review
Bcl-2 proteins: regulators of apoptosis
or of mitochondrial homeostasis?
Matthew G. Vander Heiden*† and Craig B. Thompson †‡
*Committee on Immunology, University of Chicago, Chicago, Illinois 60637, USA
†Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
‡e-mail: [email protected]
Programmed cell death (apoptosis) is used by multicellular organisms during development and to maintain
homeostasis within mature tissues. One of the first genes shown to regulate apoptosis was bcl-2. Subsequently, a
number of Bcl-2-related proteins have been identified. Despite overwhelming evidence that Bcl-2 proteins are
evolutionarily conserved regulators of apoptosis, their precise biochemical function remains controversial. Three
biochemical properties of Bcl-2 proteins have been identified: their ability to localize constitutively and/or
inducibly to the outer mitochondrial, outer nuclear and endoplasmic reticular membranes, their ability to form
heterodimers with proteins bearing an amphipathic helical BH3 domain, and their ability to form ion-conducting
channels in synthetic membranes. The discovery that mitochondria can play a key part in the induction of
apoptosis has focused attention on the role that Bcl-2 proteins may have in regulating either mitochondrial
physiology or mitochondria-dependent caspase activation. Here we attempt to synthesize our current
understanding of the part played by mitochondria in apoptosis with a consideration of how Bcl-2 proteins might
control cell death through an ability to regulate mitochondrial physiology.
n interest in the relationship between mitochondria and the
apoptotic machinery was sparked by the discovery that mitochondria are required for cytosolic extracts to induce apoptotic changes in isolated nuclei1. Fractionation of the extracts
revealed that cytochrome c, a resident protein of the mitochondrial
intermembrane space, was a necessary cofactor for activation of the
apoptotic effector proteases known as caspases2. Caspases comprise
an expanding family of cysteine proteases that exist as inactive proenzymes in viable cells3. Upon activation, caspases acquire the ability to cleave key intracellular substrates as well as activate other caspases, resulting in the induction of a protease cascade that can kill
the cell. Caspase activation is sufficient to induce all of the classical
morphological features of apoptosis.
Three other factors were also identified in the cytosolic extracts
as cofactors required for caspase activation. These factors are Apaf1, procaspase-9 and either dATP or ATP2,4,5. Cytochrome c interacts
directly with Apaf-1, leading to the ATP-dependent formation of a
macromolecular complex6–8. This complex recruits and activates
procaspase-9. Activated caspase-9 can activate additional caspase-9
molecules, as well as caspases 3 and 7, which in turn activate caspases 2, 6, 8 and 10 (ref. 9). Because cytochrome c is located in the
mitochondrial intermembrane space, the activation of this caspase
cascade is dependent on the release of cytochrome c from mitochondria (Fig. 1).
A
Mitochondrial integrity regulates apoptosis
Cytochrome c is derived from a nuclear gene and is translated as a
precursor protein that is unable to initiate apoptosis. The cytochrome c precursor is localized to the intermembrane space by a
mechanism that is distinct from that required for the localization of
most other nuclear-encoded proteins that are targeted to the mitochondria. Cytochrome c is imported into the intermembrane space
as an apoprotein, and converted to a globular protein in the intermembrane space through addition of a haem group by the enzyme
cytochrome c haem lyase10. Once formed, holo-cytochrome c cannot be released into the cytosol as long as the barrier function of the
outer mitochondrial membrane remains intact. Only haem-binding holo-cytochrome c is competent to catalyse the activation of
caspases11. Thus, passage of cytochrome c through the outer mitochondrial membrane is the critical event responsible for mitochon-
dria-dependent caspase activation and cell death.
In addition to sequestering cytochrome c, there are other ways in
which mitochondrial integrity can participate in the regulation of
caspase activation and apoptosis. A fraction of the cellular pool of
both caspase-9 and caspase-3 has been localized to the mitochondrial intermembrane space in some cell types, and caspase-2 has
also been reported to reside in mitochondria12–16. These caspases can
be released from the mitochondria to the cytosol along with cytochrome c during the induction of apoptosis. Another protein,
termed ‘apoptosis-inducing factor’ (AIF), also redistributes from
the intermembrane space of mitochondria, and induces some of the
nuclear morphology associated with apoptosis in a caspase-independent manner17. Thus, the barrier function of the outer mitochondrial membrane serves as an important regulator of
programmed cell death by maintaining the compartmentalization
of important apoptotic mediators.
