Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-x_L. Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family proteins.Bax and Bak are the key effectors of the intrinsic apoptotic pathway initiated in response to diverse stimuli including anoikis, DNA damage and growth factor withdrawal.¹ Both proteins are normally dormant in healthy cells, but upon reception of an apoptotic stimulus, they undergo conformation change that allows their self-association to form pores in the mitochondrial outer membrane (MOM).^{2, 3, 4, 5, 6, 7} The consequence of disruption of the MOM is twofold; it impairs the ability of mitochondria to generate ATP by oxidative phosphorylation and it allows the release of intermembrane proteins including cytochrome c that agonizes caspases that dismantle the cell.Bak and Bax share significant structural homology in their inactive states and have conserved mechanism of conformation change and oligomerization.^{3, 8, 9, 10} Further, genetic studies reveal that Bak and Bax perform at least partially overlapping function, with deficiency in both necessary to perturb apoptosis during embryonic development and in response to toxic insult.^{1, 11} However, whether Bak and Bax are regulated similarly is unclear. Whereas Bak is constitutively anchored in the MOM via its hydrophobic C-terminal transmembrane domain, Bax is predominantly cytosolic in the majority of non-apoptotic cells.¹² Recent evidence indicates that Bax is in a dynamic equilibrium between cytosol and mitochondria and is constantly trafficked away from the MOM in non-apoptotic cells.^{13, 14} In response to apoptotic stress this ‘retrotranslocation'' is disrupted causing Bax to accumulate at mitochondria; a hallmark of most apoptotic cells. The mechanism governing the dynamic distribution of Bax in healthy and apoptotic cells is unclear with interactions with pro-survival proteins debated.^{13, 14}Voltage-dependent anion channels (VDACs) are the major channels responsible for ion passage across the MOM. Studies have also implicated an additional role for the VDACs in the regulation of Bak or Bax apoptotic function or potentially even constituting a component of the Bak/Bax apoptotic pore.^{15, 16, 17, 18} However, these studies have provided contrasting findings relating to whether VDACs might positively or negatively regulate Bak/Bax apoptotic function.We used blue native-PAGE (BN-PAGE) to investigate how Bax oligomerizes in the MOM during apoptosis. We observed that VDAC2 is a determinant of the constitutive association of both Bax and Bak with the MOM. The defect in Bax mitochondrial localization can be bypassed by Bak-dependent recruitment during apoptosis. Thus, our data suggest that mitochondrial localization of Bax occurs via distinct mechanisms in healthy and apoptotic cells and that either VDAC2 or Bak is required for the efficient translocation of Bax and hence for the oligomerization at the MOM and Bax apoptotic function.     

A role for mitochondrial Bak in apoptotic response to anticancer drugs.

In the present study a clonal Jurkat cell line deficient in expression of Bak was used to analyze the role of Bak in cytochrome c release from mitochondria. The Bak-deficient T leukemic cells were resistant to apoptosis induced by UV, staurosporin, VP-16, bleomycin, or cisplatin. In contrast to wild type Jurkat cells, these Bak-deficient cells did not respond to UV or treatment with these anticancer drugs by membranous phosphatidylserine exposure, DNA breaks, activation of caspases, or release of mitochondrial cytochrome c. The block in the apoptotic cascade was in the mitochondrial mechanism for cytochrome c release because purified mitochondria from Bak-deficient cells failed to release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. The resistance of Bak-deficient cells to VP-16 was reversed by transduction of the Bak gene into these cells. Also, the cytochrome c releasing capability of the Bak-deficient mitochondria was restored by insertion of recombinant Bak protein into purified mitochondria. Following mitochondrial localization, low dose recombinant Bak restored the mitochondrial release of cytochrome c in response to Bax; at increased doses it induced cytochrome c release itself. The function of Bak is independent of Bid and Bax because recombinant Bak induced cytochrome c release from mitochondria purified from Bax(-/-), Bid(-/-), or Bid(-/-) Bax(-/-) mice. Together, our findings suggest that Bak plays a key role in the apoptotic machinery of cytochrome c release and thus in the chemoresistance of human T leukemic cells.     

