Mitochondrial apoptosis induced by BH3‐only molecules in the exclusive presence of endoplasmic reticular Bak

Bak and Bax are critical apoptotic mediators that naturally localize to both mitochondria and the endoplasmic reticulum (ER). Although it is generally accepted that mitochondrial expression of Bak or Bax suffices for apoptosis initiated by BH3‐only homologues, it is currently unclear whether their reticular counterparts may have a similar potential. In this study, we show that cells exclusively expressing Bak in endoplasmic membranes undergo cytochrome c mobilization and mitochondrial apoptosis in response to BimEL and Puma, even when these BH3‐only molecules are also targeted to the ER. Surprisingly, calcium was necessary but not sufficient to drive the pathway, despite normal ER calcium levels. We provide evidence that calcium functions coordinately with the ER‐stress surveillance machinery IRE1α/TRAF2 to transmit apoptotic signals from the reticulum to mitochondria. These results indicate that BH3‐only mediators can rely on reticular Bak to activate an ER‐to‐mitochondria signalling route able to induce cytochrome c release and apoptosis independently of the canonical Bak,Bax‐dependent mitochondrial gateway, thus revealing a new layer of complexity in apoptotic regulation.     

Bax dimerizes via a symmetric BH3:groove interface during apoptosis

During apoptotic cell death, Bax and Bak change conformation and homo-oligomerize to permeabilize mitochondria. We recently reported that Bak homodimerizes via an interaction between the BH3 domain and hydrophobic surface groove, that this BH3:groove interaction is symmetric, and that symmetric dimers can be linked via the α6-helices to form the high order oligomers thought responsible for pore formation. We now show that Bax also dimerizes via a BH3:groove interaction after apoptotic signaling in cells and in mitochondrial fractions. BH3:groove dimers of Bax were symmetric as dimers but not higher order oligomers could be linked by cysteine residues placed in both the BH3 and groove. The BH3:groove interaction was evident in the majority of mitochondrial Bax after apoptotic signaling, and correlated strongly with cytochrome c release, supporting its central role in Bax function. A second interface between the Bax α6-helices was implicated by cysteine linkage studies, and could link dimers to higher order oligomers. We also found that a population of Bax:Bak heterodimers generated during apoptosis formed via a BH3:groove interaction, further demonstrating that Bax and Bak oligomerize via similar mechanisms. These findings highlight the importance of BH3:groove interactions in apoptosis regulation by the Bcl-2 protein family.     

Minimal BH3 peptides promote cell death by antagonizing anti-apoptotic proteins

The pro-apoptotic "BH3 domain-only" proteins of the Bcl-2 family (e.g. Bid and Bad) transduce multiple death signals to the mitochondrion. They interact with the anti-apoptotic Bcl-2 family members and induce apoptosis by a mechanism that requires the presence of at least one of the multidomain pro-apoptotic proteins Bax or Bak. Although the BH3 domain of Bid can promote the pro-apoptotic assembly and function of Bax/Bak by itself, other BH3 domains do not function as such. The latter point raises the question of whether, and how, these BH3 domains induce apoptosis. We show here that a peptide comprising the minimal BH3 domain from Bax induces apoptosis but is unable to stimulate the apoptotic activity of microinjected recombinant Bax. This relies on the inability of the peptide to directly induce Bax translocation to mitochondria or a change in its conformation. This peptide nevertheless interferes with Bax/Bcl-xL interactions in vitro and stimulates the apoptotic activity of Bax when combined with Bcl-xL. Similarly, a peptide derived from the BH3 domain of Bad stimulates Bax activity only in the presence of Bcl-xL. Thus, BH3 domains do not necessarily activate multidomain pro-apoptotic proteins directly but promote apoptosis by releasing active multidomain pro-apoptotic proteins from their anti-apoptotic counterparts.     

