Bcl-XL inhibits membrane permeabilization by competing with Bax

Abstract

Although Bcl-XL and Bax are structurally similar, activated Bax forms large oligomers that permeabilize the outer mitochondrial membrane, thereby committing cells to apoptosis, whereas Bcl-XL inhibits this process. Two different models of Bcl-XL function have been proposed. In one, Bcl-XL binds to an activator, thereby preventing Bax activation. In the other, Bcl-XL binds directly to activated Bax. It has been difficult to sort out which interaction is important in cells, as all three proteins are present simultaneously. We examined the mechanism of Bax activation by tBid and its inhibition by Bcl-XL using full-length recombinant proteins and measuring permeabilization of liposomes and mitochondria in vitro. Our results demonstrate that Bcl-XL and Bax are functionally similar. Neither protein bound to membranes alone. However, the addition of tBid recruited molar excesses of either protein to membranes, indicating that tBid activates both pro- and antiapoptotic members of the Bcl-2 family. Bcl-XL competes with Bax for the activation of soluble, monomeric Bax through interaction with membranes, tBid, or t-Bid-activated Bax, thereby inhibiting Bax binding to membranes, oligomerization, and membrane permeabilization. Experiments in which individual interactions were abolished by mutagenesis indicate that both Bcl-XL-tBid and Bcl-XL-Bax binding contribute to the antiapoptotic function of Bcl-XL. By out-competing Bax for the interactions leading to membrane permeabilization, Bcl-XL ties up both tBid and Bax in nonproductive interactions and inhibits Bax binding to membranes. We propose that because Bcl-XL does not oligomerize it functions like a dominant-negative Bax in the membrane permeabilization process.

Figure 1. Bax and tBid Cooperate To Induce Liposome Permeabilization, Which Is Inhibited by Bcl-XL

(A) Liposomes encapsulated with ANTS and DPX were incubated with 100 nM Bax, 20 nM tBid, or both (left panel) or with 100 nM Bax, 20 nM tBid, and the indicated concentrations of Bcl-XL (right panel). Membrane permeabilization was assayed by an increase of ANTS fluorescence. (B) Liposome binding of tBid, Bax, and Bcl-XL. The proteins, at the indicated concentrations, were incubated with liposomes. Membrane-bound proteins were separated from soluble proteins by Sepharose CL-2B gel filtration chromatography. Individual fractions were analyzed by immunoblotting (IB) using Bid, Bax, or Bcl-XL antibodies, as indicated. (C) Mitochondria from bak knockout mice were incubated with tBid (20 nM), Bax (100 nM), and Bcl-XL (100 nM), as indicated. Permeabilization was assayed by pelleting the mitochondria and analyzing both the pellet (P) and the supernatant (S) fractions by immunoblotting using an α-cytochrome c antibody. (D and E) Mitochondria were incubated with the indicated proteins (concentrations as in (C)) and the levels of (D) Bax or (E) Bcl-XL assayed in the mitochondrial pellet. Integration of Bax or Bcl-XL into mitochondrial membranes was assayed by carbonate extraction, using Hsp60 (a soluble matrix protein) as a control.

Figure 3. Bcl-XL Binds Both tBid and Bax To Prevent Membrane Permeabilization

(A) Liposomes encapsulated with ANTS and DPX were incubated with 100 nM Bax, 20 nM tBid or tBid-mt1, and increasing concentrations of Bcl-XL, Bcl-XL Y101K, or Bcl-XL ΔBH4. Membrane permeabilization was assayed as in Figure 1A and is presented as percentage of ANTS/DPX release mean ± standard deviation for at least three independent experiments. (B) Mitochondria isolated from bak knockout mouse livers (bak –/– MLM) were incubated with 200 nM Bax, 250 pM tBid or tBid-mt1, and increasing concentrations of Bcl-XL or Bcl-XL Y101K, as indicated. Permeabilization was assayed by pelleting the mitochondria and analyzing both the pellet (P) and the supernatant (S) fractions by immunoblotting using an α-cytochrome c antibody. (C) Results from experiments as in (B) quantified as percentage of cytochrome c release mean ± standard deviation for at least three independent experiments. (D) Mitochondria isolated from wild-type mouse livers (wt MLM) were assayed as in (B) except that the concentration of tBid was reduced 10-fold to 25 pM. Permeabilization was quantified as in (C).