Bcl-2 proteins regulate cytochrome c release
Proteins of the Bcl-2 family are either anti-apoptotic (Bcl-2-like
proteins, such as Bcl-2 and Bcl-xL) or pro-apoptotic (for example,
Bax and Bak). These proteins regulate apoptosis in part by affecting
the mitochondrial compartmentalization of cytochrome c. Expression of Bcl-2 or Bcl-xL prevents the redistribution of cytochrome c
in response to multiple death-inducing stimuli18–21, while Bax promotes cytochrome c release22–25. In mixing experiments using cellfree systems, Bcl-2 could prevent cytochrome c release only from
the mitochondria on which it resided18. Direct addition of Bax to
isolated mitochondria induces cytochrome c release24. These data
indicate that Bcl-2 proteins regulate cytochrome c redistribution
from the mitochondria on which the proteins are localized.
Mitochondria can also influence cell-death decisions by regulating changes in the mitochondrial membrane potential, the cytoplasmic pH, and the cellular redox state. Changes in mitochondrial
function may be responsible for the increased levels of reactive oxygen species and cytoplasmic acidification that are reported to be
early events during apoptosis26. Bcl-2 expression inhibits the generation of reactive oxygen species27,28 and intracellular acidification29,
and stabilizes the mitochondrial membrane potential20,30. Bcl-2 can
also affect mitochondrial proton flux31. The observation that Bcl-2
proteins can regulate these metabolic events, as well as the redistri-
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bution of cytochrome c, indicates that Bcl-2 may be a general regulator of mitochondrial physiology.
Biochemical properties of Bcl-2 proteins
Bcl-2 proteins possess several biochemical properties that may be
important for their ability to regulate both mitochondrial physiology and cell death. The carboxy-terminal transmembrane tail functions to target these proteins to intracellular membranes, including
the outer mitochondrial membrane32–34. Several lines of evidence
support the idea that the regulated targeting of Bcl-2 proteins is
important for their function. The carboxy-terminal transmembrane tail of Bcl-xL is the most highly conserved portion of the protein (at the amino-acid level) between mammals and birds, and Bcl2 proteins that lack their targeting domains show a reduced ability
to either promote or inhibit death34–36. Furthermore, it has been
suggested that the regulated translocation of Bax from a cytosolic to
a mitochondrial distribution is necessary for the protein to induce
cell death36–38. This evidence indicates that Bcl-2 proteins may exert
a function at intracellular membranes.
At the membranes, the potential biochemical activity of the proteins may involve interactions with other proteins in a signal-transduction network. Bcl-2 proteins have the ability to interact
physically with other proteins. Among the numerous interacting
partners that have been reported, Bcl-2 proteins exhibit the strongest interactions with other members of the Bcl-2 family. However,
Bcl-2-like proteins can retain anti-apoptotic function despite mutaApaf-1
Cytochrome c
ATP
ADP
Apaf-1–cytochrome c
complex
formation
Regulation of cytochrome c redistribution
Caspase-9
Caspase activation
Apoptosis
Figure 1 Mitochondria-dependent caspase activation. Representation of the
pathway by which the mitochondrial intermembrane-space protein, cytochrome c,
initiates the activation of caspases. Cytochrome c and ATP induce formation of a
macromolecular complex containing a number of Apaf-1 molecules that recruits and
activates the death effector caspase-9. The pale pink area in the caspase-9 protein
represents a prodomains that is cleaved to activate the protein.
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tions that disrupt their ability to interact with other Bcl-2-family
proteins39. Nevertheless, such mutants are less efficient in preventing cell death than their wild-type counterparts, indicating that
interactions among Bcl-2 proteins play some part in the regulation
of their function39,40.