Bax-dependent regulation of Bak by voltage-dependent anion channel 2

Many studies have demonstrated a critical role of Bax in mediating apoptosis, but the role of Bak in regulating cancer cell apoptotic sensitivities in the presence or absence of Bax remains incompletely understood. Using isogenic cells with defined genetic deficiencies, here we show that in response to intrinsic, extrinsic, and endoplasmic reticulum stress stimuli, HCT116 cells show clear-cut apoptotic sensitivities in the order of Bax+/Bak+ > Bax+/Bak- > Bax-/Bak+ > Bax-/Bak-. Small interference RNA-mediated knockdown of Bak in Bax-deficient cells renders HCT116 cells completely resistant to apoptosis induction. Surprisingly, however, Bak knockdown in Bax-expressing cells only slightly affects the apoptotic sensitivities. Bak, like Bax, undergoes the N terminus exposure upon apoptotic stimulation in both Bax-expressing and Bax-deficient cells. Gel filtration, chemical cross-linking, and co-immunoprecipitation experiments reveal that different from Bax, which normally exists as monomers in unstimulated cells and is oligomerized by apoptotic stimulation, most Bak in unstimulated HCT116 cells exists in two distinct protein complexes, one of which contains voltage-dependent anion channel (VDAC) 2. During apoptosis, Bak and Bax form both homo- and hetero-oligomeric complexes that still retain some VDAC-2. However, the oligomeric VDAC-2 complexes are diminished, and Bak does not interact with VDAC-2 in Bax-deficient HCT116 cells. These results highlight VDAC-2 as a critical inhibitor of Bak-mediated apoptotic responses.     

Bak and Bax function to limit adenovirus replication through apoptosis induction

Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level.     

The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.     

Inhibition of Bak Activation by VDAC2 Is Dependent on the Bak Transmembrane Anchor

Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.     

Apoptosis is regulated by the VDAC1 N-terminal region and by VDAC oligomerization: release of cytochrome c,AIF and Smac/Diablo

Mitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF. Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H₂O₂ or selenite augmented the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1 N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-apoptotic proteins, such as HK and Bcl2.     

Natural diterpenoid compound elevates expression of Bim protein, which interacts with antiapoptotic protein Bcl-2, converting it to proapoptotic Bax-like molecule

Overwhelming evidence indicates that Bax and Bak are indispensable for mediating cytochrome c release from mitochondria during apoptosis. Here we report a Bax/Bak-independent mechanism of cytochrome c release and apoptosis. We identified a natural diterpenoid compound that induced apoptosis in bax/bak double knock-out murine embryonic fibroblasts and substantially reduced the tumor growth from these cells implanted in mice. Treatment with the compound significantly increased expression of Bim, which migrated to mitochondria, altering the conformation of and forming oligomers with resident Bcl-2 to induce cytochrome c release and caspase activation. Importantly, purified Bim and Bcl-2 proteins cooperated to permeabilize a model mitochondrial outer membrane; this was accompanied by oligomerization of these proteins and deep embedding of Bcl-2 in the membrane. Therefore, the diterpenoid compound induces a structural and functional conversion of Bcl-2 through Bim to permeabilize the mitochondrial outer membrane, thereby inducing apoptosis independently of Bax and Bak. Because Bcl-2 family proteins play important roles in cancer development and relapse, this novel cell death mechanism can be explored for developing more effective anticancer therapeutics.     

Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB⁺Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB⁺Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-X_L, the anti-apoptotic counterparts of Bim. In contrast, gzmB⁺Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB⁺Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB⁺Tc-induced death pathways.     