c-Myc functionally cooperates with Bax to induce apoptosis

c-Myc promotes apoptosis by destabilizing mitochondrial integrity, leading to the release of proapoptotic effectors including holocytochrome c. Candidate mediators of c-Myc in this process are the proapoptotic members of the Bcl-2 family. We show here that fibroblasts lacking Bak remain susceptible to c-Myc-induced apoptosis whereas bax-deficient fibroblasts are resistant. However, despite this requirement for Bax, c-Myc activation exerts no detectable effects on Bax expression, localization, or conformation. Moreover, susceptibility to c-Myc-induced apoptosis can be restored in bax-deficient cells by ectopic expression of Bax or by microinjection of a peptide comprising a minimal BH3 domain. Microinjection of BH3 peptide also restores sensitivity to c-Myc-induced apoptosis in p53-deficient primary fibroblasts that are otherwise resistant. By contrast, there is no synergy between BH3 peptide and c-Myc in fibroblasts deficient in both Bax and Bak. We conclude that c-Myc triggers a proapoptotic mitochondrial destabilizing activity that cooperates with proapoptotic members of the Bcl-2 family.     

Mcl-1 determines the Bax dependency of Nbk/Bik-induced apoptosis

B cell lymphoma 2 (Bcl-2) homology domain 3 (BH3)–only proteins of the Bcl-2 family are important functional adaptors that link cell death signals to the activation of Bax and/or Bak. The BH3-only protein Nbk/Bik induces cell death via an entirely Bax-dependent/Bak-independent mechanism. In contrast, cell death induced by the short splice variant of Bcl-x depends on Bak but not Bax. This indicates that Bak is functional but fails to become activated by Nbk. Here, we show that binding of myeloid cell leukemia 1 (Mcl-1) to Bak persists after Nbk expression and inhibits Nbk-induced apoptosis in Bax-deficient cells. In contrast, the BH3-only protein Puma disrupts Mcl-1–Bak interaction and triggers cell death via both Bax and Bak. Targeted knockdown of Mcl-1 overcomes inhibition of Bak and allows for Bak activation by Nbk. Thus, Nbk is held in check by Mcl-1 that interferes with activation of Bak. The finding that different BH3-only proteins rely specifically on Bax, Bak, or both has important implications for the design of anticancer drugs targeting Bcl-2.     

Transient binding of an activator BH3 domain to the Bak BH3-binding groove initiates Bak oligomerization

The mechanism by which the proapoptotic Bcl-2 family members Bax and Bak release cytochrome c from mitochondria is incompletely understood. In this paper, we show that activator BH3-only proteins bind tightly but transiently to the Bak hydrophobic BH3-binding groove to induce Bak oligomerization, liposome permeabilization, mitochondrial cytochrome c release, and cell death. Analysis by surface plasmon resonance indicated that the initial binding of BH3-only proteins to Bak occurred with similar kinetics with or without detergent or mitochondrial lipids, but these reagents increase the strength of the Bak-BH3-only protein interaction. Point mutations in Bak and reciprocal mutations in the BH3-only proteins not only confirmed the identity of the interacting residues at the Bak-BH3-only protein interface but also demonstrated specificity of complex formation in vitro and in a cellular context. These observations indicate that transient protein-protein interactions involving the Bak BH3-binding groove initiate Bak oligomerization and activation.     

The vaccinia virus protein F1L interacts with Bim and inhibits activation of the pro-apoptotic protein Bax

Vaccinia virus, the prototypic member of the orthopoxvirus genus, encodes the mitochondrial-localized protein F1L that functions to protect cells from apoptotic death and inhibits cytochrome c release. We previously showed that F1L interacts with the pro-apoptotic Bcl-2 family member Bak and inhibits activation of Bak following an apoptotic stimulus (Wasilenko, S. T., Banadyga, L., Bond, D., and Barry, M. (2005) J. Virol. 79, 14031-14043). In addition to Bak, the pro-apoptotic protein Bax is also capable of initiating cytochrome c release suggesting that vaccinia virus infection could also inhibit Bax activity. Here we show that F1L inhibits the activity of the pro-apoptotic protein Bax by inhibiting oligomerization and N-terminal activation of Bax. F1L expression also inhibited the subcellular redistribution of Bax to the mitochondria and the insertion of Bax into the outer mitochondrial membrane. The ability of F1L to inhibit Bax activation does not require Bak, because F1L expression inhibited cytochrome c release and Bax activation in Bak-deficient cells. No interaction between Bax and F1L was detected during infection, suggesting that F1L functions upstream of Bax activation. Notably, F1L was capable of interacting with the BH3-only protein BimL as shown by co-immunoprecipitation, and F1L expression inhibited apoptosis induced by BimL. These studies suggest that, in addition to interacting with the pro-apoptotic protein Bak, F1L also functions to indirectly inhibit the activation of Bax, likely by interfering with the pro-apoptotic activity of BH3-only proteins such as BimL.     