Bcl-2-like proteins have also been reported to interact directly
with non-Bcl-2 proteins, including the cell-death proteins CED-4
and Apaf-1 (refs 41–45). For the most part, it has not been possible to
generalize this ability to interact with other known death effector
molecules to other members of the Bcl-2 family. For instance,
although Apaf-1 has been shown to bind to Bcl-xL, it has also been
reported that it does not interact with other anti-apoptotic Bcl-2
proteins46. Thus, the interactions of Bcl-2 proteins with non-family
members may allow for the individual regulation of specific Bcl-2family members. Such interactions may allow signal-transduction
pathways to modulate the apoptotic function of specific Bcl-2 proteins. In support of this hypothesis, both Bcl-xL and Bcl-2 contain a
regulatory loop domain that can affect the ability of the proteins to
regulate death in response to specific apoptotic stimuli47. However,
studies of loop-domain-dependent interactions have failed to identify a biochemical mechanism that explains how Bcl-2 proteins function to regulate cell death.
Bcl-xL shows both structural and functional similarity to the
pore-forming domains of bacterial toxins48,49. In addition to Bcl-xL,
Bcl-2, Bax and cleaved Bid all form ion channels in planar lipid
bilayers50–52. Like the bacterial pore-forming proteins, the ion channels formed by Bcl-2 proteins tend to exhibit multiple conductance
states, and channel formation is favoured by changes in membrane
potential or pH. The channels are relatively non-selective, although
Bcl-2 and Bcl-xL channels have a preference for monovalent cations. The relevance of the ion-channel properties to the regulation
of cell death by Bcl-2 and Bcl-xL has been addressed using mutants
that swap a portion of the channel-forming domain of Bcl-2 or BclxL with that of Bax40,53. These mutants exhibit a reduced ability to
protect cells from Bax-induced death and from cell death resulting
from growth-factor withdrawal. Interestingly, the loss-of-function
Bcl-xL mutant with altered channel properties retains similar channel selectivity, but exhibits a decreased open time at negative membrane potentials40. This indicates that the ability to form functional
channels in the presence of a transmembrane potential may be
important for the anti-apoptotic properties of Bcl-xL. Whether
additional channel properties, such as ion selectivity or conductance, will also prove important remains to be determined.
The ability of Bcl-2 proteins to modulate cell death correlates in
many cases with their ability to inhibit or promote cytochrome c
redistribution. However, both the mechanism of cytochrome c
redistribution and its regulation by Bcl-2 remain controversial.
Cytochrome c release may occur through a specific channel in the
outer membrane that is either formed or regulated by Bcl-2 proteins (Fig. 2a)24,54. In this model, channel formation is a result of
apoptotic signal transduction, and the primary function of Bcl-2
proteins is to regulate apoptosis by controlling the cytochrome c
efflux channel. Alternatively, Bcl-2 proteins may regulate perturbations in mitochondrial physiology, which in turn are responsible for
the loss of outer-membrane integrity and cytochrome c redistribution (Fig. 2b)20,55.
A cytochrome c efflux pore could be either formed or regulated
by pro-apoptotic proteins. For instance, in vitro, Bax can form
large-conductance channels in synthetic lipid bilayers and cause
cytochrome c redistribution in the absence of large-amplitude
mitochondrial swelling24. Multimerization of Bax is important for
its pro-apoptotic function38. Alternatively, Bax may cooperate with
the principle outer-membrane channel, the voltage-dependent
anion channel (VDAC), to form a cytochrome c conducting
channel54. It has also been suggested that Bax can destabilize mem-
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branes directly56. Whether Bax-mediated membrane solubilization
or the formation of Bax and/or Bax-hybrid channels affects outermembrane permeability in vivo is uncertain.
Other studies have indicated that addition of a BH3 peptide
alone (the BH3 domain is found in all Bcl-2-family proteins) is sufficient to cause release of cytochrome c from isolated
mitochondria57. Amphipathic helices that insert into membranes
are thought to multimerize with their hydrophilic faces aligning to
form a central aqueous channel58. However, the BH3 domain is
located in the middle of the primary sequence of known Bcl-2related proteins. Therefore, it is not clear how this domain could
span a biological membrane, as it does not appear to be large
enough to cross a membrane twice.
