Uren RT , Dewson G , Chen L , Coyne SC , Huang DC , Adams JM , Kluck RM .

CA80188 NCI NIH HHS , Wellcome Trust , R01 CA080188 NCI NIH HHS

	Figure 1. Specificity of BH3 peptide–induced cytochrome c release. (A) Prosurvival proteins are proposed to block mitochondrial permeability by sequestering BH3-only proteins (direct activation model) or primarily Bax/Bak proteins (indirect activation model). (B) Previously identified specific interactions between members of each Bcl-2 subfamily (Fig. S1 B, available at http://www.jcb.org/cgi/content/full/jcb.200606065/DC1; Chen et al., 2005). (C) Selective permeabilization by BH3 peptides. Mitochondria isolated from mouse liver or from Xenopus eggs were incubated for 2 h at 37 (mouse liver) or 22°C (Xenopus) with peptides derived from the BH3 domains of BH3-only proteins. Equivalent volumes of supernatant (S/N) and mitochondrial pellet (Mito) were then analyzed for cytochrome c by Western blotting. The sequence of each peptide is from the human protein sequence (Fig. S1 A) except for Bmf (from mouse) and Bad (human in top panel and mouse in bottom panel). BH3 peptides derived from mouse and human Bad were equivalent in permeabilizing and binding activity (not depicted) and were used interchangeably. (D) Bcl-xl and traces of Mcl-1 are present in MLM. Bcl-xl and Mcl-1 levels in MLM and mouse embryonic fibroblasts (MEFs) were compared by Western blotting. 0.7 pmol of recombinant Bcl-xL and 0.25 pmol Mcl-1 were included where indicated. Positions of molecular weight markers are indicated. The asterisk denotes a band in MLM that aligns with the bottom band of the Mcl-1 doublet in the mouse embryonic fibroblast. Reprobing the blots for cytochrome c (bottom) provides a comparison of the mitochondrial content of each sample. (E) Mutant Bim peptides lose permeabilizing activity. Mouse BimBH3 peptides with mutations at one (L94A) or four (4E; I89E, L94E, I97E, and F101E) conserved residues were compared with wild-type (wt) peptide for the ability to permeabilize XEM as in C (residue number refers to the mBimL protein). Equivalent results were found with MLM (not depicted). (F) Mutated Noxa peptides with enhanced binding to Bcl-xL gain permeabilizing activity. NoxaBH3 peptides containing substitutions of charge (K35E), hydrophobicity (F32I), or both (K35E and F32I; Fig. S1; Chen et al., 2005) were compared with wild-type peptide for the ability to permeabilize mitochondria as in C.
	Figure 2. Permeabilization of MLM is temperature dependent. MLM were incubated with the indicated BH3 peptides (left) or tBid (right) for 2 h at different temperatures as indicated. Cytochrome c release was assessed by Western blotting as in Fig. 1 C. S/N, supernatant; Mito, mitochondrial pellet.
	Figure 3. Four prosurvival proteins block tBid-induced permeabilization of isolated mitochondria. (A) Mitochondria from XEM or MLM were preincubated with recombinant prosurvival proteins at the indicated concentrations and supplemented with tBid for a further 2 h. Supernatant and pellets were examined by Western blotting for cytochrome c (in the MLM experiment, only half of the supernatant samples were loaded compared with the pellets). (B) C-terminal truncation of hBcl-xL decreases potency. MLM were incubated as in A with full-length hBcl-xL, a C-terminally truncated form (hBcl-xLΔC24), or with Xenopus Bcl-xLΔC23. (C) High concentrations of Bcl-w can complement Bcl-xL–mediated protection. MLM were incubated as in A with Bcl-wΔC10, Bcl-xL, or both before exposure to tBid at the indicated concentrations. S/N, supernatant; Mito, mitochondrial pellet.
