Download ZAMZAMI N, KROEMER G, 2001. The mitochondrion in apoptosis

PERSPECTIVES
agonists and antagonists. Cell 96, 625–634 (1999).
16. Shimizu, S. & Tsujimoto, Y. Proapoptotic BH3-only Bcl-2
family members induce cytochrome c release, but not
mitochondrial membrane potential loss, and do not
directly modulate voltage-dependent anion channel
activity. Proc. Natl Acad. Sci. USA 97, 577–582 (2000).
17. von Ahsen, O. et al. Preservation of mitochondrial
structure and function after Bid- or Bax-mediated
cytochrome c release. J. Cell Biol. 150, 1027–1036
(2000).
18. Chai, J. C. et al. Structural and biochemical basis of
apoptotic activation by Smac/DIABLO. Nature 406,
855–862 (2000).
19. Antonsson, B., Montessuit, S., Lauper, S., Eskes, R. &
Martinou, J.-C. Bax oligomerization is required for
channel-forming activity in liposomes and to trigger
cytochrome c release from mitochondria. Biochem. J.
345, 271–278 (2000).
20. Gross, A., Jockel, J., Wei, M. C. & Korsmeyer, S. J.
Enforced dimerization of BAX results in its translocation,
mitochondrial dysfunction and apoptosis. EMBO J. 17,
3878–3885 (1998).
21. Cheng, E. H.-Y. et al. Conversion of Bcl-2 to a Bax-like
death effector by caspases. Science 278, 1966–1968
(1997).
22. Saito, M., Korsmeyer, S. J. & Schlesinger, P. H. Baxdependent transport of cytochrome c reconstitued in
pure liposomes. Nature Cell Biol. 2, 553–555 (2000).
23. Gilbert, R. J. C. et al. Two structural transitions in
membrane pore formation by pneumolysin, the poreforming toxin of Streptococcus pneumoniae. Cell 97,
647–655 (1999).
24. Bernardi, P., Scorrano, L., Colonna, R., Petronilli, V. & Di
Lisa, F. Mitochondria and cell death. Eur. J. Biochem.
264, 687–701 (1999).
25. Crompton, M. The mitochondrial permeability transition
pore and its role in cell death. Biochem. J. 341, 233–249
(1999).
26. Eskes, R., Desagher, S., Antonsson, B. & Martinou, J.-C.
Bid induces the oligomerization and insertion of Bax into
the outer mitochondrial membrane. Mol. Cell. Biol. 20,
929–935 (2000).
27. Marzo, I. et al. Bax and adenine nucleotide translocator
cooperate in the mitochondrial control of apoptosis.
Science 281, 2027–2031 (1998).
28. Shimizu, S., Narita, M. & Tsujimoto, Y. Bcl-2 family
proteins regulate the release of apoptogenic cytochrome
c by the mitochondrial channel VDAC. Nature 399,
483–487 (1999).
29. Shimizu, S., Ide, T., Yanagida, T. & Tsujimoto, Y.
Electrophysiological study of a novel large pore formed by
Bax and the voltage-dependent anion channel that is
permeable to cytochrome c. J. Biol. Chem. 275,
12321–12325 (2000).
30. Shimizu, S., Konishi, A., Kodoma, T. & Tsujimoto, Y. BH4
domain of antiapoptotic Bcl-2 family members closes
voltage-dependent anion channel and inhibits apoptotic
mitochondrial changes and cell death. Proc. Natl Acad.
Sci. USA 97, 3100–3105 (2000).
31. Vander Heiden, M. G., Chandel, N. S., Schumacker, P. T.
& Thompson, C. B. Bcl-xL prevents cell death following
growth factor withdrawal by facilitating mitochondrial
ATP/ADP exchange. Mol. Cell 3, 159–167 (1999).
32. Vander Heiden, M. G. et al. Outer mitochondrial
membrane permeability can regulate coupled respiration
and cell survival. Proc. Natl Acad. Sci. USA 97,
4666–4671 (2000).
33. Zhuang, J., Dinsdale, D. & Cohen, G. M. Apoptosis, in
human monocytic THP.1 cells, results in the release of
cytochrome c from mitochondria prior to their
ultracondensation, formation of outer membrane
discontinuities and reduction in inner membrane
potential. Cell Death Differ. 5, 953–962 (1998).
34. Kroemer, G., Dallaporta, B. & Resche-Rigon, M. The
mitochondrial death/life regulator in apoptosis and
necrosis. Annu. Rev. Physiol. 60, 619–642 (1998).
35. Gross, A. et al. Biochemical and genetic analysis of the
mitochondrial response of yeast to Bax and Bcl-xL. Mol.
Cell. Biol. 20, 3125–3136 (2000).
36. Priault, M., Chaudhuri, B., Clow, A., Camougrand, N. &
Manon, S. Investigation of Bax-induced release of
cytochrome c from yeast mitochondria. Eur. J. Biochem.