If a channel in the outer membrane were to be responsible for
cytochrome c redistribution, it must be large and nonspecific. In
addition to cytochrome c, several other proteins and small molecules from the mitochondrial intermembrane space redistribute to
the cytosol upon the induction of apoptosis12,15–17,59–61. In fact, every
intermembrane-space molecule that has been studied so far redistributes concomitantly with cytochrome c. Furthermore, both antibodies and cytochrome c introduced into the cytosol are able to
cross the outer membrane after cytochrome c has been released20.
Thus the lesion responsible for cytochrome c redistribution must be
bidirectionally permeable to molecules with relative molecular
masses (Mr values) as large as 160,000 (160K).
a
?
?
VDAC
Cyt. c
Other
proteins
Specific channel
b
Alteration in
mitochondrial
function
The permeability-transition pore
The redistribution of all intermembrane-space proteins during
apoptosis indicates that cytochrome c release may result from a
nonspecific disruption of the outer membrane. One model that has
been proposed as an explanation for nonspecific rupture of the
outer membrane is opening of the mitochondrial permeabilitytransition pore62. This pore is a large-conductance channel proposed to span both mitochondrial membranes63. Opening of the
permeability-transition pore has been reported to be affected by
changes in calcium, voltage, pH and/or the redox environment. It
has been proposed that Bcl-2 proteins regulate gating of this pore64.
Opening of the pore is characterized by an abrupt increase in the
permeability of the inner mitochondrial membrane to molecules as
large as 1.5K. Apoptosis can be induced by agents that cause opening of the permeability-transition pore, including calcium, GD3
ganglioside and atractyloside64–66. Further evidence for the permeability-transition-pore model comes from experiments using
cyclosporin A or bongkrekic acid to prevent both pore opening and
cell death67. However, these data are controversial, because under
some conditions cyclosporin A appears to prevent cell death, while
in others it has no effect68. In addition, although cyclosporin A is a
transient inhibitor of permeability transition in vitro, it affects several cellular targets, raising the question as to whether its effects are
specific to mitochondria when used in whole cells.
The sudden increase in inner-membrane permeability caused
by the opening of the permeability-transition pore is predicted
to cause mitochondrial depolarization, the chemical equilibration of solutes across the membranes, and large-amplitude
swelling of the mitochondrial matrix. As the surface area of the
inner mitochondrial membrane is greater than the surface area
of the outer membrane, matrix swelling could rupture the outer
membrane 20,62.
As predicted by the permeability-transition-pore model, a disruption of the mitochondrial membrane potential is observed in
dying cells. However, a loss of the mitochondrial membrane potential, as well as all other actively generated gradients across cellular
membranes, is eventually observed in all dead cells. Although mitochondrial depolarization and the acute loss of ATP synthesis would
be expected to result in cell death, it remains uncertain whether the
loss of mitochondrial membrane potential occurs as an initiator,
rather than an effect, of apoptosis.
Bax
Loss of outer-membrane integrity
Figure 2 Proposed mechanisms to account for cytochrome c redistribution
during apoptosis. a, Intermembrane-space proteins, including cytochrome c (red),
may cross the outer membrane through a specific channel formed as the result of
an apoptotic signal. This channel may involve the voltage-dependent anion channel
(VDAC) and/or pro-apoptotic Bcl-2-family members such as Bax. b, Alternatively,
intermembrane-space proteins may be released from the intermembrane space as
a result of a nonspecific disruption of the outer mitochondrial membrane. This
disruption of the outer membrane could occur following alterations in mitochondrial
physiology that are the consequence of an apoptotic stimulus.
Several reports have shown that cytochrome c release can occur
before mitochondrial depolarization18–21. In addition, cell-sorting
experiments have shown that cells committed to die continue to
exhibit a mitochondrial membrane potential26. To account for these
observations, it has been proposed that the permeability-transition
pore may ‘flicker’ between an open and closed state, resulting in
rupture of the outer membrane while allowing the inner membrane
to restore a transmembrane potential62. However, although transient opening of the permeability-transition pore does occur69–71, it
does not appear to result in either cytochrome c redistribution or
cell death72. Alternative approaches by which to assess the opening
of the permeability-transition pore have been used in the study of
tumour necrosis factor (TNF)-mediated apoptosis: a cytosolic
fluorophore of Mr 623 gained access to mitochondria following
TNF-induced mitochondrial depolarization 73. However, it has also
been reported that, at the time of cytochrome c redistribution,
matrix proteins are not released and the inner membrane remains
impermeant to small molecules60,61. These data indicate that the primary structural perturbation in mitochondria that accompanies
cytochrome c release may be a disruption of the outer membrane,
not the inner membrane. As the permeability-transition pore is
proposed to span both mitochondrial membranes at sites of contact
between the two membranes, and to connect the matrix directly
with the cytosol74, it is unlikely to be the channel through which
cytochrome c gains access to the cytosol from the intermembrane
space. Further clarification of these important issues awaits a more
complete characterization of the permeability-transition pore.
