Volume 16, Issue 5, Pages 807-818 (December 2004) The First α Helix of Bax Plays a Necessary Role in Its Ligand-Induced Activation by the BH3-Only Proteins Bid and PUMA  Pierre-François Cartron, Tristan Gallenne, Gwenola Bougras, Fabien Gautier, Florence Manero, Patricia Vusio, Khaled Meflah, François M. Vallette, Philippe Juin  Molecular Cell  Volume 16, Issue 5, Pages 807-818 (December 2004) DOI: 10.1016/j.molcel.2004.10.028

Figure 1 Role of Bax Hα1 and Bid BH3 in the Interaction of Bax with Bid (A) Interaction of Bid mutants with Baxα and Bax Hα1 in cell-free assays. Experiments were performed as described in Table 2, using IVTR Baxα or Hα1-RFP and the granzyme B cleavage product of the indicated His-tagged Bid variant (wt, wild-type). Data are mean (±SEM) of three independent experiments. (B) Interaction between Bid and Bax mutants in glioblastoma cells. Bax-deficient human glioblastoma (BdGBM) cells stably expressing the indicated Bax variant were transiently transfected with a plasmid encoding for His-tagged p13-Bid. Cellular extracts were immunoprecipitated (IP) with the 2D2 anti-Bax antibody, the immunoprecipitated fractions were separated on SDS-PAGE, and immunoblots (WB) were analyzed for the presence of Bax (detected with the 2D2 antibody) and for the presence of p13-Bid (detected with the anti-His antibody). Data illustrated are representative of three independent experiments. (C) Overlay sensograms for surface plasmon resonance analysis of BH3Bid peptides binding to Hα1Bax peptides. Avidin bound biotin, biotinylated BH3Bid, or BH3Bid R84G was examined for real-time binding to immobilized Hα1Bax (left) or Hα1Bax D33A (right) as described in Experimental Procedures. Molecular Cell 2004 16, 807-818DOI: (10.1016/j.molcel.2004.10.028)

Figure 2 Role of Bax Hα1 in the Effect of BH3-Only Proteins and BH3 Peptides on Bax Conformation and Mitochondrial Activity (A) Cell-free immunoprecipitation assays with a conformation-specific anti-Bax antibody. The indicated IVTR Bax variant (4 fmol) was incubated with p13-Bid, PUMA (0.5 nM), BH3Bid, or BH3PUMA (200 nM) in the absence or in the presence of Hα1Bax or BH4Bcl-xL (200 nM) prior to immunoprecipitation with the indicated antibodies as described in Experimental Procedures. Untreated or Tx100 (0.5%)-treated IVTR Baxα were used as a negative or positive control, respectively, for immunoprecipitation with the 6A7 antibody. (B and C) Effect of Bid, PUMA, and BH3 peptides on the interaction of Bax variants with isolated mitochondria. (B) Mitochondrial targeting. 4 fmol of the indicated 35S-Bax variant were incubated with mitochondria in the absence (unt.) or in the presence of p13-Bid, PUMA (0.5 nM), BH3Bid, BH3PUMA, or BH3Bad (200 nM). Where indicated, 35S-Bax was preincubated with an equimolar amount of recombinant Bcl-xL or Bcl-2 prior to the addition of BH3Bad (200 nM). The amount of mitochondrial bound (top), and of alkaline-resistant mitochondrial bound 35S-Bax (bottom) is shown for each condition. Data are mean (±SEM) of at least three independent experiments. (C) Cytochrome c release. Experiments were performed as described in (B). The amount of cytochrome c in the mitochondrial supernatant was analyzed by Western blotting. The initial amount of plain mitochondria (Mit.), and the supernatant thereof (S), were also loaded. Molecular Cell 2004 16, 807-818DOI: (10.1016/j.molcel.2004.10.028)