260, 684–691 (1999).
37. Kissova, I. et al. The cytotoxic action of Bax on yeast cells
does not require mitochondrial ADP/ATP carrier but may
be related to its import to the mitochondria. FEBS Lett.
471, 113–118 (2000).
38. Priault, M., Camougrand, N., Chaudhuri, B., Schaeffer, J.
& Manon, S. Comparison of the effects of Bax-expression
in yeast under fermentative and respiratory conditions:
investigation of the role of adenine nucleotides carrier and
cytochrome c. FEBS Lett. 456, 232–238 (1999).
39. Pastorino, J. G., Chen, S.-T., Tafani, M., Snyder, J. W. &
Farber, J. L. The overexpression of Bax produces cell
death upon induction of the mitochondrial
permeability transition. J. Biol. Chem. 273, 7770–7775
(1998).
40. Bossy-Wetzel, E., Newmeyer, D. D. & Green, D. R.
Mitochondrial cytochrome c release in apoptosis occurs
upstream of DEVD-specific caspase activation and
independently of mitochondrial transmembrane
depolarization. EMBO J. 17, 37–49 (1998).
41. Zhivotovsky, B., Orrenius, S., Brustugun, O. T. &
Doskeland, S. O. Injected cytochrome c induces
apoptosis. Nature 391, 449–450 (1998).
42. Deshmukh, M. & Johnson, E. M. Evidence of a novel
event during neuronal death: development of
competence-to-die in response to cytoplasmic
cytochrome c. Neuron 21, 653–655 (1998).
43. Green, D. Apoptotic pathways: papers wraps stone
blunts scissors. Cell 102, 1–4 (2000).
44. Desagher, S. et al. Bid-induced conformational change of
Bax is responsible for mitochondrial cytochrome c release
during apoptosis. J. Cell Biol. 144, 891–901 (1999).
45. Knudson, C. M. & Korsmeyer, S. J. Bcl-2 and Bax
function independently to regulate cell death. Nature
Genet. 16, 358–363 (1997).
46. Kane, D. J., Ord, T., Anton, R. & Bredesen, D. E.
Expression of Bcl-2 inhibits necrotic neuronal cell death.
J. Neurosci. Res. 40, 269–275 (1995).
Acknowledgements.
We thank B. Antonsson, S. Desagher, M. Koco–Vilbois, K.
Maundrell, S. Montessuit, O. Terradillos and X. Roucou for
critical reading of the manuscript.
OPINION
The mitochondrion in apoptosis:
how Pandora’s box opens
Naoufal Zamzami and Guido Kroemer
There is widespread agreement that
mitochondria have a function in apoptosis,
but the mechanisms behind their
involvement remain controversial. Here we
suggest that opening of a multiprotein
complex called the mitochondrial
permeability transition pore complex is
sufficient (and, usually, necessary) for
triggering apoptosis.
As with Pandora’s box, the mitochondrion is
full of potentially harmful proteins and biochemical reaction centres. Loss of subcellular
and submitochondrial compartmentalization,
then, may liberate a flood of toxic compounds,
such as reactive oxygen species and proteins
that can activate catabolic hydrolases. In most
pathways leading to apoptosis, permeabilization of the inner and outer mitochondrial
membranes (IMM and OMM, respectively) is
critical1,2 (BOX 1). This culminates in the release
of certain inactive caspase precursors (procaspases), cytochrome c (a caspase activator),
Smac (second mitochondria-derived activator
of caspase, also known as DIABLO, which is a
caspase co-activator) and apoptosis-inducing
factor (AIF; a nuclease activator), as well as a
plethora of other proteins from the intermembrane space. All of these proteins must pass
through the OMM3, but the IMM is also
affected (although its permeabilization may be
transient, and is partial for solutes up to 1.5
kDa, allowing proteins in the mitochondrial
matrix to be retained).
Box 1 | Evidence for the involvement of mitochondria in controlling cell death
• Mitochondrial membrane permeabilization (MMP), which can affect both the inner and
outer mitochondrial membranes, precedes the signs of necrotic or apoptotic cell death,
including the apoptosis-specific activation of caspases.
• MMP is a more accurate predictive parameter for cell death than caspase activation
(which often is not required for cell death to occur and, in contrast, may also participate
in positive signalling).
• Many pro-apoptotic proteins and second messengers act on mitochondria to induce MMP.
Such signalling molecules include the pro-apoptotic members of the Bcl-2 family,
phosphatases and kinases acting on Bcl-2-like proteins, as well as transcription factors
(for example, p53 and TR3/Nur-77/NGFI-B).
• Bcl-2 and related anti-apoptotic proteins are present in mitochondrial membranes and
prevent apoptosis by suppressing MMP.
• MMP-mediated release of caspase and nuclease activators is required for full-blown apoptotic
cell death. Such activators are normally sequestered in the intermembrane space and include
cytochrome c, Smac/DIABLO and apoptosis-inducing factor (AIF).