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Cytoplasm
Cytoplasm
ATP
ADP
Bcl-2
HK
Bcl-2
VDAC
?
Glucose
VDAC
CK
ANT
S
Glucose-6-P
HK
S
Cr
CK
Cph D
Cr-P
H+
CsA
ANT
F0
ATP
Matrix
F1
Matrix
ADP
The permeability-transition pore
Figure 3 Proposed functions for the ANT and the VDAC in formation of the
permeability-transition pore and in oxidative phosphorylation. Left, the
adenine-nucleotide translocator (ANT) and the voltage-dependent anion channel
(VDAC) are proposed to be specialized components of the permeability-transition
pore, a large-conductance channel that promotes chemical equilibration between the
mitochondrial matrix and the cytoplasm. Other proteins proposed to be components
Components of the permeability-transition pore
Although the conductance properties of the permeability-transition pore have been studied for several years, the identity of its
molecular components remains controversial. It has been suggested
that the core components are the adenine-nucleotide translocator
(ANT) and the VDAC63. Each of these proteins has a defined function in mitochondrial adenine-nucleotide exchange that, when disrupted, might be expected to result in cell death independently of
an involvement in the activity of the permeability-transition pore
(Fig. 3). Other proteins have been proposed as regulatory components of the permeability-transition pore on the basis primarily of
their association with either the ANT or the VDAC74,75. These proteins include the intermembrane-space protein creatine kinase,
cytosolic hexokinase, and matrix cyclophilin D. These proteins have
metabolic functions that may account for their association with the
ANT and/or the VDAC. For instance, creatine kinase functions to
generate creatine phosphate from ATP as a cellular high-energy
phosphate buffer76. An association with the ANT and the high
ATP:ADP ratio in the intermembrane space facilitate this function.
Similarly, as hexokinase catalyses one of the ATP-dependent, ratelimiting steps in glycolysis, an association between hexokinase and
the VDAC would facilitate access of the enzyme to ATP77.
There have been numerous mechanisms proposed by which Bcl2, Bcl-xL and Bax might act as regulators of the permeability-transition pore. For instance, despite their apparent localization to different membranes, it has been reported that Bax can interact
directly with the ANT and promote opening of the pore78. Furthermore, Bax can be co-purified with the putative components of the
permeability-transition pore, while Bcl-2 and Bcl-xL act as peripheral components that regulate pore opening79. It has also been suggested that Bax and Bak can regulate cytochrome c release and the
permeability-transition pore by binding the VDAC directly80. This
model has been extended to propose that Bcl-xL binding maintains
the VDAC in a cytochrome c impermeant configuration, while Bax
induces a larger open state for the VDAC that allows cytochrome c
release and triggers opening of the permeability-transition pore54.
Regulation of mitochondrial homeostasis by Bcl-2
An alternative to considering the permeability-transition pore as a
physiological control point for apoptosis was suggested by the
observation that Bcl-xL could affect the regulation of ATP/ADP
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Conventional properties of the ANT and VDAC
of the permeability-transition pore include hexokinase (HK), creatine kinase (CK) and
cyclophilin D (Cph D). Right, the more established role of the VDAC and ANT in
adenine-nucleotide transport, and the more conventional function of HK in the
phosphorylation of glucose to glucose-6-phosphate (glucose-6-P), and of CK in the
phosphorylation of creatine (Cr) to creatine phosphate (Cr-P).
exchange between mitochondria and the cytosol26. In these studies,
mitochondrial ATP/ADP exchange was impaired in cells that were
deprived of growth factors. The cytosolic ATP:ADP ratio fell while
the mitochondrial ATP:ADP ratio increased. This result was the
opposite from what would be predicted if the permeability-transition pore were opening, or even flickering, as pore opening should
function to dissipate chemical gradients between the cytosol and
the matrix. These data were more consistent with the traditional
view that the VDAC and ANT are the outer and inner membrane
proteins responsible for adenine-nucleotide transport between the
cytosol and the matrix. A perturbation in the function of one or the
other would lead to the ATP/ADP imbalance observed following
growth-factor withdrawal.