	Figure 4. Mcl-1 and BHRF1 can even block the action of those BH3 peptides that they cannot bind. (A) Permeability of XEM induced by BadBH3 is efficiently blocked by prosurvival proteins that cannot bind Bad. XEM were preincubated for 20 min with the indicated concentrations of Mcl-1ΔC11 or BHRF1ΔC16 and were supplemented with mBadBH3 peptide for a further 2 h. Supernatant and pellets were then examined for cytochrome c by Western blotting. Similar experiments involving Mcl-1 and BHRF1-mediated protection from hBad, Bmf, Hrk, Bim, and Puma BH3 peptides are shown in Fig. S4 (available at http://www.jcb.org/cgi/content/full/jcb.200606065/DC1. (B) Permeability of MLM induced by mBad, Bmf, and Hrk BH3 peptides is efficiently blocked by Mcl-1 and BHRF1. MLM were incubated as in A with the indicated combinations of prosurvival proteins and BH3 peptides. (C) Model of how MLM are permeabilized by Bad (top) and protected by Mcl-1 (bottom). For simplicity, all of the Bak in the healthy cell is depicted as bound to Bcl-xL, but the indirect activation model postulates that only a proportion of the Bak molecules is in that form. (D) Permeabilization of mitochondria lacking both Bim and Bid by the sensitizer BH3 peptides Bad and Bmf. In an experiment like that in B, the absence of both of these putative activators does not prevent cytochrome c release or affect the protection conveyed by Mcl-1 and BHRF1, which do not bind Bad or Bmf. S/N, supernatant; Mito, mitochondrial pellet; Wt, wild type.
	Figure 5. Specific binding of BH3 peptides to prosurvival proteins results in mitochondrial permeability. (A and B) Mcl-1– or Bcl-xL–mediated protection can be overcome only by BH3 peptides that bind that prosurvival protein. XEM (A) or MLM (B) were preincubated with either no addition or with Mcl-1ΔC11 or Bcl-xL and were supplemented with tBid together with Bad (human in A and mouse in B), Noxa, Bim, or Puma peptides as indicated for a further 2 h. (C) Specific binding of BH3 peptides to BHRF1 and Bcl-w results in mitochondrial permeability. XEM were preincubated with no addition, BHRF1ΔC16, or Bcl-wΔC10 and supplemented with tBid together with hBad, Noxa, or Puma peptides as indicated for a further 2 h. Supernatant and pellets were examined by Western blotting for cytochrome c. S/N, supernatant; Mito, mitochondrial pellet.
	Figure 6. Complementation between Bad and Noxa BH3 peptides is needed to permeabilize mitochondria protected by both Mcl-1 and Bcl-xL–like prosurvival proteins. XEM (which functional experiments indicate contain predominantly Bcl-xL–like prosurvival proteins) were preincubated with 300 nM Mcl-1ΔC11 to produce dual-protected mitochondria. Noxa and mBadBH3 peptides were then added alone or in combination as indicated. Supernatant and pellets were examined by Western blotting for cytochrome c. Similar results were obtained with MLM (not depicted). Note that unlike in Fig. 5, tBid is not included. S/N, supernatant; Mito, mitochondrial pellet.
	Figure 7. Specific binding of BH3 ligands to Mcl-1 and Bcl-xL disrupts their binding of Bak. (A) The ability of BH3 peptides to permeabilize MLM corresponds with their disruption of Bak binding to Mcl-1 or Bcl-xL. MLM were treated as in Fig. 5 B, and aliquots of the supernatant and pellet fractions were analyzed by Western blotting for cytochrome c and Bak (top three panels). The remaining mitochondrial pellet fractions underwent immunoprecipitation for Mcl-1 or Bcl-xL before Western blotting for Bak, Mcl-1, and Bcl-xL (bottom three panels). The Noxa BH3 peptide used was K35E. (B) Bad and Noxa expressed in HeLa cells compete with Bak for association with Mcl-1 and Bcl-xL. Mcl-1 or Bcl-xL was immunoprecipitated from HeLa cells stably expressing HA-tagged Bad or HA-tagged Noxa. Immunoprecipitates and cell lysates were examined by Western blotting for Bak, Mcl-1, Bcl-xL, and HA. The asterisk marks the IgG light chain, which runs just above Bcl-xL. IP, immunoprecipitation; S/N, supernatant; Mito, mitochondrial pellet.

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