Necroptosis Interfaces with MOMP and the MPTP in Mediating Cell Death

During apoptosis the pro-death Bcl-2 family members Bax and Bak induce mitochondrial outer membrane permeabilization (MOMP) to mediate cell death. Recently, it was shown that Bax and Bak are also required for mitochondrial permeability transition pore (MPTP)-dependent necrosis, where, in their non-oligomeric state, they enhance permeability characteristics of the outer mitochondrial membrane. Necroptosis is another form of regulated necrosis involving the death receptors and receptor interacting protein kinases (RIP proteins, by Ripk genes). Here, we show cells or mice deficient for Bax/Bak or cyclophilin D, a protein that regulates MPTP opening, are resistant to cell death induced by necroptotic mediators. We show that Bax/Bak oligomerization is required for necroptotic cell death and that this oligomerization reinforces MPTP opening. Mechanistically, we observe mixed lineage kinase domain-like (MLKL) protein and cofilin-1 translocation to the mitochondria following necroptosis induction, while expression of the mitochondrial matrix isoform of the antiapoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), is significantly reduced. Some of these effects are lost with necroptosis inhibition in Bax/Bak1 double null, Ppif^-/-, or Ripk3^-/- fibroblasts. Hence, downstream mechanisms of cell death induced by necroptotic stimuli utilize both Bax/Bak to generate apoptotic pores in the outer mitochondrial membrane as well as MPTP opening in association with known mitochondrial death modifying proteins.     

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.     

Metaxins 1 and 2, two proteins of the mitochondrial protein sorting and assembly machinery,are essential for Bak activation during TNF alpha triggered apoptosis

The proteins Bax and Bak are central in the execution phase of apoptosis; however, little is known about the partners involved in the control of this complex process. Here, we show that mitochondrial Bak is incorporated into a VDAC2/Mtx1/Mtx2 multi-protein complex in both resting and dying cells. VDAC2 is a porin that has previously been described as a partner of Bak while Mtx1 and Mtx2 are two proteins of the mitochondrial sorting and assembly machinery (SAM) that have been implicated in TNF-induced apoptosis. We show that, after the induction of apoptosis, Bak switches from its association with Mtx2 and VDAC2 to interact with Mtx1.     

Bax and Bak independently promote cytochrome C release from mitochondria

Pro-apoptotic Bax and Bak have been implicated in the regulation of p53-dependent apoptosis. We assessed the ability of primary baby mouse kidney (BMK) epithelial cells from bax(-/-), bak(-/-), and bax(-/-) bak(-/-) mice to be transformed by E1A alone or in conjunction with dominant-negative p53 (p53DD). Although E1A alone transformed BMK cells from p53-deficient mice, E1A alone did not transform BMK cells from bax(-/-), bak(-/-), or bax(-/-) bak(-/-) mice. Thus, the loss of both Bax and Bak was not sufficient to relieve p53-dependent suppression of transformation in epithelial cells. To test the requirement for Bax and Bak in other death signaling pathways, stable E1A plus p53DD-transformed BMK cell lines were derived from the bax(-/-), bak(-/-), and bax(-/-) bak(-/-) mice and characterized for their response to tumor necrosis factor-alpha (TNF-alpha)-mediated apoptosis. The loss of both Bax and Bak severely impaired TNF-alpha-mediated apoptosis, but the presence of either Bax or Bak alone was sufficient for cell death. Cytochrome c was released from mitochondria, and caspase-9 was activated in Bax- or Bak-deficient cells in response to TNF-alpha but not in cells deficient in both. Thus, either Bax or Bak is required for death signaling through mitochondria in response to TNF-alpha, but both are dispensable for p53-dependent transformation inhibition.     

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.     