An explanation for cytochrome c release in this model is based
on the observation that mitochondria swell, and therefore may rupture, when the rate of electron transport decreases81, and that mitochondrial swelling is inhibited by expression of Bcl-xL20. A
decreased rate of electron transport has been observed in growthfactor-deprived cells before they undergo apoptosis20. Electron
transport stalls when the ability of the electron-transport chain to
pump protons into the intermembrane space becomes limited by
hyperpolarization of the inner mitochondrial membrane. Innermembrane hyperpolarization has been reported to occur in
response to multiple apoptotic stimuli20,82,83.
A relative increase in mitochondrial membrane potential during
apoptosis could be explained by a defect in mitochondrial ATP/
ADP exchange26. If ADP return to the mitochondrial matrix
becomes limiting, the ATP synthase, because of a lack of its substrate (ADP), cannot use the electrochemical potential across the
inner mitochondrial membrane. Limitation of the substrate of the
ATP synthase will thus result in mitochondrial hyperpolarization.
This, in turn, would cause an increase in the generation of reactive
oxygen species from the electron-transport chain. Falling cytosolic
ATP levels would also stimulate glycolysis, resulting in increased
lactate generation and cytosolic acidification. Persistent hyperpolarization would ultimately lead to matrix swelling and cytochrome
c redistribution.
Several factors are important in the regulation of mitochondrial
volume. The negative-inside mitochondrial-membrane potential
requires mechanisms by which to limit the accumulation of matrix
cations and subsequent mitochondrial swelling as a result of electrogenic uptake. To achieve this, the inner membrane has a low per-
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meability to charged molecules. In addition, mitochondria have
evolved mechanisms by which to carry out the electoneutral
exchange of protons for cations, the most important of which is
potassium (K+). Thus, mechanisms that regulate the accumulation
of K+ ions are important in the control of mitochondrial volume84,85.
However, mitochondrial swelling can only occur as a result of the
net movement of osmoles, and an osmotically important movement of ions across a closed membrane must involve electrically
neutral uptake of both cations and anions.
Although the proton gradient present across the mitochondrial
inner membrane can be used to drive the efflux of matrix K+ ions,
this gradient also provides a pathway that allows the accumulation
of anions in the matrix. The accumulation of protons in the intermembrane space results in the neutralization of weak acids, which,
in a non-charged state, become permeant to the inner membrane.
Once across the membrane, the acids are deprotonated in the alkaline environment of the matrix. In mitochondria undergoing coupled respiration these weak organic acids are metabolized in the
matrix. As a result, the accumulation of K+ ions in the matrix is limited by a lack of compensatory anion movement. Thus, during
active respiration, the concentration of K+ ions and matrix volume
can be effectively regulated by protogenic efflux from the matrix.
However, if electron transport is stalled as a result of ADP limitation and hyperpolarization, unmetabolized anions accumulate in
the matrix. Osmotic stress occurs because of the accompanying
increase in the leak of cations across the membrane as a result of
hyperpolarization. Although this process might initially be limited
by H+/K+ exchange across the membrane, over time persistent
hyperpolarization will result in the accumulation of weak acids in
the matrix and increased retention of cations to maintain charge
balance. Unlike the large-amplitude swelling reported to occur after
opening of the permeability-transition pore, the osmotic swelling
associated with membrane hyperpolarization occurs gradually,
with a time course consistent with the onset of apoptosis, which
usually occurs several hours after the apoptotic stimulus.
