from the pellet fraction that contains mitochondria. On the other hand, BAXT182I remained in the pellet, and presumably stayed associated with mitochondria, due to slow retrotranslocation.