Introduction

Mitochondria became the subject of intensive scientific investigation in the mid-twentieth century when their role as cellular energy producers was discovered. This culminated in the discovery and characterization of oxidative phosphorylation as the mechanism by which most cellular ATP is produced. Free exchange of substrate and ATP/ADP between mitochondria and the cytosol was proposed to provide the homeostatic mechanism by which glycolysis and oxidative phosphorylation are coupled to maintain the intracellular ATP/ADP ratio.1,2 In the last few years, there has been a resurgence of interest in mitochondria following the discovery that mitochondria play a crucial role in the regulation of programmed cell death, or apoptosis.3,4,5,6,7,8,9 A number of molecules involved in the execution of apoptosis normally reside in mitochondria, safely sequestered from their targets and co-factors.10,11,12,13,14,15,16 Following an apoptotic stimulus, these proteins can be released from mitochondria and initiate the activation of the downstream effectors of apoptosis, caspases. The mechanisms by which these apoptosis initiators are released from mitochondria remain controversial. Elucidating these mechanisms, however, is crucial to the understanding of how and why a cell makes the decision to undergo apoptosis.

The mitochondrion is an organelle encompassed by two membranes (Figure 1). The inner membrane, which surrounds the mitochondrial matrix and is usually tightly folded in cristae, holds the molecular complexes of the electron transport chain. It is across the inner membrane that the electron transport chain generates the hydrogen ion gradient necessary to make ATP. In order to maintain this crucial gradient, transport across the inner membrane is tightly regulated by many different, specific transporters for the metabolites that need to cross the inner membrane, such as the phosphate carrier, the dicarboxylate carrier and the tricarboxylate carrier.17 The inner membrane also contains protein import channels, as well as the adenine nucleotide translocator (ANT) that exchanges ADP and ATP between the mitochondrial matrix and the intermembrane space. The outer mitochondrial membrane (OMM) surrounds the inner membrane, creating the intermembrane space where many of the characterized apoptosis promoting proteins reside, including cytochrome c, apoptosis inducing factor (AIF) and some pro-caspases. Transport across the outer mitochondrial membrane is thought to be less tightly regulated than transport across the inner membrane. The high permeability of the outer membrane is mediated by VDAC, which is the most common protein in the outer membrane and is permeable to molecules of up to 5000 daltons in its open configuration.18 Nevertheless, the outer membrane is impermeable to holo-cytochrome c and other apoptosis promoting proteins in the intermembrane space and this impermeability is crucial to the regulation of apoptosis. For example, if holo-cytochrome c is released into the cytosol, it can complex with Apaf-1 and ATP or dATP to activate pro-caspase 9, thus beginning the proteolytic cascade that results in the morphological features of apoptosis.19

Figure 1
figure 1

Mitochondria are producers of ATP and regulators of apoptosis. Oxidative phosphorylation occurs across the mitochondrial inner membrane via the electron transport chain, including cytochrome c (c), and the F1F0-ATPase (F1F0). ATP made in the matrix is exchanged for cytosolic ADP through the adenine nucleotide translocator (ANT) in the inner membrane and the voltage dependent anion channel (VDAC) in the outer membrane. Bcl-2 family proteins regulate mitochondrial physiology and apoptosis. Bcl-xL is constitutively localized to the outer membrane and may be inhibited through interactions with BH3-only proteins including Bad, Bim, Bik and Hrk. Bax translocates to the outer membrane after an apoptotic signal and can be toxic in the absence of a physical interaction with Bcl-xL

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Maintenance of the permeability of the outer membrane to metabolic anions is crucial for cell survival. Free ATP/ADP exchange, as well as the movement of other metabolic anions such as creatine phosphate, succinate and pyruvate between the cytosol and the matrix, is dependent on passage through the outer membrane, primarily through VDAC.20,21 Paradoxically, the continued exchange of metabolic anions across the outer membrane is required to maintain the integrity of this membrane and to prevent the release of apoptosis promoting proteins. Decreased passage of metabolites across the membrane would tend to favor inner membrane hyperpolarization, increased reactive oxygen species (ROS) production and matrix swelling, all of which could compromise the integrity of the outer membrane.

