Bax promotes cell death by permeabilizing mitochondrial outer membranes by an unresolved mechanism. However, in cells lacking the gene c-myc, membrane permeabilization by Bax is blocked by changes in the mitochondria that prevent Bax oligomerization. Drug-treated c-myc null cells and cells expressing Myc were used to map the topology of Bax in membranes prior to and after mitochondrial permeabilization. Chemical labeling of single cysteine mutants of Bax using a membrane bilayer impermeant cysteine-specific modifying agent revealed that Bax inserted both the 'pore domain' (helices alpha5-alpha6), and the tail-anchor (helix alpha9) into membranes prior to oligomerization and membrane permeabilization. Additional topology changes for Bax were not required in Myc-expressing cells to promote oligomerization and cytochrome c release. Our results suggest that unlike most pore-forming proteins, Bax membrane permeabilization results from oligomerization of transmembrane monomers rather than concerted insertion of the pore domains of a preformed oligomer.

Bcl‐2 inhibits apoptosis by regulating the release of cytochrome c and other proteins from mitochondria. Oligomerization of Bax promotes cell death by permeabilizing the outer mitochondrial membrane. In transfected cells and isolated mitochondria, Bcl‐2, but not the inactive point mutants Bcl‐2‐G145A and Bcl‐2‐V159D, undergoes a conformation change in the mitochondrial membrane in response to apoptotic agonists such as tBid and Bax. A mutant Bcl‐2 with two cysteines introduced at positions predicted to result in a disulfide bond that would inhibit the mobility of α5–α6 helices (Bcl‐2‐S105C/E152C) was only active in a reducing environment. Thus, Bcl‐2 must change the conformation to inhibit tBid‐induced oligomerization of integral membrane Bax monomers and small oligomers. The conformationally changed Bcl‐2 sequesters the integral membrane form of Bax. If Bax is in excess, apoptosis resumes as Bcl‐2 is consumed by the conformational change and in complexes with Bax. Thus, Bcl‐2 functions as an inhibitor of mitochondrial permeabilization by changing conformation in the mitochondrial membrane to bind membrane‐inserted Bax monomers and prevent productive oligomerization of Bax.

Interactions among Bcl-2 family proteins mediated by Bcl-2 homology (BH) regions transform apoptosis signals into actions.The interactions between BH3 region-only proteins and multi-BH region proteins such as Bax and Bcl-2 have been proposed to be the dominant interactions required for initiating apoptosis. Experimental evidence also suggests that both homo-and hetero-interactions are mediated primarily by the BH3 regions in all Bcl-2 family proteins and contribute to commitment to or inhibition of apoptosis. We found that a peptide containing the BH3 helix of Bax was not sufficient to activate recombinant Bax to permeabilize mitochondria. However, an extended peptide containing the BH3 helix and additional downstream sequences activated Bax to permeabilize mitochondria and liposomes. Bcl-2 inhibited the membrane-permeabilizing activity of peptide-activated Bax. This activity of Bcl-2 was inhibited by the extended but not the BH3-only peptide despite both peptides binding to Bcl-2 with similar affinity. Further, membrane-bound Bax activation intermediates directly activated soluble Bax further permeabilizing the membrane. Bcl-2 inhibited Bax auto-activation. We therefore propose that Bax auto-activation amplifies the initial death signal produced by BH3-only proteins and that Bcl-2 functions as an inhibitor of Bax auto-activation.The dominant form of programmed cell death, apoptosis, plays an essential role in embryogenesis and tissue homeostasis of multicellular organisms by removing unwanted or damaged cells. Impaired regulation of apoptosis is implicated in a variety of diseases including cancer and stroke. Two kinds of signals can trigger apoptosis, death signals received by death receptors on cell surface, and stress signals such as structural and genotypic damage. The latter apoptotic signals mainly route through mitochondria to provoke activation of caspases and nucleases that cleave critical proteins and DNAs thereby dismantling the cell (1, 2).Apoptosis is regulated by a complicated series of interactions between Bcl-2 family proteins. These interactions include hetero-and homo-interactions of proteins containing either multiple Bcl-2 homology (BH) 3 regions (BH1-3 or BH1-4) or a single (BH3) region. After receiving certain apoptotic stimuli, pro-apoptotic BH3-only proteins induce the release of cytochrome c and other pro-apoptotic proteins from mitochondria by triggering permeabilization of the mitochondrial outer membrane (MOM) by the multi-BH domain proteins Bax and Bak (3, 4). One proposed mechanism of MOM permeabilization involves BH3-only protein-induced conformational changes in Bax and/or Bak leading to their oligomerization in the MOM (5-7).BH3-only proteins are found in a variety of different subcellular locations and share very limited sequence similarity, often only in the short BH3 region. There is insufficient sequence similarity in BH3 region sequences to permit unambiguous identification from sequence alone. It appears that BH3 region proteins act as sensors within the cell, reporting on...

In healthy cells the antiapoptotic protein Bcl-2 adopts a topology typical of tail-anchored proteins with only the hydrophobic carboxyl terminus inserted into the membrane, as shown by labeling cell lysates with a membrane-impermeant sulfhydryl-specific reagent. Induction of apoptosis in cells triggered a change in the conformation of Bcl-2 such that cysteine 158 near the base of helix 5 inserted into the lipid bilayer of both endoplasmic reticulum and mitochondria where it was protected from labeling. Addition of a peptide corresponding to the BH3 domain of the proapoptotic protein Bim to cell lysates triggered a similar conformational change in Bcl-2, demonstrating that preexisting, membrane-bound Bcl-2 proteins change topology.