A unified model of mammalian BCL-2 protein family interactions at the mitochondria

Abstract

During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two "modes" whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins ("MODE 1") or by binding active BAX and BAK ("MODE 2"). Notably, we found that MODE 1 sequestration is less efficient and more easily derepressed to promote MOMP than MODE 2. Further, MODE 2 sequestration prevents mitochondrial fusion. We provide a unified model of BCL-2 family function that helps to explain otherwise paradoxical observations relating to MOMP, apoptosis, and mitochondrial dynamics.

Figure 1. tBID^BAX-BH3 and tBID^BAK-BH3 directly activate BAX and BAK

(A) Sequence alignment of the BH3 domains used to generate the tBID^BH3 chimeras. (B) HeLa cells were co-transfected with mitochondrial matrix targeted dsRed (mito-dsRed) and the indicated GFP-tBID^BH3 chimera in presence of Q-VD-OPH (20 μM) and imaged after 24 hr by live-cell confocal microscopy. Representative micrographs are shown. (C) HeLa cells were transfected with the indicated tBID^BH3 chimeras and apoptotic cell death was quantified after 24 hr by Annexin V staining and flow cytometry analysis. (D) HeLa cells stably expressing Omi-mCherry were transfected with tBID^BH3 chimeras and scored for mitochondrial versus cytosolic Omi-mCherry localization after 24 hr. (E) Mitochondria-enriched heavy membrane (MHM) fractions extracted from bak^−/− C57BL/6 mouse liver were incubated with 10 nM purified recombinant BAX and IVTT (in vitro transcription and translation) from wheat germ extract expressing the indicated tBID^BH3 chimera for 1 hr at 37°C. Supernatant (S) and pellet (P) fractions were then separated by centrifugation and cytochrome c release was monitored by Western blot. (F) MHM extracted from WT C57BL/6 mouse liver were treated as in (E) in the absence of recombinant BAX. (G) Large Unilamelar Vesicles (LUV) containing Fuorescein-Dextran were incubated with IVTT of the indicated tBID^BH3 chimera in presence of 120 nM purified recombinant BAX. LUV permeabilization is shown as % release of Fluorescein-Dextran. (H) LUV were incubated as in (G) in presence of 500 nM purified recombinant BAK-ΔC (lacking the C-terminus). Error bars represent standard deviation from triplicate data. (See also Figure S1)

Figure 2. Anti-apoptotic BCL-2 proteins inhibit MOMP by sequestering BH3-only and effector proteins

(A/B) HEK-293T cells were transiently transfected with plasmid encoding HA-tagged BCL-2, BCL-xL and MCL-1 together with FLAG-tagged BIM-s, BAX, BAK (A) or the indicated FLAG-tBID^BH3 chimera (B). Cells were lysed in 0.5% NP40 and protein complexes were immunoprecipitated with an anti-FLAG antibody. Protein interactions were analyzed by Western blot using anti-HA and anti-FLAG antibodies (Lys: total cell lysate, IP: immunoprecipitation). (C) Interaction profile of tBID^BH3 chimeras and effector proteins with anti-apoptotic BCL-2 proteins as experimentally determined by immunoprecipitation in (A) and (B). (D) Schematic representation of possible scenarios for MOMP inhibition by anti-apoptotic BCL-2 proteins. (E) MHM fractions extracted from bak^−/− C57BL/6 mouse liver were incubated for 1 hr at 37°C with IVTT of the indicated tBID^BH3 chimera in presence of 10 nM purified recombinant BAX and 1 μM purified recombinant BCL-2-ΔC, BCL-xL-ΔC or MCL-1-ΔC. Supernatant (S) and pellet (P) fractions were then separated by centrifugation and cytochrome c release was monitored by Western blot. The boxes’ colors correspond to the individual scenarios predicted in (D). (F) MHM extracted from WT C57BL/6 mouse liver were treated as in (D) in the absence of recombinant BAX.