Figure 3 Role of Bax Hα1 in the Induction of Bax Apoptotic Activity by BH3-Only Proteins and BH3 Peptides (A) Influence of Bax and Bak expression on the sensitivity of human glioblastoma cells to apoptosis induced by BH3-only proteins. BeGBM, BdGBM, BeGBM (Bak AS), and BdGBM (BakAS) cells (Bax+/Bak+, Bax−/Bak+, Bax+/Bak−, and Bax−/Bak− GBM cells, respectively) were transiently transfected with empty plasmid (−) or with plasmids encoding for the indicated BH3-only protein (5 or 10 μg, as indicated). The activation of DEVDase following transfection of the indicated cells with plasmids encoding for the indicated BH3-only protein was assayed as described in Experimental Procedures. Data are mean (±SEM) of at least three independent experiments. (B) Induction of apoptosis by BH3-only proteins in human glioblastoma cells expressing Bax mutants. BeGBM, BdGBM, or BdGBM cells stably expressing the indicated Bax variant were transiently transfected with an empty vector (cont) or with plasmids encoding for the indicated BH3-only protein (5 μg). The activation of DEVDase (top), and the loss of cell viability (bottom) following transfection were assayed as described in Experimental Procedures. Data are mean (±SEM) of at least three independent experiments. (C) Immunocytochemical analysis. The staining of BdGBM cells expressing the indicated Bax variant and transiently transfected with empty vector (Cont) or with plasmids encoding for the indicated BH3-only protein, as described in (B), was performed as described in Experimental Procedures. Mitochondria (Mito.) were detected with MitoTracker Green (green), and Bax and cytochrome c (Cyto c) were immunodetected using Alexa 568- and 633-conjugated secondary antibodies (red and blue, respectively). Overlays of the green and red (Mito + Bax) and of the green and blue (Mito + Cyto c) images are also shown. (D) Induction of apoptosis by BH3 peptides in human glioblastoma cells. The indicated cells were microinjected with the indicated peptides (1 μM), and cell death was assayed at the indicated time following injection. Data are mean (±SEM) of three independent experiments. Molecular Cell 2004 16, 807-818DOI: (10.1016/j.molcel.2004.10.028)

Figure 4 Functional Interaction between BidR84D and BaxD33R (A) Interaction of BidR84D with BaxD33R in cell-free assays. Experiments were performed as described in Table 2, using the indicated radiolabeled Bax variant and His-tagged p13-Bid variants. 1 fmol (i25%) 35S-Bax was loaded besides 35S-Bax that dimerized with His-tagged proteins (Dim). (B) Interaction of BidR84D with BaxD33R in glioblastoma cells. BdGBM cells expressing the indicated Bax variant were transiently transfected with a plasmid encoding for the indicated His-tagged p13-Bid. Cellular extracts were immunoprecipitated (IP) with the anti-His antibody and the presence of Bax (detected with the 2D2 antibody) and p13-Bid (detected with the anti-His antibody) in the immunoprecipitated fractions were detected by immunoblotting (WB) as described in Figure 1B. Data illustrated are representative of three independent experiments. (C) Effect of BidR84D on BaxD33R conformation in a cell-free system. Experiments were performed as described in Figure 2A, using the indicated 35S-Bax variants and the indicated His-tagged p13-Bid variants. Untreated IVTR Bax (Unt.) or Tx100 (0.5%)-treated IVTR Baxα (T) were used as a negative or positive control, respectively, for immunoprecipitation with the 6A7 antibody. (D and E) Effect of BidR84D on the interaction of BaxD33R with isolated mitochondria. The effect of the indicated Bid variants on the association of the indicated 35S-Bax with mitochondria (D) and on the ability of the indicated 35S-Bax to induce cytochrome c release (E) were analyzed as described in Figures 2B and 2C respectively. Molecular Cell 2004 16, 807-818DOI: (10.1016/j.molcel.2004.10.028)

Figure 5 Induction of BaxD33R Apoptotic Activity by BidR84D (A) Induction of apoptosis by p13-BidR84D in BaxD33R-expressing cells. Experiments were performed as described in Figure 3B using BdGBM cells expressing the indicated Bax variant transiently transfected with plasmids encoding for the indicated Bid variant. Data are mean (±SEM) of three independent experiments. (B) Immunocytochemical analysis. Experiments were performed as described in Figure 3C. Mitochondria were detected with MitoTracker Green (green), and Bax was immunodetected using the indicated primary antibody and Alexa 568-conjugated secondary antibody (red). An overlay of the green and red images is shown. (C) Induction of BaxD33R apoptotic activity by BidR84D in the absence of Bak. BdGBM (Bak AS) GBM cells (Bax−/Bak− cells) were first transfected with plasmids encoding for the indicated Bax variant (5 μg), and the resulting cells, together with untreated Bax−/Bak− GBM cells, were transfected with an empty plasmid (−) or with plasmids encoding for the indicated p13-Bid variant (5 μg). The activation of DEVDase induced in the indicated cells by the indicated p13-Bid variant was assayed as described in Figure 3A. Data are mean (±SEM) of at least three independent experiments. Molecular Cell 2004 16, 807-818DOI: (10.1016/j.molcel.2004.10.028)