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
VOLUME 2 | JANUARY 2001 | 6 7
© 2001 Macmillan Magazines Ltd
PERSPECTIVES
Many proteins from the Bcl-2/Bax family
regulate apoptosis when locally present in
mitochondrial membranes (see BOX 1 in the
article by Martinou and Green on page 63).
As a rule, close homologues of Bcl-2 (for
example, Bcl-xL, Bcl-W, Mcl1 and A1) reside
in the mitochondria and stabilize the barrier function of the mitochondrial membranes. Pro-apoptotic proteins can shuttle
between a non-mitochondrial localization
(the cytosol for Bax, Bad and Bid; microtubules for Bim and mitochondria). Upon
induction of apoptosis, these proteins insert
into mitochondrial membranes and cause
them to be permeabilized.
The controversy surrounds the mode of
action of these Bcl-2 family members2,4,5. Do
they induce permeabilization of mitochondrial membranes in an autonomous fashion,
without the need for interactions with sessile
mitochondrial proteins? Or do they act on a
multiprotein complex called the permeability
transition pore complex (PTPC)?
The PTPC regulates cell death
The PTPC — or mitochondrial megachannel — is formed at the contact site between
the IMM and OMM (FIG. 1). Its core components are the adenine nucleotide translocator (ANT, found in the IMM) and the volt-
Box 2 | The mitochondrial permeability transition
Definition. The mitochondrial permeability transition involves a sudden (and initially
reversible) increase in permeability of the IMM to solutes up to 1.5 kDa. It is commonly defined
by its inhibition by cyclosporin A or derivatives of this compound that bind to mitochondrial
cyclophilin (peptidyl-prolyl-cis–trans-isomerase) such as N-methyl-Val-4-cyclosporin A.
Cyclosporin A-mediated inhibition of the permeability transition is transient (lasting 60 min).
Regulation. The permeability transition pore complex (PTPC) functions as a sensor for:
• Voltage: The PTPC decodes voltage changes into variations of the probability (the ‘gating
potential’) at which pore opening occurs. Pore agonists shift the gating potential to more
negative values (physiological = 200 mV, negative inside), favouring pore opening, whereas pore
antagonists favour its closure.
• Divalent cations: Matrix Ca2+ increases the probability of pore opening. Matrix Mg2+ or Mn2+,
and external divalent metal ions including Ca2+ all decrease the probability of pore opening.
• Matrix pH: The permeability transition pore is closed at neutral or acidic pH owing to
reversible protonation of histidine residues and/or inhibition of the interaction between matrix
cyclophilin and the ANT. Alkalinization is permissive for pore opening with a maximum effect
at a matrix pH of ~7.3.
• Thiol oxidation: Oxidation (disulphide formation) of a critical mitochondrial dithiol
(presumably cysteine 56 of the ANT dimer) increases the probability of pore opening. The
redox status of this dithiol is in equilibrium with that of matrix glutathione.
• Oxidation/reduction state of pyridine nucleotides (NADH/NAD+ and NADPH/NADP+).
Oxidation of pyridine nucleotides favours permeability transition.
• ANT ligands: The endogenous ANT ligand ADP as well as bongkrekate inhibit permeability
transition. Atractyloside, another ANT ligand, induces permeability transition.
• Metabolites: Glucose and creatine inhibit permeability transition, presumably through their
action on hexokinase and creatine kinase. Ubiquinone O (coenzyme Q) also inhibits
permeability transition. Long-chain fatty acids, ceramide and ganglioside GD3 favour
permeability transition.
• Anti- and pro-apoptotic members of the Bcl-2 family.
Metabolic consequences. Full-blown permeability transition causes uncoupling of the
respiratory chain with collapse of the electrochemical proton gradient ∆Ψm and cessation of ATP
synthesis, matrix Ca2+ outflow, depletion of reduced glutathione, depletion of NADPH,
hypergeneration of superoxide anion, and mitochondrial release of intermembrane proteins.
Several of the consequences of permeability transition themselves favour opening of the
permeability transition pore, implying that permeability transition is a self-amplifying process.
Physiological function. Periodic reversible opening of the permeability transition pore allows for
the release of Ca2+ from the mitochondrial matrix, thereby participating in Ca2+ homeostasis
and/or the generation of Ca2+ waves (Ca2+-induced Ca2+-release). A role in neuronal plasticity has
been suggested. The ANT/VDAC couple (and its interacting proteins hexokinase and creatine
kinase) may also participate in regulating ATP/ADP transport/synthesis. Irreversible permeability
transition triggers mitochondrial autophagy (a process by which cells digest parts of their
cytoplasm), apoptosis or necrosis.
68
| JANUARY 2001 | VOLUME 2
age-dependent anion channel (VDAC,
located in the OMM).