Bcl-2 family

Members of the Bcl-2 family of proteins may regulate OMM integrity and function. Bcl-2 proteins can localize to the outer membrane and are established regulators of apoptosis.5,6,22 The family can be divided into three different groups based on Bcl-2 homology (BH) domains and function.23 The anti-apoptotic members, such as Bcl-2 and Bcl-xL, typically have BH1 through BH4 domains. The pro-apoptotic members can be divided into two groups: those with BH1, BH2 and BH3 domains, such as Bax and Bak, and those with only BH3 domains, such as Bad, and Bim.24,25,26 An exception to these categories may be Bcl-xS, a pro-apoptotic protein, which is an alternate splice form of Bcl-xL and contains BH3 and BH4 domains.27 These domains have functional and structural significance. The BH3 domain of pro-apoptotic family members can interact with the hydrophobic cleft formed by the BH1, BH2 and BH3 domains of anti-apoptotic proteins, and this interaction may be an important regulatory mechanism.28,29 Structural studies of Bcl-xL have shown a striking similarity between Bcl-xL and the colicin family of pore forming proteins,30 and functional studies have shown that Bcl-xL, as well as Bcl-2, Bax, and Bak, are capable of forming channels in synthetic lipid bilayers.31,32,33,34 This functional property of Bcl-2 family proteins may be even more significant than originally appreciated, since it was recently shown that cleaved Bid, which shares primary sequence homology with Bcl-xL only in the BH3 domain, has a similar three-dimensional structure to Bcl-xL and can also form channels in synthetic lipid bilayers.35,36,37 The ability of Bcl-2 family members to form channels in association with intracellular membranes appears to be important to their function in apoptosis, as does their ability to localize to the OMM.22,29 Thus a number of groups have sought to determine whether Bcl-2 family proteins may perform their functions by regulating the permeability of the outer mitochondrial membrane. Furthermore, by regulating the permeability of the outer mitochondrial membrane, Bcl-2 family members could potentially be regulating mitochondrial homeostasis in general, rather than being solely involved in apoptosis regulation.

The mechanisms by which the outer mitochondrial membrane increases in permeability during apoptosis, and the mechanisms by which Bcl-2 proteins regulate this process have remained controversial. A number of models for outer membrane permeabilization have been proposed, such as the non-specific rupture of the outer membrane or the formation of a specific channel. Bcl-2 family proteins may regulate both of these processes, via both physical interactions and channel forming capabilities. In any case, it is clear that the disruption in the OMM during apoptosis must be large and freely permeable, since every intermembrane space molecule that has been measured appears to pass through the channel or hole that ultimately forms.38

Non-specific outer membrane rupture

Non-specific rupture of the outer mitochondrial membrane is one possible mechanism for the increase in outer membrane permeability following an apoptotic stimulus. This is an attractive model to explain the non-selective release of intermembrane space proteins and the simultaneous accessibility of the inner membrane to large cytosolic proteins. Rupture of the outer mitochondrial membrane could be secondary to swelling of the matrix, which can expand to a greater surface area than the outer membrane due to the extensive folding of the inner membrane. Mitochondrial swelling has been reported to occur following many apoptotic stimuli, including growth factor withdrawal, heat shock, sustained increase in intracellular calcium levels, TNF treatment and ischemia.39,40,41,42,43,44 Matrix swelling that leads to outer membrane rupture could occur in at least two ways. One model hypothesizes that different apoptotic stimuli have downstream metabolic effects on mitochondria that induce matrix swelling, while another model invokes PT pore opening as the cause of swelling.

Matrix swelling and OMM rupture in response to metabolic changes

Matrix swelling is known to occur in response to a reduction in the rate of electron transport.45 A reduction in the rate of electron transport can occur in the presence of mitochondrial poisons, or due to mitochondrial substrate limitation,39 which can occur when a cell has inadequate nutrients, oxygen, or cannot transport substrates into the mitochondria.46 These conditions are associated with the induction of apoptosis in multiple systems.47,48