Bcl-xL and/or Bcl-2 promote mitochondrial adenine-nucleotide
exchange and prevent mitochondrial hyperpolarization26. Bcl-xL
appears to accomplish this by maintaining mitochondrial membrane permeability; however, the predicted diameter of the Bcl-xL
pore is not large enough to allow permeability to either ADP or
ATP. Therefore, if the channel properties of Bcl-xL function to
maintain mitochondrial ATP/ADP transport, the effect must be
indirect. An attractive candidate for an outer-membrane protein
that Bcl-xL could affect indirectly is the VDAC. When the VDAC
adopts a closed configuration, it exhibits a decreased permeability
to complex anions, including adenine nucleotides86,87. Interestingly,
the same transmembrane potential that would induce VDAC closure would also favour the formation of Bcl-xL channels. Therefore,
the ability of Bcl-xL to dissipate electrochemical gradients may
function to maintain the VDAC and/or other outer-membrane
channels in a configuration competent to pass anions such as ADP.
The ability of Bcl-xL to regulate the open state of VDAC electrophysiologically would not require a direct physical interaction
between the proteins. However, the pro-apoptotic protein Bax can
interact with the VDAC to form a cytochrome c permeant
channel54. In these experiments, Bcl-xL prevented the Bax-induced
permeability of the VDAC to cytochrome c. Although this study
identified the VDAC as a potential binding target for the Bcl-2 family of proteins, it leaves open the question of whether or not Bcl-xL
regulates the function of the VDAC or works by antagonizing the
effects of Bax.
What is the point of no return during apoptosis?
A feature of Bcl-2 proteins is their ability to regulate cell death
upstream of the commitment to death88. The fact that Bcl-2 proteins can control mitochondrial homeostasis indicates that mitochondrial damage may be a fatal cellular lesion. The ability of
mitochondria to function normally is critical to meet the energy
demands of most eukaryotic cells. Classically, necrotic death has
been associated with mitochondrial perturbations89. The discovery
that Bcl-2 proteins can protect cells from necrotic stimuli, including
direct mitochondrial poisons, supports the idea that mitochondrial
damage might represent a commitment point in some forms of
apoptotic death. Their ability to inhibit necrotic cell death further
suggests that Bcl-2 proteins regulate a more fundamental aspect of
mitochondrial physiology than just the redistribution of cytochrome c20,90.
Substrate
Substrate
Limited
Mitochondrial
Damage
Extensive
mitochondrial
damage
OR
Autophagy
Cytochrome c
release
Inhibitory factors
(IAP proteins? others?)
ATP
Necrosis
Survival
Caspase activation
Apoptosis
Figure 4 Possible consequences for cell survival following mitochondrial
damage. Mitochondrial homeostasis is dependent on a continuous supply of
substrates for the regulation of oxidative phosphorylation.Limited mitochondrial
damage may not result in apoptosis if damaged mitochondria (red) are removed
before they release cytochrome c (by the process of autophagy), or if caspase
activation is prevented after cytochrome c is released. In contrast, extensive
mitochondrial damage may be incompatible with cell survival and the mechanism of
death determined by the availability of ATP.
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Cell-sorting experiments have shown that cells with evidence
of mitochondrial damage following growth-factor withdrawal are
committed to die26. In addition, caspase inhibitors are unable to
prevent cell death under all circumstances20,91,92. In these cases, caspase inhibitors are sufficient to inhibit the development of apoptotic morphology but are unable to rescue cell death. On the other
hand, the ability of cytochrome c to activate caspases through
Apaf-1 does contribute to the initiation of cell death under some
circumstances. For instance, Apaf-1-deficient mouse embryonic
fibroblasts form tumours with increased frequency in vivo, indicating that Apaf-1-dependent caspase activation may help to prevent inappropriate cell survival93. In addition, two studies have
shown that sympathetic neurons can still be recovered after cytochrome c has been released, provided that caspases are not
activated94,95. Perhaps, in some cells, mechanisms have evolved
that enable cell survival despite brief periods of stress that result in
mitochondrial damage. Thus, although enough mitochondrial
damage may be sufficient to cause cell death, caspase activation is
required for death when the cell has sustained sublethal mitochondrial damage.
This raises the question of how much mitochondrial damage is
necessary to kill the cell. Limited mitochondrial damage might
release an amount of cytochrome c that, although not fatal by itself,
might result in cell death if the caspase cascade is initiated (Fig. 4).