Mitochondrial permeabilization relies on BH3 ligands engaging multiple prosurvival Bcl-2 relatives, not Bak

The Bcl-2 family regulates apoptosis by controlling mitochondrial integrity. To clarify whether its prosurvival members function by sequestering their Bcl-2 homology 3 (BH3)-only ligands or their multidomain relatives Bak and Bax, we analyzed whether four prosurvival proteins differing in their ability to bind specific BH3 peptides or Bak could protect isolated mitochondria. Most BH3 peptides could induce temperature-dependent cytochrome c release, but permeabilization was prevented by Bcl-x(L), Bcl-w, Mcl-1, or BHRF1. However, their protection correlated with the ability to bind Bak rather than the added BH3 peptide and could be overcome only by BH3 peptides that bind directly to the appropriate prosurvival member. Mitochondria protected by both Bcl-x(L)-like and Mcl-1 proteins were disrupted only by BH3 peptides that engage both. BH3-only reagents freed Bak from Bcl-x(L) and Mcl-1 in mitochondrial and cell lysates. The findings support a model for the control of apoptosis in which certain prosurvival proteins sequester Bak/Bax, and BH3-only proteins must neutralize all protective prosurvival proteins to allow Bak/Bax to induce mitochondrial disruption.     

Releasing the spindle assembly checkpoint without tension

Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim''s activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim''s proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.     

Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins

Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax^-/-Bak^-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.     

Differential regulation of Bax and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1

The pro-apoptotic members of the Bcl-2 family include initiator proteins that contain only BH3 domains and downstream effector multi-BH domain-containing proteins, including Bax and Bak. In this report, we compared the ability of the six human anti-apoptotic Bcl-2 family members to suppress apoptosis induced by overexpression of Bax or Bak, correlating findings with protein interactions measured by three different methods: co-immunoprecipitation, glutathione S-transferase pulldown, and fluorescence polarization assays employing synthetic BH3 peptides from Bax and Bak. Bcl-B and Mcl-1 showed strong preferences for binding to and suppression of Bax and Bak, respectively. In contrast, the other anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-X(L), Bcl-W, and Bfl-1) suppressed apoptosis induced by overexpression of either Bax or Bak, and they displayed an ability to bind both Bax and Bak by at least one of the three protein interaction methods. Interestingly, however, full-length Bax and Bak proteins and synthetic Bax and Bak BH3 peptides exhibited discernible differences in their interactions with some anti-apoptotic members of the Bcl-2 family, cautioning against reliance on a single method for detecting protein interactions of functional significance. Altogether, the findings reveal striking distinctions in the behaviors of Bcl-B and Mcl-1 relative to the other anti-apoptotic Bcl-2 family members, where Bcl-B and Mcl-1 display reciprocal abilities to bind and neutralize Bax and Bak.     

BH3 domains other than Bim and Bid can directly activate Bax/Bak

Bcl-2 family proteins regulate a critical step in apoptosis referred to as mitochondrial outer membrane permeabilization (MOMP). Members of a subgroup of the Bcl-2 family, known as the BH3-only proteins, activate pro-apoptotic effectors (Bax and Bak) to initiate MOMP. They do so by neutralizing pro-survival Bcl-2 proteins and/or directly activating Bax/Bak. Bim and Bid are reported to be direct activators; however, here we show that BH3 peptides other than Bim and Bid exhibited various degrees of direct activation of the effector Bax or Bak, including Bmf and Noxa BH3s. In the absence of potent direct activators, such as Bim and Bid, we unmasked novel direct activator BH3 ligands capable of inducing effector-mediated cytochrome c release and liposome permeabilization, even when both Bcl-xL- and Mcl-1-type anti-apoptotic proteins were inhibited. The ability of these weaker direct activator BH3 peptides to cause MOMP correlated with that of the corresponding full-length proteins to induce apoptosis in the absence of Bim and Bid. We propose that, in certain contexts, direct activation by BH3-only proteins other than Bim and Bid may significantly contribute to MOMP and apoptosis.     