Matrix swelling necessarily leads to a reduction in intermembrane space volume. The intermembrane space contains a number of proteins that are impermeant to the outer membrane. Because these proteins cannot equilibrate across the outer membrane, maintaining a chemical equilibrium in the face of intermembrane space volume changes could involve the movement of ions, thus generating an electrical gradient across the outer membrane (Donnan potential). This electrical gradient would favor closure of VDAC, which could exacerbate the initial swelling. By limiting exchange of ADP for ATP across the outer membrane, VDAC closure would stall the electron transport chain by limiting the ability of the ATP synthase to utilize the hydrogen ion gradient to generate ATP from ADP. Under such conditions, osmotic influx of weak organic acids will lead to slow swelling of the matrix. Eventually, as the mitochondria become more polarized, and the rate of electron transport slows, the matrix space would be predicted to swell beyond the confines of the outer membrane, resulting in outer membrane rupture and the release of pro-apoptotic proteins into the cytosol. Tears in the outer mitochondrial membrane have been observed by electron microscopy in response to growth factor withdrawal.39,49 An ability to maintain VDAC in an open configuration despite a charge across the outer membrane or an ability to prevent the build-up of charge across the outer membrane would allow the outer membrane to withstand the stress associated with matrix swelling, and it has been suggested that anti-apoptotic Bcl-2 proteins may play such a role.50

Consistent with this model, it has been demonstrated that following growth factor withdrawal, there is a build-up of ADP in the cytosol, and a build-up of ATP in the mitochondria.48 These conditions could result from a decrease in exchange between the mitochondria and the cytosol. In addition, mitochondria also accumulate creatine phosphate following growth factor withdrawal. Creatine phosphate is made in the intermembrane space from ATP and creatine in a reaction requiring creatine kinase. Creatine phosphate then diffuses into the cytosol to buffer falling ATP levels.51 The accumulation of creatine phosphate in mitochondria therefore suggests that transport across the OMM is impaired, while the continued production of creatine phosphate implies that ATP transport across the inner membrane is intact.21 The defect in exchange across the OMM, with a subsequent stalling of the F1F0-ATPase's utilization of the hydrogen ion gradient, could also be responsible for the mitochondrial hyperpolarization that has been reported in response to multiple apoptotic stimuli.29,39,52,53 In addition, reduced function of the ATP synthase could lead to increased ROS production and oxidation of outer membrane lipids.

Anti-apoptotic Bcl-2 proteins prevent disruption of mitochondrial metabolism

Anti-apoptotic Bcl-2 family members have been shown to prevent the matrix swelling, ROS damage, release of cytochrome c and loss of membrane potential associated with apoptosis.39,54,55,56 They have also been shown to prevent the disruption in ATP/ADP exchange between the mitochondrial matrix and the cytosol.48 The defect in exchange has been localized to the outer mitochondrial membrane where both VDAC and Bcl-2 family proteins are localized.21 Therefore, one way Bcl-2 family proteins could modulate ATP/ADP exchange is through regulation of VDAC (Figure 2). VDAC can exist in both open and closed conformations. In closed conformations, VDAC is impermeable to ATP and other metabolic anions (Figure 2A).21,57 By maintaining VDAC in an open conformation, Bcl-2 family proteins would be able to maintain ATP/ADP exchange, thus preventing mitochondrial hyperpolarization, swelling and rupture (Figure 2B).

Figure 2
figure 2

Bcl-xL regulates outer membrane permeability via regulation of VDAC. (A) Under normal conditions, VDAC exchanges metabolic anions, including ATP and ADP, between the cytosol and the intermembrane space (IMS). In the absence of Bcl-xL, apoptotic signals can lead to VDAC closure, which disrupts the transport of metabolic anions across the outer mitochondrial membrane. This leads to mitochondrial dysfunction, including swelling, which can eventually result in outer membrane rupture and cytochrome c release. (B) The presence of Bcl-xL at the outer membrane during apoptotic signal transduction allows for maintenance of VDAC in the open configuration and prevention of mitochondrial dysfunction and apoptosis