The VDAC is normally permeable to
solutes of up to 5 kDa, thereby allowing the
free exchange of respiratory-chain substrates
such as NADH, FADH and ATP/ADP
between the mitochondrial intermembrane
space and the cytosol. In contrast, the IMM is
almost impermeable — a feature that is essential for generation of the electrochemical proton gradient (∆ψm) used for oxidative phosphorylation. But the permeability of both
membranes may suddenly increase in vitro —
in isolated mitochondria, for instance, after
the addition of atractyloside (which is a ligand
of the ANT) — and this ‘permeability transition’6 is mediated by the PTPC (BOX 2).
As well as the abundant IMM and OMM
proteins — ANT and VDAC respectively —
the PTPC contains the peripheral benzodiazepine receptor (located in the OMM), creatine kinase (located in the intermembrane
space), hexokinase II (tethered to the VDAC
on the cytosolic face of the OMM), cyclophilin
D (located in the mitochondrial matrix), as
well as Bax/Bcl-2-like proteins (FIG. 1).
Direct interactions have been shown for
the ANT and cyclophilin D, as well as between
the ANT and VDAC6,7. As a consequence of
such interactions, changes in the conformation of the ANT (which are indirectly affected
by cyclosporin A, which affects the cyclophilin
D–ANT interaction) may indirectly impinge
on the function of the VDAC, or vice versa. So
the PTPC may simultaneously control permeability of the OMM and the IMM.
Alternatively, initial permeabilization of
the IMM may cause the mitochondrial
matrix to swell (through osmosis), leading to
rupture of the OMM. (The IMM, with its
folded christae, does not burst as its surface
area is greater than that of the OMM.)
Ultrastructural evidence for matrix swelling
or rupture of the OMM has been obtained in
many models of cell death, including hepatocyte apoptosis induced by injection of an
antibody recognizing the CD95 ‘death receptor’ in the plasma membrane8. This particular
phenotype is deficient in knockout mice that
lack either of the two pro-apoptotic proteins
from the Bcl-2 family (Bid or Bak) or overexpress a Bcl-2 transgene in the liver9. However,
mitochondrial swelling and membrane rupture is not a universal feature of apoptosis10–13,
suggesting alternative possibilities of membrane permeabilization, including the formation of pores in the OMM.
Many reports show that apoptosis correlates with signs of a permeability transition,
such as loss of the mitochondrial transmembrane potential (∆ψm); that induction of a
www.nature.com/reviews/molcellbio
© 2001 Macmillan Magazines Ltd
PERSPECTIVES
permeability transition is enough to trigger
cell death; and that, by inhibiting this permeability transition, cell death can be prevented
(for reviews see REFS 6,14). For example,
cyclosporin A (and N-methyl-4-Valcyclosporin A, which is a non-immunosuppressive derivative that acts on cyclophilin
D) can inhibit apoptosis induced in vivo by
brain trauma15, by ischaemia reperfusion
damage6, or by injection of an antibody
against CD95 (which mainly affects hepatocytes)8. Moreover, bongkrekate, an ANT ligand that inhibits the PTPC, prevents apoptosis induced by diverse stimuli including
glucocorticoids (in thymocytes)14, excitotoxins (in neurons)16 and tumour necrosis factor (in hepatocytes)17. However, such pharmacological inhibitors are not universally
cytoprotective, implying that permeabilization of the mitochondrial membrane may
involve individual PTPC components (for
example, the VDAC without the
ANT/cyclophilin D) or occur in a completely
PTPC-independent fashion.
Several viral or bacterial proteins that modulate apoptosis also interact with components
of the PTPC: for example, Vpr from HIV-1
(which binds the ANT and is pro-apoptotic)18;
vMIA/UL37 from cytomegalovirus (which
binds the ANT and is anti-apoptotic)19; the
hepatitis virus B X-protein (which binds the
VDAC and is pro-apoptotic)20; and porin B
from Neisseria meningidis (which binds the
VDAC and is anti-apoptotic)21.
Bcl-2 and Bax interact with the PTPC
Co-immunoprecipitation studies indicate
that Bcl-2 and Bax can interact — directly
or indirectly — with the VDAC in the
OMM22. Bcl-2, Bcl-xL, Bax and Bak interact
directly with the ANT, as shown by three
independent methods: co-purification, coimmunoprecipitation and yeast two-hybrid
screening23. The two-hybrid screen revealed
that a short stretch of human ANT2 (amino
acids 105–156) suffices for the interaction
with Bcl-2-related proteins23. The apoptosisregulatory potential of this portion of the
ANT has been confirmed by deletion mapping: overexpression of full-length mouse
Ant1 (which has 95% amino-acid identity
with human ANT2) or its amino-terminal
half (amino acids 1–141) induces apoptosis,
but expression of an even shorter truncation
mutation (amino acids 1–101) does not. So
the Bcl-2/Bax-binding site (amino acids
105–156) overlaps with the apoptosis-regulatory region of ANT (amino acids
102–141), as well as with its Vpr-binding
consensus motif (WXXF; amino acids
109–113). Interestingly, a substitution in the
Glucose-6phosphate
+ ADP
Glucose
+ ATP
Kinases
Phosphatases
pH change
Caspases
HK II
HVB-X
Porin B
BH3 peptides
Bax
Bcl-2
PBR
OMM
VDAC
VDAC
Creatine
+ ATP
mtCK
Creatine-P
+ ADP
vMIA
Vpr
Atractyloside/palmitate
Bongkrekate/ATP/ADP
ANT
ANT
∆ψm
IMM
Cardiolipin
Ca2+/oxidation?