For example, growth-factor-deprived neurons appear to survive
having most of their mitochondria release cytochrome c, if cytochrome c dependent caspase activation is prevented94. Similarly, this
might also explain how Apaf-1 acts as a tumour supressor in vivo93.
Loss of Apaf-1 function may allow potential tumour cells to adapt
to glycolysis-dependent survival when mitochondria are damaged
as a result of tumour anoxia, nutrient deprivation or growth-factor
limitation. Although endogenous caspase inhibitors, such as the
inhibitor of apoptosis (IAP) proteins, are candidates for influencing
a cell’s sensitivity to cytochrome c96, the factor(s) that determines
how much cytochrome c redistribution is required for death is
unknown.
The idea that a threshold amount of mitochondrial damage is
required for cell death indicates that cell survival might also be controlled by the removal of damaged organelles. Autophagy, or programmed organelle degradation, might affect cell-death regulation
by removing damaged mitochondria97,98. The programmed removal
of mitochondria might also contribute to cell survival under conditions of metabolic suppression, such as growth-factor withdrawal.
A decrease in substrate available to individual mitochondria as a
result of reduced cellular metabolism is one possible trigger for
mitochondrial dysfunction that might lead to cell death. If the
number of mitochondria could be decreased in proportion to the
overall decrease in cellular metabolism, then substrate limitation of
the remaining mitochondria would be alleviated. In this way, the
autophagy of mitochondria may influence cell survival by promoting homeostasis of the remaining mitochondria (Fig. 4).
Apoptosis versus necrosis and the role of mitochondria
The idea that the bioenergetic processes fundamental to support life
are linked to the cell-death pathway is a satisfying one. It is perhaps
not surprising that apoptosis often involves damage to mitochondria. In fact, the morphology of death — that is, whether it is apoptotic or necrotic — is probably itself linked to metabolism. Cellular
ATP levels can influence whether a dying cell exhibits the features
characteristic of apoptosis or necrosis99,100. Consistent with the
observation that both caspase activity and ATP are required for
apoptotic cell death, ATP or dATP (which are interconverted by a
single enzymatic reaction) is required for cytochrome c/Apaf-1dependent caspase activation6–8. Necrosis and apoptosis may be
more similar than currently believed. For example, if mitochondrial
cytochrome c release occurs in a cell with high enough ATP levels,
caspase-9 activation results and apoptotic death is initiated. HowE214
ever, in a cell depleted of ATP, cytochrome c release may lead to a
permanent impairment in oxidative phosphorylation and necrosis.
Conclusions
Cell death results when the processes necessary to support life can
no longer be sustained. Mitochondrial function is necessary for survival. Therefore, a disruption of mitochondrial function results in
death. A link between the initiation of apoptosis and the release of
an essential mitochondrial protein may have been evolutionarily
selected for, because it allows the cell to recognize the impairment
in mitochondrial function and initiate death in an orderly fashion.
All of the components necessary to initiate apoptosis are locally
concentrated in and around the outer mitochondrial membrane.
Bcl-2 proteins are strategically poised at the outer mitochondrial
membrane to enable the coupling of cytosolic metabolism to mitochondrial physiology. By promoting mitochondrial adaptation to
perturbations in cellular metabolism, Bcl-2-like proteins have the
ability to promote cell survival.
The involvement of mitochondria in cell death indicates that
maintenance of the symbiotic relationship between the mitochondrion and the cytosol is a requirement for eukaryotic cell survival.
The ability to couple cytosolic energy demands to mitochondrial
function may therefore reflect a requirement for Bcl-2 proteins in
the establishment of mitochondrial endosymbiosis. A regulated
exchange of ions must occur not only across the mitochondrial
membranes but also across all intracellular membranes to allow for
proper cellular physiology. Bcl-2 proteins also reside in the endoplasmic reticular and outer nuclear membranes, and mutant proteins targeted exclusively to these sites still function to inhibit cell
death under some conditions101. Perhaps the true cellular function
of Bcl-2 proteins is to establish homeostasis across intracellular
membranes, and perhaps it is this ability that also allows these proteins to contribute to the regulation of cell survival.
h
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ACKNOWLEDGEMENTS
We thank members of the Thompson laboratory for their critique of the manuscript and S. Kerns for
expert editorial assistance.
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