Direct Interaction of Bax and Bak Proteins with Bcl-2 Homology Domain 3 (BH3)-only Proteins in Living Cells Revealed by Fluorescence Complementation

The key event in the mitochondrial pathway of apoptosis is the activation of Bax and Bak by BH3-only proteins through a molecular mechanism that is still a matter of debate. Here we studied interactions among anti- and proapoptotic proteins of the Bcl-2 family in living cells by using bimolecular fluorescence complementation analysis. Our results indicate that the antiapoptotic proteins Mcl-1 and Bcl-x_L bind preferably to the BH3-only proteins Bim, PUMA, and Noxa but can also bind to Bak and Bax. We also found a direct interaction between Bim, PUMA, or Noxa with either Bax or Bak during apoptosis induction. In HeLa cells, interaction of Bim with Bax occurs in cytosol, and then Bim-Bax complexes translocate to mitochondria. Complexes of either PUMA or Noxa with Bax or Bak were always detected at mitochondria. Overexpression of Bcl-x_L or Mcl-1 delayed Bim/Bax translocation to mitochondria. These results reveal the ability of main BH3-only proteins to directly activate Bax and Bak in living cells and suggest that a complex network of interactions regulate the function of Bcl-2 family members during apoptosis.     

How the Bcl-2 family of proteins interact to regulate apoptosis

Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.     