Assembly of the Mitochondrial Apoptosis-induced Channel, MAC

Although Bcl-2 family proteins control intrinsic apoptosis, the mechanisms underlying this regulation are incompletely understood. Patch clamp studies of mitochondria isolated from cells deficient in one or both of the pro-apoptotic proteins Bax and Bak show that at least one of the proteins must be present for formation of the cytochrome c-translocating channel, mitochondrial apoptosis-induced channel (MAC), and that the single channel behaviors of MACs containing exclusively Bax or Bak are similar. Truncated Bid catalyzes MAC formation in isolated mitochondria containing Bax and/or Bak with a time course of minutes and does not require VDAC1 or VDAC3. Mathematical analysis of the stepwise changes in conductance associated with MAC formation is consistent with pore assembly by a barrel-stave model. Assuming the staves are two transmembrane α-helices in Bax and Bak, mature MAC pores would typically contain ∼9 monomers and have diameters of 5.5–6 nm. The mitochondrial permeability data are inconsistent with formation of lipidic pores capable of transporting megadalton-sized macromolecules as observed with recombinant Bax in liposomes.Permeabilization of the mitochondrial outer membrane is the commitment step in intrinsic apoptosis. This process is tightly regulated by Bcl-2 family proteins that control formation of the megachannel mitochondrial apoptosis-induced channel (MAC)² in this membrane. MAC formation correlates with release of pro-apoptotic factors, including cytochrome c from the intermembrane space into the cytosol, and initiates apoptosis (1–7).MAC is absent from normal mitochondria but forms in the outer membrane early in apoptosis, reaching peak conductances of 1.5–5 nS. This channel is formed in the presence of the multidomain pro-apoptotic proteins Bax and/or Bak (8–13), and may be composed of these proteins along with other components (14, 15). Unlike Bax, Bak is normally a resident of the mitochondrial outer membrane and is bound to VDAC2, another outer membrane protein (16). However, Bak is not available for oligomerization until another pro-apoptotic protein, like t-Bid, disrupts the interaction of Bak with VDAC2. In contrast, most Bax is located in the cytoplasm until an apoptotic signal induces the translocation of Bax to the outer membrane of mitochondria and eventual Bax oligomerization in this same membrane (14, 17).Bax and Bak have multiple putative transmembrane domains; the amphipathic helices 5 and 6 of Bax are predicted to form, at least in part, the pore of the cytochrome c release channel (18). Bax lacking helices 5 and 6 does not translocate to mitochondria nor cause cytochrome c release (19, 20). Given the structural similarities between Bax and Bak, the same helices may be important in formation of the MAC pore by both proteins (21). Although Bax and Bak are certainly involved in MAC formation, the exact molecular composition of this channel remains unknown.In this study we report that Bax and Bak are functionally redundant with regard to MAC formation and cytochrome c release in mouse embryonic fibroblasts (MEF). This is true despite the fact that Bak normally resides in the outer membrane, whereas Bax is generally translocated to this membrane to induce MAC formation. Our experimental design bypasses Bax translocation and any underlying autocatalytic mechanism that might be involved (22). Instead, it focuses on formation of the MAC pore. Early MAC-associated conductance increments are relatively small, suggesting that Bax-dependent formation of the cytochrome c-permeable pore does not occur prior to membrane insertion of Bax. Mathematical modeling of the conductance changes indicates that, if MAC is a circular pore assembled by sequential addition of helices 5 and 6 from Bax and/or Bak monomers, the mature, cytochrome c transport-competent pore is likely a 9–10-mer of these proteins.     

Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.     

Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release

ATP depletion induced by hypoxia or mitochondrial inhibitors results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured rat proximal tubule cells. Translocated Bax undergoes further conformational changes to oligomerize into high molecular weight complexes (Mikhailov, V., Mikhailova, M., Pulkrabek, D. J., Dong, Z., Venkatachalam, M. A., and Saikumar, P. (2001) J. Biol. Chem. 276, 18361-18374). Here we report that following Bax translocation in ATP-depleted rat proximal tubule cells, Bak, a proapoptotic molecule that normally resides in mitochondria, also reorganizes to form homo-oligomers. Oligomerization of both Bax and Bak occurred independently of Bid cleavage and/or translocation. Western blots of chemically cross-linked membrane extracts showed nonoverlapping "ladders" of Bax and Bak complexes in multiples of approximately 21 and approximately 23 kDa, respectively, consistent with molecular homogeneity within each ladder. This indicated that Bax and Bak complexes were homo-oligomeric. Nevertheless, each oligomer could be co-immunoprecipitated with the other, suggesting a degree of affinity between Bax and Bak that permitted co-precipitation but not cross-linking. Furthermore, dissociation of cross-linked complexes by SDS and renaturation prior to immunoprecipitation did not prevent reassociation of the two oligomeric species. Notably, expression of Bcl-2 prevented not only the oligomerization of Bax and Bak, but also the association between these two proteins in energy-deprived cells. Using Bax-deficient HCT116 and BMK cells, we show that there is stringent Bax requirement for Bak homo-oligomerization and for cytochrome c release during energy deprivation. Using Bak-deficient BMK cells we further show that Bak deficiency is associated with delayed kinetics of Bax translocation but does not affect either the oligomerization of translocated Bax or the leakage of cytochrome c. These results suggest a degree of functional cooperation between Bax and Bak in this form of cell injury, but also demonstrate an absolute requirement of Bax for mitochondrial permeabilization.     