Cyclosporin A
Cyp-D
Figure 1 | Hypothetical molecular architecture of the permeability transition pore complex and its
regulation. The permeability transition pore complex (PTPC) involves several transmembrane proteins:
the adenine nucleotide translocator (ANT), the voltage-dependent anion channel (VDAC) and the
peripheral benzodiazepine receptor (PBR). It also involves members of the Bax/Bcl-2 family, as well as
associated proteins such as hexokinase II (HK II), mitochondrial creatine kinase (mtCK) and the
peptidyl–prolyl isomerase cyclophilin D (Cyp-D). The VDAC functions as a nonspecific pore, allowing
diffusion of solutes up to 5 kDa. The ANT is responsible for exchange of ATP and ADP on the inner
membrane. The exact function of the PBR is unknown. Agents or metabolites labelled in green facilitate
PTPC opening; agents in red inhibit pore opening. Proteins or peptides carrying the Bcl-2 homology
region-3 (BH3) motif may act on either Bax or Bcl-2 (or their homologues) in the outer mitochondrial
membrane. Ca2+ has been postulated to act on ANT-associated cardiolipin molecules; cyclosporin A acts
on cyclophilin D. HIV-1 Vpr, the viral mitochondrial inhibitor of apoptosis (vMIA), hepatitis virus B X-protein
(HVB-X), and Neisseria meningitidis porin B also act on the PTPC. (OMM, outer mitochondrial membrane;
IMM, inner mitochondrial membrane; ∆ψm electrochemical proton gradient.)
highly conserved alanine residue 114 in
ANT1 causes a dominant human mitochondriopathy24. Whether this pathology is
related to a deficient control of apoptosis,
however, remains unclear.
There is still controversy about where
the Bcl-2-like proteins act. Most authors
suggest that these proteins are found mainly in the OMM, although a few reports
indicate that Bcl-2 (REFS 25,26) and Bax23 are
localized to the IMM. Pro-apoptotic stimuli
may cause Bax to translocate from the
OMM to the IMM23 or cause Bcl-2 to move
from the IMM to the OMM26, using as-yetunknown mechanisms. It is also conceivable that protruding domains of Bcl-2 and
Bax, which are anchored to the OMM, bind
to the IMM within the OMM–IMM contact sites. Indeed, Bcl-2 is particularly
enriched at these contact sites.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
Bcl-2 and Bax act on the PTPC
The biochemical features of pore-forming
proteins can be studied by reconstituting
them into synthetic lipid bilayers, either in
proteoliposomes or in planar membranes. In
response to various pro-apoptotic agents,
including atractyloside23, Ca2+, the thiol
crosslinker diamide27 and the HIV-1 protein
Vpr18, ANT proteoliposomes become permeabilized to hydrophilic compounds with a relative molecular mass of less than 1.5 kDa.
Proteoliposomes containing both the ANT
and recombinant Bax show higher permeabilization responses to atractyloside23or Vpr18
than do proteoliposomes containing either
ANT or Bax alone. In contrast, Bcl-2 prevents
the ANT-mediated permeabilization of liposomes responding to atractyloside23 or Vpr18.
Analogous results have been obtained for
VDAC-containing proteoliposomes (which
VOLUME 2 | JANUARY 2001 | 6 9
© 2001 Macmillan Magazines Ltd
PERSPECTIVES
Bcl-2
Bax
Nonspecific
Autonomous channel formation
sets off local ion imbalances and
indirectly stabilizes OMM/IMM
Specific
Nonspecific
Cooperative channel formation
within the PTPC or with PTPC
components (ANT, VDAC), in
OMM and/or IMM
Membrane insertion and
oligomerization leads to
formation of protein-permeable
conduits in OMM
MMP and cell death
Figure 2 | Alternative hypotheses for the modus operandi of Bcl-2/Bax-like proteins. Although Bax
and Bcl-2 heterodimerize and inhibit each other, both proteins can act independently in regulating
apoptosis. According to one hypothesis (left), Bcl-2 forms protein-impermeable channels, thereby
preventing local ion imbalances and non-physiological closure of the voltage-dependent anion channel
(VDAC) in the outer mitochondrial membrane (OMM). Bcl-2 and Bax may also interact with the
permeability transition pore complex (PTPC) and regulate channel formation by this two-membranespanning protein complex, regulating the permeability of the inner mitochondrial membrane (IMM) and
OMM (middle). Bax-like proteins have also been proposed to oligomerize in the OMM, upon interaction
with a truncated form of Bid known as tBid, thereby causing the formation of cytochrome c-permeable
conduits. (ANT, adenine nucleotide translocator; MMP, mitochondrial membrane permeabilization.)