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Recent in vitro data demonstrate that recombinant Bcl-xL can maintain VDAC in an open conformation. Studies in planar phospholipid membranes showed that when physiologic voltages of between −25 and +25 mV are applied to a membrane containing VDAC, addition of rBcl-xL favors the maintenance of VDAC in its high conductance state. In addition, isolated mouse liver mitochondria in the presence of a VDAC inhibitor (β-NADH) consumed ADP at a faster rate after rBcl-xL addition, a process requiring more efficient transport of ADP across the outer membrane. Finally, mitochondria isolated from growth factor withdrawn cells had accumulated creatine phosphate due to a defect in outer membrane permeability. These mitochondria were able to release creatine phosphate, but not cytochrome c, in response to added rBcl-xL.58 Together, these data suggest that Bcl-xL can maintain VDAC in the open state. How Bcl-xL maintains the open state of VDAC is unclear. Bcl-xL could form a direct physical interaction with VDAC, or Bcl-xL insertion could maintain the VDAC open state by affecting the local lipid concentration or distribution. Alternatively, Bcl-xL could dissipate charge on the outer membrane by acting as a channel and thus indirectly affect VDAC. However Bcl-xL functions to maintain the open state of VDAC, this property would sustain metabolite exchange and prevent rupture of the outer mitochondrial membrane and release of apoptosis promoting proteins.

Pro-apoptotic Bcl-2 proteins disrupt mitochondrial metabolism

The pro-apoptotic Bcl-2 family members themselves may be capable of inducing the mitochondrial transport problems that can lead to matrix swelling. Many of these proteins have been shown to change conformation and translocate to the mitochondrial membrane in response to various apoptotic stimuli. Some data suggests that there may be a lag time between Bax translocation and cell death,59 suggesting that the pro-apoptotic family members may not always release cytochrome c directly, but rather may contribute to a mitochondrial dysfunction that leads to cytochrome c release. A direct impact of Bax on membrane permeability was suggested by recent studies in the yeast S. cerevisiae, where a mutant impaired in transport across the outer mitochondrial membrane due to a deficiency of yeast VDAC was more susceptible than wild-type yeast to Bax induced toxicity.60 This suggests that Bax and VDAC may have opposing functions on the permeability of the outer membrane. Bax over-expression in S. cerevisiae has been shown to induce mitochondrial hyperpolarization and metabolic arrest, which is also suggestive of a block in substrate transport or in the activity of the F1F0-ATPase.53

Other groups, however, have shown that Bax and Bid are capable of promoting cytochrome c release in the absence of observed matrix swelling.38 These data, along with data demonstrating the ability of Bax to multimerize and form larger membrane channels, suggests that Bax may directly induce cytochrome c releasing channels in the mitochondrial outer membrane without prior alteration in mitochondrial physiology.

Reactive oxygen species

The redox state of a cell has been shown to play an important role in many types of apoptosis, including death induced by TNF, UV, growth factor withdrawal and ceramide.61 Bcl-2 and Bcl-xL expression can protect cells from oxidant-induced apoptosis, although it has remained unclear whether the protective effect is due to a scavenging function or to a regulation of the production of ROS.54,56,61 How does this function of Bcl-2 family proteins tie in with Bcl-2 family regulation of outer mitochondrial membrane permeability? Mitochondrial ROS can be produced when there is a backup in the electron transport chain, particularly a block after complex III, which is thought to produce most mitochondrial ROS. As described above, a decrease in the permeability of the outer mitochondrial could cause a backup in the electron transport chain as ADP transport into the mitochondria becomes limiting. Therefore, a block in mitochondrial transport of ADP could lead to mitochondrial hyperpolarization as well as production of ROS. Bcl-xL can restore outer mitochondrial membrane permeability, thus relieving the block in electron transport and preventing both ROS production and hyperpolarization. Hyperpolarization in combination with ROS production has been seen in response to Fas signaling and Bax over-expression.53,62 Mitochondrial hyperpolarization may be unstable and progress to a loss in membrane potential, which can lead to further ROS production. Recent work has shown that in response to treatment with TNF-α, 2B4 cells, a murine T-cell hybridoma cell line, show a decreased mitochondrial membrane potential that is accompanied by ROS production. Bcl-xL expression prevented both the drop in membrane potential and the increase in ROS production while delaying the kinetics of apoptosis. However Bcl-xL was not acting as a scavenger since the basal levels of ROS production were the same in both Bcl-xL expressing and control cells.63 These data suggest that Bcl-xL is able to both attenuate ROS production and delay apoptosis by regulating mitochondrial physiology. Further work will be required to understand the etiology and the impact of the ROS induced during apoptosis.