Figure 3. BAK conformational change upon activation is detected by limited calpain digestion

(A) HEK-293T cells stably expressing FLAG-BAK were transiently transfected with plasmids encoding HA-BCL-2, HA-BCL-xL, HA-MCL-1 or empty vector. FLAG-BAK was immunoprecipitated from cell lysates with anti-FLAG-agarose in 2% CHAPS or 0.5% NP40 as indicated and incubated with calpain (20 nM) in presence of CaCl₂ (0.5 mM) for 30 min at room temperature. BAK cleavage was monitored by Western blot using anti-BAK-G23 antibody. Full length BAK (BAK FL) and two cleavage fragments (BH4 Cl and BH3 Cl) are indicated with arrows (B) Schematic representation of the position of the two calpain cleavage sites in active BAK. (C) MHM fractions extracted from C57BL/6 mouse liver were incubated for 1 hr at 37°C with IVTT from wheat germ extract expressing tBID, BIM-s, BAD or NOXA and left untreated (upper panel) or incubated with calpain (20 nM) in presence of CaCl₂ (0.5 mM) for 30 min at room temperature (middle panel). BAK cleavage was monitored by Western blot using anti-BAK-G23 antibody. Supernatants (S) and mitochondrial pellets (P) were also analyzed for cytochrome c by Western blot (lower panel). (D) MHM fractions from C57BL/6 mouse liver were incubated with IVTT of tBID in the presence or absence of 50 nM purified recombinant BCL-xL-ΔC (lacking the C-terminus) and treated as in (C) for calpain proteolysis (upper panel) or monitored for cytochrome c release (lower panel). (E) MHM fractions extracted from C57BL/6 mouse liver were incubated with IVTT of the indicated tBID^BH3 chimeras in presence of 1 μM purified recombinant BCL-2-ΔC, BCL-xL-ΔC or MCL-1-ΔC and treated as in (C) for calpain proteolysis. The boxes’ colors correspond to the scenarios predicted in Figure 2D. (See also Figure S2)

Figure 4. Inhibition of MOMP is less efficient in MODE 1 than in MODE 2

(A) MHM fractions from bak^−/− (upper panels) or wild type C57BL/6 mouse liver (lower panels) were incubated with IVTT from wheat germ extract expressing the indicated tBID^BH3 chimeras, 10 nM recombinant BAX (upper panels only) and increasing concentrations of recombinant BCL-2-ΔC or MCL-1-ΔC (0, 4, 12, 37,111, 333 and 1000 nM) for 1 hr at 37°C. Supernatant (S) and pellet (P) fractions were then separated by centrifugation and cytochrome c release was monitored by Western blot. (B) MHM fractions extracted from wild type C57BL/6 mouse liver were left untreated (right panel) or incubated with 1 μM recombinant BCL-2-ΔC or MCL-1-ΔC (left panel). When indicated, reactions were supplemented with IVTT of tBID^BAX-BH3 for 1h at 37°C. Reactions were then de-repressed with addition of BAD-BH3 or NOXA-BH3 peptide (5 or 50 μM) for 1 hr at 37°C and monitored for cytochrome c release as in (A). Lower panel shows a schematic representation of MODE 1 (red) versus MODE 2 de-repression (purple) used in this assay.

Figure 5. BCL-xL blocks MOMP sequentially through MODE 1 and MODE 2

(A) MHM fractions extracted from C57BL/6 mouse liver were incubated for 1 hr at 37°C with 100 nM full length recombinant BCL-xL and the indicated concentrations of cleaved recombinant BID (n/cBID). MHM were then incubated with calpain (20 nM) in presence of CaCl₂ (0.5 mM) for 1hr at room temperature. BAK cleavage was monitored by Western blot using anti-BAK-G23 antibody. Full-length BAK (BAK FL) and two cleavage fragments (BH4 Cl and BH3 Cl) are indicated with arrows. Supernatants (S) and mitochondrial pellets (P) were also analyzed for cytochrome c by Western blot. (B) HeLa cells stably over-expressing BCL-xL or an empty vector were transiently transfected with the indicated amount of tBID for 24 hr in presence of Q-VD-OPH (20 μM), permeabilized with digitonin (200 μg/ml) and incubated with calpain (20 nM) in presence of CaCl₂ (0.5 mM) for 30 min at room temperature. BAK cleavage was monitored by Western blot using anti-BAK-G23 antibody. Presence of cytochrome c was analyzed in pellet fractions (P) only as cytosolic components were lost during digitonin permeabilization. (C) HeLa cells stably over-expressing BCL-xL or an empty vector were exposed to increasing UV intensity (0, 2.5, 5, 10, 20 and 40 mJ/cm²) in presence of Q-VD-OPH (20 μM). After 16 hr, cells were treated as in (B) for calpain proteolysis (upper panel) or monitored for cytochrome c release (middle panel). Apoptosis was assessed on intact cells by Annexin V staining and flow cytometry analysis (lower panel). Error bars represent standard deviation from triplicate data. (D) MHM fractions from C57BL/6 mouse liver were incubated for 1 hr at 37°C with full-length recombinant BCL-xL (100 nM) and n/cBID (10 nM). Reactions were then de-repressed with the indicated concentrations of BAD-BH3 peptide or ABT-737 for 1 hr at 37°C and treated as in (C) for calpain proteolysis (upper panel) or monitored for cytochrome c release (lower panel). (See also Figure S3)