are permeable to sucrose)22. Bcl-2, as well as
peptides derived from the BH4 domain of
Bcl-2 (where BH4 stands for Bcl-2 homology
domain 4, and is a domain that is missing in
pro-apoptotic Bcl-2-like proteins), reduces
the permeability of such VDAC proteoliposomes to sucrose, whereas Bax enhances their
permeability22. Bax/VDAC liposomes (but
not liposomes containing VDAC or Bax
alone) are permeable to cytochrome c (14.5
kDa), but impermeable to a 50 kDa protein22.
nel is qualitatively different from that formed
by Bax alone. With a combination of ANT
and Bcl-2 (in a molar ratio of 1:1), atractyloside-induced ion movements are no longer
seen, indicating the closure of both the ANT
and the Bcl-2 channels28.
Electrophysiological experiments also
indicate cooperative pore formation by the
VDAC plus Bax, and inhibition of VDAC
channel formation by Bcl-2 (REF. 29). The
VDAC and Bax cooperatively create a large
pore, with conductance levels fourfold and
Bax, ANT and VDAC channels
Bax and the ANT (or VDAC) can cooperate
to generate channels with higher conductance levels as well as higher opening probabilities than either of the two compounds
alone. Moreover, Bcl-2 and the ANT (or
VDAC) mutually inhibit the formation of ion
channels. Single-channel current measurements involving proteins reconstituted into
planar lipid bilayers indicate that the ANT
can form large channels with multiple subconductance states (70 to 600 pS) in response
to Ca2+. The ANT can also form small channels (30 pS) in response to atractyloside28. A
mixture of Bax and the ANT (in a molar ratio
of 1:4) has a higher probability of atractyloside-induced pore opening than the ANT
alone and shows two conductance levels (30
and 80 pS), as well as cation specificity. At low
concentrations (1 nM), Bax does not yield
any pronounced macroscopic conductance,
unless combined with ANT treated with
atractyloside28. So channel formation is more
efficient for the combination of the ANT,
atractyloside and Bax than for combinations
of the ANT plus atractyloside, the ANT and
Bax, or atractyloside and Bax. Moreover, the
channel formed by Bax at high concentrations is anion specific. So, the ANT/Bax chan-
70
“The intricate relationship
between Bcl-2/Bax-like
proteins and mitochondrial
proteins adds another level
of complexity to regulation
of cell death.”
tenfold greater than those of the VDAC and
Bax channels, respectively. Although the
VDAC and Bax channels both show ion selectivity and voltage-dependent modulation of
their activity, the VDAC–Bax channel has neither of these properties. Cytochrome c reportedly passes through a single VDAC–Bax channel, but not through the VDAC or Bax
channels in a planar lipid bilayer29. Anti-apoptotic Bcl-xL and its BH4 oligopeptide reportedly close the VDAC channel30.
Studies in isolated mitochondria
When incorporated into mitochondrial
membranes, Bcl-2 and its anti-apoptotic
homologues enhance the resistance of these
| JANUARY 2001 | VOLUME 2
mitochondria to a permeability transition.
Similarly, microinjection of Bcl-2 into the
cytoplasm of intact cells prevents both permeabilization of their mitochondrial membranes and nuclear apoptosis induced by the
ANT ligand atractyloside31. If added to purified mitochondria in vitro, recombinant Bax
or Bak induce membrane permeabilization,
and this is inhibited by various PTPC
inhibitors, including Koenig’s polyanion,
cyclosporin A, N-methyl-4-Val-cyclosporin A
and bongkrekate23,31–33. These are controversial findings, however, as some groups10,11
have not detected any effect of cyclosporin A.
Oligomycin, an inhibitor of the F0–F1-ATPase
(which, on theoretical grounds, might indirectly affect the ANT), also inhibits mitochondrial membrane permeabilization
induced by Bax31–33.
At low doses, Bax causes signs of outermembrane permeabilization (that is, release
of cytochrome c) but not of inner-membrane
permeabilization (for instance, matrix
swelling and loss of the ∆ψm). But higher
doses of Bax affect both the OMM and the
IMM33, and cyclosporin A prevents the effects
of Bax on both membranes23,31–33.