Matrix swelling and OMM rupture in response to PT pore opening

Opening of the permeability transition (PT) pore is another model that postulates that outer membrane rupture is responsible for cytochrome c release (Figure 3).64,65 The PT pore is proposed to be a large multi-protein complex that can form a large channel between the cytosol and the mitochondrial matrix, with the ability to pass proteins up to 1.5 kD, in response to a number of mitochondrial stresses.66 Opening of the PT pore correlates with swelling of the matrix and eventual rupture of the outer mitochondrial membrane with the release of cytochrome c and other proteins from the intermembrane space. Proposed components of the PT pore include VDAC, the ANT, creatine kinase, hexokinase, the benzodiazapine receptor and cyclophilin D.66,67,68 Molecules that bind different components of the PT pore, such as cyclosporin A, bongkrekic acid, or Bcl-2 family members themselves, may regulate pore function.69 These compounds also prevent apoptosis in response to some stimuli, indicating that opening of the PT pore may be an early event in apoptosis.64,69 A model in which opening of the PT pore commits the cell to apoptosis would predict an initial increase in the passage of molecules between the mitochondrial matrix and the cytosol. In fact, some reports have shown that large molecules previously impermeant to mitochondria do gain access to the matrix.70 Another prediction of the PT pore model is a rapid depolarization of the inner membrane as the pore opens. While depolarization eventually does occur in all apoptosing cells, its role as an initiating event is called into question by multiple reports of mitochondrial hyperpolarization and subsequent matrix alkalinization as an early event in apoptosis.29,39,52,53,71 Furthermore, studies done with FL5.12 cells have shown that cells that have undergone hyperpolarization are doomed to die.48 Hyperpolarization could precede the opening of the PT pore, but this would place hyperpolarization rather than depolarization at the commitment point to apoptosis.

Figure 3
figure 3

The proposed permeability transition pore. The PT pore is made up of a number of mitochondrial proteins and can be regulated by Bcl-2 proteins. Opening of the pore requires interactions between VDAC, the ANT, hexokinase (HK), creatine kinase (CK) and cyclophilin D (CD). Bax may regulate opening of the pore via an interaction with the ANT. Opening of the pore leads to equilibration between the matrix and the cytosol

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Release of pro-apoptotic proteins through specific OMM channels

Another mechanism for the release of cytochrome c and other pro-apoptotic proteins from the mitochondrial intermembrane space is the formation of specific channels in the OMM. These channels must be large enough to release intermembrane space proteins non-specifically. One way these channels have been hypothesized to form is through interactions betwen Bax and VDAC to form hybrid channels. Another possibility is the multimerization of pro-apoptotic proteins to form a single large channel.

Specific channel formed by Bax and the voltage-dependent anion channel

One model for the disruption of OMM permeability by specific channels postulates that VDAC and Bax together can form a hybrid channel capable of releasing cytochrome c (Figure 4). Electrophysiology studies on VDAC and Bax suggest that together these proteins can form a channel with a larger pore size than either VDAC or Bax alone.72 In support of the hypothesis that this interaction between Bax and VDAC is required for Bax activity, experiments using mitochondria isolated from S. cerevisiae deficient in yeast VDAC showed that these mitochondria did not release cytochrome c in response to Bax.73 Other studies, however, have shown that VDAC-deficient yeast are at least as sensitive, if not more sensitive, to Bax toxicity as wild-type yeast.53,60 Thus, the observed interaction between Bax and VDAC does not appear to be required for Bax toxicity in vivo.