In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins

Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak.The regulated elimination of cells by apoptosis is a key mechanism of development, tissue homeostasis and defense. In vertebrates, apoptosis is regulated through two pathways, the death receptor-mediated (extrinsic) and the mitochondrial (intrinsic) pathway, which is activated by numerous apoptotic stimuli. Mitochondrial apoptosis is characterized by loss of mitochondrial outer membrane integrity and the release of mitochondrial intermembrane space proteins, most notably cytochrome c, which leads to the activation of the caspase-9 and effector caspases.¹Release of cytochrome c is governed by proteins of the B-cell lymphoma 2 (Bcl-2) family.² The Bcl-2 family consists of three groups, whose expression and interaction decide cell survival. The anti-apoptotic Bcl-2 proteins include Bcl-2, Bcl-X_L (B-cell lymphoma-extra large), Bcl-w (Bcl-2-like protein 2), Mcl-1 (myeloid cell leukemia sequence 1) and A1 (Bcl-2-related protein A1). The pro-apoptotic group of BH3-only proteins (containing a BH3-domain: Bim (Bcl-2-interacting mediator of cell death), Bid (BH3-interacting domain death agonist), Puma (p53-upregulated modulator of apoptosis), Noxa (Phorbol-12-myristate-13-acetate-induced protein 1), Bad (Bcl-2-associated death promoter), Bik (Bcl-2-interacting killer) and Hrk (activator of apoptosis hara-kiri)) activate the pro-apoptotic effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak). Bax and Bak can replace each other in most situations, but the presence of one of them is required for mitochondrial apoptosis. Upon activation Bax and Bak form oligomers in the outer mitochondrial membrane and cause the release of cytochrome c. How Bax and Bak are activated is still under debate. Different activation models have been proposed and investigated.According to the direct activation model BH3-only proteins can directly, by physical interaction activate Bax and Bak.³ The model was derived in studies investigating synthetic BH3-domain peptides in in vitro systems, that is, isolated mitochondria or liposomes, where peptides encompassing the BH3-domains of Bim or Bid (‘activator'' BH3-only proteins) were able to activate Bax. Peptides derived from the BH3-only proteins Bad, Bik, Hrk, Noxa or Puma did not activate Bax directly. However, these peptides can bind to anti-apoptotic Bcl-2 proteins with varying preferences.⁴ As this may neutralize a combination of anti-apoptotic proteins it may facilitate Bax/Bak activation by activator BH3-only proteins. Consequently, this group of BH3-only proteins has been named ‘sensitizer'' or ‘derepressor'' BH3-only proteins.^{3, 5, 6, 7} The direct activation model has received recent support by structural studies of activator BH3-domains bound to Bax.⁸ That study also found that the BH3-only peptides used previously lacked a residue that is important in the activation of Bax, and the previous results may have to be reconsidered. Indeed, a recent study illustrates that placing the BH3-domain from the various BH3-only proteins into intact Bid protein enhances Bax/Bak-activating capacity of the BH3-domains of Bid, Bim, Puma, Bmf (Bcl-2-modifying factor), Bik and Hrk.⁹The displacement (or indirect activation) model on the other hand posits that Bax and Bak are held in check by anti-apoptotic Bcl-2 proteins and auto-activate when this interaction is broken by BH3-only proteins (displacement). BH3-only proteins can bind to anti-apoptotic Bcl-2 proteins and upon apoptotic stimulation may cause the displacement of these proteins from Bax and Bak, which may lead to the activation of effectors. BH3-peptides derived from Bim and Puma can bind to all anti-apoptotic Bcl-2 proteins and its corresponding proteins exert killing upon overexpression, whereas Bad, Bmf, Bid, Bik, Hrk and Noxa display binding patterns restricted to certain anti-apoptotic Bcl-2 proteins.⁴ It was therefore suggested that Bax/Bak activation requires the neutralization/displacement of several anti-apoptotic proteins, which may be achieved by one BH3-only protein with broadly binding characteristics (such as Bim) or by the combination of BH3-only proteins with restricted binding capabilities (for instance Bad plus Noxa).^{10, 11}The models have been further refined; the ‘embedded together'' model additionally considers the dynamic interaction of the proteins with the mitochondrial membrane,¹² and it has been proposed that the models can be unified by taking two ‘modes'' of inhibition into account: anti-apoptotic Bcl-2 proteins have a dual function in inactivating both, BH3-only proteins and effectors. Pro-apoptotic signals cause the release of activator BH3-only proteins from sequestration with anti-apoptotic Bcl-2 proteins. Free BH3-only proteins directly activate effectors, however, cell death may still not be initiated because the effectors are then held in check by anti-apoptotic Bcl-2 proteins. Free activator BH3-only proteins are required to activate effectors.¹³This model unifies the two above models in the sense that it incorporates aspects of both, inhibition and displacement as well as direct activation. However, the core difference between the (direct) activation and the displacement model appears to be irreconcilable: in the activation model Bax and Bak are inactive unless receiving a stimulus from BH3-only proteins whereas in the displacement model they are active unless bound to anti-apoptotic proteins. Thus, in the absence of all other proteins one model predicts that Bax/Bak are active, the other that they are inactive. Obviously they cannot be both.The direct activation model has initially been established with Bax and the displacement model with Bak. The data are very strong that Bax is activated by direct interaction with BH3-only proteins. Recombinant Bak can also be directly activated by recombinant tBid,¹⁴ and Bid/BH3-chimaeras can activate recombinant Bak missing its C terminus.⁹ However, since Bak is normally inserted into the outer mitochondrial membrane where it may be bound to numerous other Bcl-2-family members, it has been difficult directly to test activation of Bak in the physiological situation.One possibility to ‘unify'' the original models may be in a model where Bax is physiologically activated by direct activation (Bax is inactive until receiving a signal through BH3-only proteins) whereas Bak is activated indirectly (auto-activates when the inhibition by Bcl-2-like proteins is relieved). Here we test this possibility of indirect Bak activation. We targeted anti-apoptotic Bcl-2 family proteins using RNAi. In this setting, protein concentrations and conditions are physiological, which avoids some of the problems associated with overexpression or cell-free experiments. Non-malignant cells may respond differently to the loss of anti-apoptotic Bcl-2 proteins compared with tumor cells.¹⁵ In this study, using non-malignant cells, we targeted all anti-apoptotic Bcl-2 molecules in combinations of two. In the absence of apoptotic stimuli we observed that the combined loss of Bcl-X_L and Mcl-1 was sufficient to induce apoptosis. The direct activator proteins Bid, Bim and Puma were not needed. These observations provide evidence for indirect activation of Bak.     