Studies in intact cells
Similar inhibitory profiles to those seen in
vitro have been obtained when Bax is
microinjected into the cytoplasm of intact
cells. In control cells, Bax causes loss of the
∆Ψm and nuclear apoptosis. Our group has
found23 that treatment with cyclosporin A,
N-methyl-4-Val-cyclosporin
A
or
bongkrekate abolishes both the mitochondrial and the nuclear signs of Bax-induced
apoptosis. However, other data10 show that
cyclosporin A does not inhibit apoptosis
(and the associated mitochondrial membrane permeabilization) induced by transfection with the Bax gene.
Proteolytic activation of Bid by caspase-8
(yielding a truncated form of Bid known as
tBid) normally leads to the permeabilization
of mitochondrial membranes. This permeabilization (and subsequent apoptosis) can
be prevented by the ANT-targeted viral
mitochondrial inhibitor of apoptosis
(vMIA)19. Killing by another pro-apoptotic
protein from the Bcl-2 family, BNIP3, is prevented by cyclosporin A and bongkrekate34.
Obviously, however, it may be argued that
pharmacological inhibition experiments are
not truly conclusive — drugs may affect not
only components of the PTPC, but also
other molecules in the cell — and that
genetic interventions on components of the
PTPC would be more informative.
In yeast cells, several mitochondrial defects
www.nature.com/reviews/molcellbio
© 2001 Macmillan Magazines Ltd
PERSPECTIVES
reduce Bax-induced killing, including deletion
of the three ANT isoforms23,35, and mutants
inactivating the F0–F1-ATPase35,36. These data
do not support the idea that Bax kills cells
through simple lysis, indicating that Bcl-2-like
proteins might modulate cell death through a
functional interaction with the PTPC.
cell type. Whatever the answer to this conundrum, it seems clear that the intricate relationship between Bcl-2/Bax-like proteins and
mitochondrial proteins adds another level of
complexity to regulation of cell death.
Naoufal Zamzami and Guido Kroemer are at the
Centre National de la Recherche Scientifique,
UMR1599, Institut Gustave Roussy, 39 rue
Camille-Desmoulins, F-94805 Villejuif, France.
Correspondence to G.K. e-mail: [email protected]
Do Bax/Bcl-2 always act on the PTPC?
Recombinant Bax, as well as tBid (but not
Bcl-2), has been reported to destabilize lipid
bilayers without causing the appearance of
ion channels37,38. Alternatively, Bcl-2, Bax and
tBid may form channels, with defined levels
of conductivity and variable requirements of
pH or the local presence of acidic phospholipids39,40. When added to liposomes, Bax can
insert into the membranes, oligomerize (presumably to tetramers) and form cytochrome
c-permeant conduits with an estimated pore
size of around 30 Å (REF. 40). It has been proposed that Bid aids the insertion or oligomerization of Bax12 or Bak13, which would form
pores without any interaction with VDAC or
ANT. Moreover, recombinant tBid (in contrast to Bax or Bak), added to isolated mitochondria, has been reported41 to cause an
exclusive permeabilization of the OMM that
is not affected by PTPC inhibitors.
All these data may be interpreted to mean
that proteins from the Bcl-2/Bax family can
permeabilize membranes through a nonspecific effect — that is, without the need for interactions with other proteins from the PTPC. This
interpretation is supported by the observation
that, during apoptosis, cytochrome c can be
released from mitochondria that have an
apparently normal ultrastructure and conserve
an inner transmembrane potential11,12.
How can we reconcile the evidence for
these nonspecific effects of Bax/Bcl-2 with the
many reports that suggest specific, PTPCdependent effects? One possibility would be to
assume that, at physiological concentrations,
Bax and Bcl-2 act through these specific
effects; for instance, by modulating the probability of channel formation by other proteins.
But at higher concentrations, Bax would
become able to oligomerize and to exert
autonomous, nonspecific effects, involving
either channel formation or membrane destabilization. This implies a hierarchic superposition of two levels of regulation. Alternatively,
the two pathways could come into action as a
function of the local abundance of PTPC
components, their isoforms, or PTPC
inhibitors/activators (FIG. 2). Indeed, a similar
mechanism has been proposed for CD95induced signalling, in which the same primary
signal can trigger cell death through completely distinct mechanisms, as a function of the
Links
DATABASE LINKS Smac | Bcl-2 | Bcl-xL | Bcl-W |
Mcl1 | A1 | Bax | Bad | Bid | Bim | creatine
kinase | hexokinase II | cyclophilin D | CD95 |
ANT2 | mouse Ant1 | BNIP3
ENCYCLOPEDIA OF LIFE SCIENCES Apoptosis:
molecular mechanisms
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Green, D. R. & Reed, J. C. Mitochondria and apoptosis.
Science 281, 1309–1312 (1998).
Kroemer, G. & Reed, J. C. Mitochondrial control of cell
death. Nature Med. 6, 513–519 (2000).
Budijardjo, I., Oliver, H., Lutter, M., Luo, X. & Wang, X.