Figure 4
figure 4

Bcl-2 family proteins may induce novel open and closed states of VDAC. (A) In the absence of Bcl-2 family regulation, VDAC functions normally. (B) Bcl-xL can physically interact with VDAC, which leads to VDAC closure via interactions between VDAC and the BH4 domain. (C) Bax and VDAC together form a novel hybrid channel with the capacity to release cytochrome c

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This model also postulates regulation of VDAC by Bcl-xL, but attributes a different effect of this regulation on OMM permeability. In this case, Bcl-xL closes VDAC, preventing the release of cytochrome c. In a recent study, co-immunoprecipitation experiments identified VDAC as a Bcl-xL-interacting protein. Further experiments tested the sucrose uptake of synthetic liposomes containing either VDAC alone, VDAC with rBcl-xL or VDAC with rBax. Liposomes with VDAC alone took up sucrose, whereas those with both VDAC and rBcl-xL were not able to do so and those with VDAC and rBax showed enhanced sucrose uptake. Furthermore, although liposomes with either VDAC alone or Bax alone were not permeable to cytochrome c, the addition of rBax or rBak to VDAC-containing liposomes resulted in cytochrome c release.73

Electrophysiology experiments to evaluate Bcl-xL and VDAC interaction in bilayers support the hypothesis that Bcl-xL can close VDAC.72 These results are different from the results described above suggesting that Bcl-xL can maintain VDAC in the open configuration.58 The differences between the findings of the groups that have performed electrophysiology studies on VDAC and Bcl-xL may be due to the disparity in the voltages applied to the membranes. The study by Vander Heiden et al.58 focused on potentials between +25 and −25 mV, while the study by Shimizu et al. typically used a voltage of +30 mV. The potential difference attained across the outer membrane preceding apoptosis in vivo is not known, although the outer membrane is thought to carry little or no voltage under most conditions, and transmembrane voltages of 20 mV or more are usually sufficient to close VDAC in vitro.74,75

Additional work has shown that the BH4 domain alone of Bcl-2 or Bcl-xL is capable of inhibiting VDAC-dependent cytochrome c release.76 It is interesting to consider this result in light of previous results indicating that caspase cleavage of Bcl-2 or Bcl-xL yields both a BH3 containing pro-apoptotic protein that localizes to mitochondria and a BH4 containing peptide.77,78 Also, Bcl-xS, a pro-apoptotic protein, contains a BH4 domain, so the possession of a BH4 domain alone may not be the sole determinant of anti-apoptotic activity.

It is difficult to reconcile the concept of the pro-survival protein Bcl-xL closing VDAC with the physiologic necessity of metabolite transport across the outer membrane. Perhaps this effect is only transient, or only affects a small proportion of VDAC molecules at once. On the other hand, it is easier to see how facilitation of the continued transport of metabolites by Bcl-xL could promote survival. Although the differences between these models remain to be worked out, the regulation of VDAC, and thus of outer membrane permeability, by Bcl-2 family proteins seems likely to play a role in the regulation of cell survival.

Specific OMM channels formed by pro-apoptotic Bcl-2 protein

Another possible mechanism for the disruption in permeability of the outer mitochondrial membrane during apoptosis is the formation of large channels by pro-apoptotic Bcl-2 family members themselves (Figure 5).79 Bcl-2 family members have been shown to be capable of inserting into the outer mitochondrial membrane, and presumably then function at this subcellular location. The anti-apoptotic members typically reside on the outer mitochondrial membrane all of the time. Some pro-apoptotic members, however, normally reside in the cytosol, and translocate to the outer membrane in response to various stress signals.80,81,82

Figure 5
figure 5

Bax forms multimeric complexes capable of releasing cytochrome c (c). In response to apoptotic signals such as cleaved Bid or a rise in pH, cytosolic Bax can multimerize and form large channels at the mitochondrial outer membrane. The subsequent cytochrome c release may not be accompanied by matrix swelling

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The pro-apoptotic Bcl-2 family member that has been most studied in this regard is Bax. Under normal cellular conditions, Bax is found in the cytosol as a monomer,59,83 although there have also been reports that Bax is constitutively localized to the outer mitochondrial membrane.84 After an apoptotic stimulus, however, Bax has been shown to change its conformation to a pro-apoptotic form and to translocate to the outer mitochondrial membrane,82,83 perhaps as a result of interactions with Bid or pH changes.59,84 This change in conformation leads to the exposure of the N-terminal domain and the C-terminal transmembrane domain. These conformational studies have been performed by using mutation studies and antibodies to specific regions of the protein as well as by assessing protease sensitivity.59,81,82,84,85 It has also been shown using enforced dimerization and cross-linking that, while in the cytosol Bax exists as a monomer, in its pro-apoptotic form in the outer membrane, Bax may exist in an oligomer.86 Furthermore, multimerization has been shown to be sufficient for Bax translocation.83 Oligomeric Bax has been shown to be capable of releasing cytochrome c from isolated mitochondria under conditions in which monomeric Bax was unable to do so.87