Figure 6. In the presence of BCL-xL, activated BAX translocates to the mitochondria without inducing MOMP

(A) HeLa cells stably expressing Venus-BAX and Omi-mCherry in the presence or absence of over-expressed BCL-xL, were exposed to 40 mJ/cm² UV plus Q-VD-OPH (20 nM) and imaged by live-cell confocal microscopy. Representative confocal micrographs are shown with the time of each frame relative to the time of stimulation. See also Movies S1 and S2. (B) To quantify BAX translocation in the absence of MOMP, co-localization between Venus-BAX and Omi-mCherry was measured by Pearson’s correlation coefficient. Heavy lines represent the average of 15 individual cells (light lines) for each condition. Measurements were stopped when individual cells underwent MOMP. (C) HeLa cells stably expressing Venus-BAX and Omi-mCherry in the presence or absence of over-expressed BCL-xL, were exposed to 0, 2.5, 5, 10, 20 and 40 mJ/cm² UV in the presence of Q-VD-OPH (20 nM) for 30 hr. Cells were permeabilized with digitonin (200 μg/ml) and analyzed by flow cytometry. Representative dot plots are presented. (D) The percentage of cells representing mCherry^high/Venus^low (cyto BAX), mCherry^high/Venus^high (mito BAX/no MOMP) and mCherry^low/Venus^high (mito BAX/MOMP) groups is shown for HeLa cells (upper graph) and HeLa-BCL-xL cells (lower graph). Error bars represent standard deviation from triplicate data. (E) HeLa cells stably over-expressing BCL-xL or empty vector were exposed to 0, 5 or 40 mJ/cm² UV in presence of Q-VD-OPH (20 nM). After 30 hr, MHM fractions were extracted from the cells and analyzed by Western blot for cytochome c (mitochondrial pellet) or subjected to carbonate extraction and analyzed by Western blot for BAX and BAK (inserted fraction). (See also Figure S4)

Figure 7. MODE 2 sequestration promotes mitochondrial fragmentation and affects the kinetics of MOMP upon de-repression

(A) HeLa cells stably expressing Cerulean-OMM (Cerulean protein targeted to the outer mitochondrial membrane by fusion to the C-terminal region of BCL-xL) and over-expressing BCL-xL were transfected with the indicated concentrations of mCherry-tBID plasmid for 30 hr. Representative confocal micrographs are shown. (B) Mitochondrial fragmentation was monitored by fluorescence recovery after photobleaching (FRAP) of the Cerulean-OMM. Each line of the graph is an average of 30 individual cells, error bars represent standard deviation. (C) HeLa cells expressing Omi-mCherry and over-expressing BCL-xL were transfected with GFP-tBID and treated after 30 hr with ABT-737 (20 μM) plus Q-VD-OPH (20 μM) and imaged every 10 min by live-cell confocal microscopy. Representative confocal micrographs are shown. The time of each frame relative to the time of ABT-737 addition is shown. (D) Cells were treated as in (C). GFP-tBID levels preceding ABT-737 treatment in individual cells were quantified by measuring the mean GFP signal intensity. Time of onset to MOMP was determined by monitoring Omi-mCherry release and was plotted as a function of GFP-tBID intensity. The graph represents the analysis of 300 individual cells from three independent experiments (E) Cells were treated as in (C); prior to ABT-737 treatment cells were scored for mitochondrial morphology (tubular vs. fragmented) and onset of MOMP was monitored as in (D). Error bars represent standard deviation from triplicate data. (See also Figure S5)