Disruption of the VDAC2–Bak interaction by Bcl-xS mediates efficient induction of apoptosis in melanoma cells

The proapoptotic B-cell lymphoma (Bcl)-2 protein Bcl-x_S encloses the Bcl-2 homology (BH) domains BH3 and BH4 and triggers apoptosis via the multidomain protein Bak, however, the mechanism remained elusive. For investigating Bcl-x_S efficacy and pathways, an adenoviral vector was constructed with its cDNA under tetracycline-off control. Bcl-x_S overexpression resulted in efficient apoptosis induction and caspase activation in melanoma cells. Indicative of mitochondrial apoptosis pathways, Bcl-x_S translocated to the mitochondria, disrupted the mitochondrial membrane potential and induced release of cytochrome c, apoptosis-inducing factor and second mitochondria-derived activator of caspases. In melanoma cells, Bcl-x_S resulted in significant Bak activation, and Bak knockdown as well as Bcl-x_L overexpression abrogated Bcl-x_S-induced apoptosis, whereas Mcl-1 (myeloid cell leukemia-1) knockdown resulted in a sensitization. With regard to the particular role of voltage-dependent anion channel 2 (VDAC2) for inhibition of Bak, we identified here a notable interaction between Bcl-x_S and VDAC2 in melanoma cells, which was proven in reciprocal coimmunoprecipitation analyses. On the other hand, Bcl-x_S showed no direct interaction with Bak, and its binding to VDAC2 appeared as also independent of Bak expression. Suggesting a new proapoptotic mechanism, Bcl-x_S overexpression resulted in disruption of the VDAC2–Bak interaction leading to release of Bak. Further supporting this pathway, overexpression of VDAC2 strongly decreased apoptosis by Bcl-x_S. New proapoptotic pathways are of principle interest for overcoming apoptosis deficiency of melanoma cells.     

Pancreatic β-Cell Death due to Pdx-1 Deficiency Requires Multi-BH Domain Protein Bax but Not Bak

Diabetes develops in Pdx1-haploinsufficient mice due to an increase in β-cell death leading to reduced β-cell mass and decreased insulin secretion. Knockdown of Pdx1 gene expression in mouse MIN6 insulinoma cells induced apoptotic cell death with an increase in Bax activation and knockdown of Bax reduced apoptotic β-cell death. In Pdx1 haploinsufficient mice, Bax ablation in β-cells increased β-cell mass, decreased the number of TUNEL positive cells and improved glucose tolerance after glucose challenge. These changes were not observed with Bak ablation in Pdx1-haploinsufficient mice. These results suggest that Bax mediates β-cell apoptosis in Pdx1-deficient diabetes.     

Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

Bax and Bak play a redundant but essential role in apoptosis initiated by the mitochondrial release of apoptogenic factors. In addition to their presence at the mitochondrial outer membrane, Bax and Bak can also localize to the ER. Agents that initiate ER stress responses can induce conformational changes and oligomerization of Bax on the ER as well as on mitochondria. In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells. In bax-/-bak-/- cells, introduction of Bak mutants selectively targeted to either mitochondria or the ER can induce apoptosis. However, ER-targeted, but not mitochondria-targeted, Bak leads to progressive depletion of ER Ca2+ and induces caspase 12 cleavage. In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak. These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.