Thus, these data suggest that in response to a cellular stress, Bax changes conformation and then inserts into the outer mitochondrial membrane where it is capable of forming multimeric channels large enough to release cytochrome c and other apoptosis promoting proteins. In this model, protection by anti-apoptotic Bcl-2 family members could be mediated by binding to Bax and thus preventing Bax-complex formation, or by changing the dynamics of the outer mitochondrial membrane in such a way as to disfavor the formation of Bax complexes or insertion. A model in which Bcl-xL is not required to bind to Bax in order to protect from cell death may be more likely because Bcl-xL has been shown to retain its protective effects in the absence of physical interactions with Bax.29

The BH3-only proteins also play a significant role in the regulation of outer mitochondrial membrane permeability. Addition of recombinant Bid to isolated mitochondria has been shown to induce cytochrome c release38 as well as Bax oligomerization and insertion.86 Bid can be cleaved by caspase 8 in vivo after caspase 8 activation following death receptor stimulation. This allows for mitochondrial amplification of the apoptotic cascade after ligation of Fas or TNF receptors. Also, other BH3 family proteins, such as Bad, Bim, Harakiri and Bik, can translocate to mitochondria and bind to anti-apoptotic Bcl-2 family proteins in response to various apoptotic stimuli.25,88,89,90

Conclusion

Much progress has been made in the field of mitochondrial regulation of apoptosis since the observation that mitochondria were required for apoptotic activity in cell extracts.8,9 Progress has also been made in the field of Bcl-2 family regulation of mitochondria since the observation that Bcl-2 proteins localize to the outer mitochondrial membrane.3,5,6,7 It is now known that mitochondrial release of cytochrome c is a crucial event in caspase activation and that anti-apoptotic Bcl-2 proteins can prevent this release while pro-apoptotic family members can promote release. Mitochondria are crucial for eukaryotic life and severe mitochondrial damage invariably leads to death. In addition to preventing cytochrome c release, anti-apoptotic Bcl-2 proteins are able to maintain mitochondrial homeostasis, preventing the kind of damage to mitochondria that will kill a cell even when the apoptotic cascade is disabled. Furthermore, pro-apoptotic Bcl-2 proteins can kill eukaryotic cells in the absence of an apoptotic cascade.91,92

Bcl-2 family proteins have recently been shown to regulate the integrity of the mitochondrial outer membrane via regulation of the outer membrane protein VDAC. The outer membrane has not been appreciated as a regulatory site for mitochondrial energy generation, but a change in its permeability in either direction has the potential to cause severe perturbations of mitochondrial physiology. If the outer membrane loses permeability after VDAC closure, metabolic anions such as ADP and malate will no longer be available to the mitochondria. This would slow down processes like ATP synthesis, the citric acid cycle and NAD+/NADH shuttles, leading to falling ATP and NAD+ levels, adversely affecting the energetic capacity of the cell, as well as leading to a drop in CO2 levels, which could affect the pH balance of the cell. These perturbations in mitochondrial physiology will eventually lead to increased ROS production and damage to membranes, swelling and rupture of the outer membrane with concomitant release of cytochrome c. A large increase in outer membrane permeability would lead directly to cytochrome c release, which could decrease the efficiency of the electron transport chain in addition to activating the apoptotic cascade.

There is now evidence that Bcl-2 family proteins can participate in the regulation of outer membrane permeability. Bcl-xL has been shown to regulate VDAC in its open configuration under conditions that would normally result in VDAC closure. Bax may be able to cause a decrease in permeability of the outer membrane in some conditions, resulting in sensitivity to VDAC deficiency, ROS production and mitochondrial hyperpolarization. In other conditions, Bax appears to form large pores in the outer membrane capable of releasing cytochrome c. The regulation of outer mitochondrial membrane permeability is clearly a complex issue with wide-ranging effects on cellular physiology, impacting both cell survival decisions and bioenergetic capacity.