Figure 4. Bcl-XL Binding to Membranes and Bcl-XL-Mediated Inhibition of Bax Membrane Binding Are Mediated by Interactions with tBid and Bax

(A) Quantification of Bax binding to membranes. Bax (100 nM) and tBid (20 nM) or tBid-mt1 were incubated with liposomes in the presence of increasing concentrations of Bcl-XL, Bcl-XL Y101K, or Bcl-XL ΔBH4. Membrane-bound protein was separated from soluble protein by Sepharose CL-2B gel filtration chromatography and quantified by immunoblotting. Results are presented as mean ± standard deviation for at least three independent experiments. (B) Quantification of Bcl-XL and Bcl-XL mutant protein binding to membranes. Bcl-XL, Bcl-XL Y101K, or Bcl-XL ΔBH4 (100 nM) was incubated with the indicated proteins and analyzed as above. Results are presented as mean ± standard deviation for at least three independent experiments. Black bars indicate experiments directly comparable between (A) and (B).

Figure 2. Bcl-XL Binds to Membrane-Bound tBid and Bax

(A) Bax (100 nM) and/or tBid (20 nm) were incubated with 100 nM Bcl-XL (left panel) or 20 nM Bcl-XL (right panels) and liposomes. Samples were immunoprecipitated (IP) in either 2% CHAPS or 0.2% NP-40, as indicated, using an antibody with the indicated specificity and immunoblotted (IB) for the indicated protein. (B–D) Mutations prevent the binding of Bcl-XL to tBid, Bax, or both. (B and D) Bcl-XL (20 nM), or the indicated Bcl-XL mutants, (C) Bcl-XL Y101K or (D) Bcl-XL ΔBH4, were incubated with (C and D) 20 nM tBid or (B) tBid-mt1 with or without Bax (100 nM) and with liposomes. Immunoprecipitations and immunoblotting were performed as in (A).

Figure 6. Bcl-XL Functions Like a Dominant-Negative Bax

(A) Cytoplasmic Bax undergoes a conformational change after interacting with membranes (step 1). Interaction of this peripheral membrane (indicated by the shadow) Bax with membrane-bound tBid causes a further conformational change such that Bax integrates in the membrane in an oligomerization competent form (step 2). Conversely, cytoplasmic Bax may interact with other activator proteins to integrate into membranes, or spontaneously active Bax molecules may integrate into the membrane without binding an activator protein. A single tBid molecule activates multiple peripheral membrane Bax molecules, and/or the activated integral membrane Bax recruits more cytoplasmic Bax to the membrane (autoactivation, step 3). Bax oligomerizes. (B) Bcl-XL exists in a cytoplasmic and/or a peripheral membrane-bound form (step 1). Membrane-bound tBid triggers membrane binding and activation of Bcl-XL (step 2). One tBid molecule can mediate the membrane binding and activation of multiple Bcl-XL molecules (step 3). Bcl-XL does not oligomerize. (C) Membrane-bound Bcl-XL sequesters tBid and thereby prevents the activation of Bax (step 4). Bcl-XL binds to membrane-bound Bax, preventing Bax oligomerization (step 5) and the recruitment of further peripheral Bax by autoactivation (step 6). In steps 4–6, Bcl-XL functions as a dominant-negative Bax. (D) Bcl-XL inhibits the conformational change of Bax (step 7, indicated as a change in equilibrium) that is elicited by peripheral membrane binding (step 1 in (A)).

Figure 5. Membrane-Bound Bcl-XL Inhibits the Liposome-Induced Conformational Change in Bax

(A) Bax (100 nM) was incubated in the presence of liposomes and in the absence or presence of tBid (20 nM). Conformation-altered Bax was immunoprecipitated using the 6A7 antibody with or without the addition of 2% CHAPS to solubilize the liposomes prior to immunoprecipitation and analyzed by immunoblotting using an α-Bax antibody. The asterisk denotes the light chain of the 6A7 antibody. (B) Bax was incubated in the presence of liposomes, tBid, and increasing concentrations of Bcl-XL. Immunoprecipitations and immunoblotting were performed as in (A) without the addition of 2% CHAPS. (C) Bax was incubated for 2 h with liposomes (without tBid) at increasing concentrations of Bcl-XL or Bcl-XL Y101K. Immunoprecipitations and immunoblotting were performed as in (B). (D and E) Bcl-XL inhibits liposome-induced cross-linking of Bax. (D) Bax (100 nM) was incubated with liposomes for 2 h either alone (left panel), with 20 nM tBid (middle panel), or with 20 nM tBid and 100 nM Bcl-XL (right panel). Cross-linking with DSS was performed for 30 min at room temperature with or without 2% CHAPS to solubilize the liposomes prior to cross-linking, as indicated. Results were analyzed by immunoblotting. (E) Bax (100 nM) was incubated with or without liposomes for 2 h. Cross-linking and immunoblotting were performed as in (D). (F) Membrane-bound Bcl-XL inhibits the liposome-induced Bax conformational change with 50 μM m1Bid but not with 50 μM Bak BH3 peptide. Immunoprecipitations and immunoblotting were performed as in (B).