Opa1-mediated cristae opening is Bax/Bak and BH3 dependent, required for apoptosis, and independent of Bak oligomerization

Controversy surrounds the role and mechanism of mitochondrial cristae remodeling in apoptosis. Here we show that the proapoptotic BH3-only proteins Bid and Bim induced full cytochrome c release but only a subtle alteration of crista junctions, which involved the disassembly of Opa1 complexes. Both mitochondrial outer membrane permeabilization (MOMP) and crista junction opening (CJO) were caspase independent and required a functional BH3 domain and Bax/Bak. However, MOMP and CJO were experimentally separable. Pharmacological blockade of MOMP did not prevent Opa1 disassembly and CJO; moreover, expression of a disassembly-resistant mutant Opa1 (Q297V) blocked cytochrome c release and apoptosis but not Bax activation. Thus, apoptosis requires a subtle form of Opa1-dependent crista remodeling that is induced by BH3-only proteins and Bax/Bak but independent of MOMP.     

BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly

Using a Bax-dependent membrane-permeabilization assay, we show that peptides corresponding to the BH3 domains of Bcl-2 family "BH3-only" proteins have dual functions. Several BH3 peptides relieved the inhibition of Bax caused by the antiapoptotic Bcl-x(L) and/or Mcl-1 proteins, some displaying a specificity for either Bcl-x(L) or Mcl-1. Besides having this derepression function, the Bid and Bim peptides activated Bax directly and were the only BH3 peptides tested that could potently induce cytochrome c release from mitochondria in cultured cells. Furthermore, Bax activator molecules (cleaved Bid protein and the Bim BH3 peptide) synergistically induced cytochrome c release when introduced into cells along with derepressor BH3 peptides. These observations support a unified model of BH3 domain function, encompassing both positive and negative regulation of other Bcl-2 family members. In this model, the simple inhibition of antiapoptotic functions is insufficient to induce apoptosis unless a direct activator of Bax or Bak is present.     

BH3-only proteins trigger cytochrome c release, but how?

The mitochondrial apoptosis pathway has been neatly ordered. Mitochondrial apoptosis is governed by Bcl-2 family proteins, and their respective contributions determine the release of cytochrome c. It is clear that, among the Bcl-2 family, BH3-only proteins are the triggers: activation of BH3-only proteins by apoptotic stimuli initiates the process. BH3-only proteins cause cytochrome c release by activating Bax and/or Bak, and the anti-apoptotic group of Bcl-2-like proteins prevents this. However, it is curiously uncertain how BH3-only proteins activate Bax/Bak. Current models suggest that this is either through direct interaction--although this interaction is not detectable experimentally--or by the neutralisation of Bcl-2-like proteins. Here we discuss the context in which these models are placed and attempt to weigh the evidence.     

Assembly of the Bak Apoptotic Pore: A CRITICAL ROLE FOR THE BAK PROTEIN α6 HELIX IN THE MULTIMERIZATION OF HOMODIMERS DURING APOPTOSIS*

Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins.     

BH3-only protein Bim inhibits activity of antiapoptotic members of Bcl-2 family when expressed in yeast

Proteins of the Bcl-2 family regulate programmed cell death in mammals by promoting the release of cytochrome c from mitochondria in response to various proapoptotic stimuli. The mechanism by which BH3-only members of the family activate multidomain proapoptotic proteins Bax and Bak to form a pore in mitochondrial membranes remains under dispute. We report that cell death promoting activity of BH3-only protein Bim can be reconstituted in yeast when both Bax and antiapoptotic protein Bcl-X(L) are present, suggesting that Bim likely activates Bax indirectly by inhibiting antiapoptotic proteins.