Biochemical pathways of caspase activation during
apoptosis. Annu. Rev. Cell Dev. Biol. 15, 269–290 (1999).
Vander Heiden, M. G. & Thompson, C. B. Bcl-2 proteins:
Inhibitors of apoptosis or regulators of mitochondrial
homeostasis? Nature Cell Biol. 1, E209–E216 (1999).
Gross, A., McDonnell, J. M. & Korsmeyer, S. J. Bcl-2
family members and the mitochondria in apoptosis.
Genes Dev. 13, 1899–1911 (1999).
Crompton, M. The mitochondrial permeability transition
pore and its role in cell death. Biochem. J. 341, 233–249
(1999).
Woodfield, K., Ruck, A., Brdiczka, D. & Halestrap, A. P.
Direct demonstration of a specific interaction between
cyclophilin-D and the adenine nucleotide translocase
confirms their role in the mitochondrial permeability
transition. Biochem. J. 336, 287–290 (1998).
Feldmann, G. et al. Opening of the mitochondrial
permeability transition pore causes matrix expansion and
outer membrane rupture in Fas-mediated hepatic
apoptosis in mice. Hepatology 31, 674–683 (2000).
Yin, X.-M. et al. Bid-deficient mice are resistant to Fasinduced hepatocellular apoptosis. Nature 400, 886–891
(1999).
Eskes, R. et al. Bax-induced cytochrome c release from
mitochondria is independent of the permeability transition
pore but highly dependent on Mg2+ ions. J. Cell Biol. 143,
217–224 (1998).
von Ahsen, O. et al. Preservation of mitochondrial
structure and function after Bid- or Bax-mediated
cytochrome c release. J. Cell Biol. 150, 1027–1036
(2000).
Eskes, R., Desagher, S., Antonsson, B. & Martinou, J. C.
Bid induces the oligomerization and insertion of Bax into
the outer mitochondrial membrane. Mol. Cell. Biol. 20,
929–935 (2000).
Wei, M. C. et al. tBID, a membrane-targeted death
ligand, oligomerizes BAK to release cytochrome c.
Genes Dev. 14, 2060–2071 (2000).
Kroemer, G., Dallaporta, B. & Resche-Rigon, M. The
mitochondrial death/life regulator in apoptosis and
necrosis. Annu. Rev. Physiol. 60, 619–642 (1998).
Sullivan, P. G., Thompson, M. B. & Scheff, S. W.
Cyclosporin A attenuates acute mitochondrial
dysfunction following traumatic brain injury. Exp. Neurol.
160, 226–234 (1999).
Budd, S. L., Tenneti, L., Lishnak, T. & Lipton, S. A.
Mitochondrial and extramitochondrial apoptotic signaling
pathways in cerebrocortical neurons. Proc. Natl Acad.
Sci. USA 97, 6161–6166 (2000).
Tafain, M., Schneider, T. G., Pastorino, J. G. & Farber,
J. L. Cytochrome c-dependent activation of caspase-3
by tumor necrosis factor requires induction of the
mitochondrial permeability transition. Am. J. Pathol. 156,
2111–2121 (2000).
Jacotot, E. et al. The HIV-1 viral protein R induces
apoptosis via a direct effect on the mitochondrial
permeability transition pore. J. Exp. Med. 191, 33–45
(2000).
Goldmacher, V. S. et al. A cytomegalovirus-encoded
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
mitochondria-localized inhibitor of apoptosis structurally
unrelated to Bcl-2. Proc. Natl Acad. Sci. USA 96,
12536–12541 (1999).
Rahmani, Z., Huh, K. W., Lasher, R. & Siddiqui, A.
Hepatitis B virus X protein colocalizes to mitochondria
with a human voltage-dependent anion channel,
HVDAC3, and alters its transmembrane potential. J. Virol.
74, 2840–2846 (2000).
Massari, P., Ho, Y. & Wetzler, L. M. Neisseria meningitidis
porin PorB interacts with mitochondria and protects cells
from apoptosis. Proc. Natl Acad. Sci. USA 97,
9070–9075 (2000).
Shimizu, S., Narita, M. & Tsujimoto, Y. Bcl-2 family
proteins regulate the release of apoptogenic cytochrome
c by the mitochondrial channel VDAC. Nature 399,
483–487 (1999).
Marzo, I. et al. Bax and adenine nucleotide translocator
cooperate in the mitochondrial control of apoptosis.
Science 281, 2027–2031 (1998).
Kaukonen, J. et al. Role of adenine nucleotide
translocator 1 in mtDNA maintenance. Science 289,
782–785 (2000).
Motoyama, S. et al. Bcl-2 is located predominantly in the
inner membrane and crista of mitochondria in rat liver.
Biochem. Biophys. Res. Commun. 249, 628–636 (1998).
Gotow, T. et al. Selective localization of Bcl-2 to the inner
mitochondrial and smooth endoplasmic reticulum
membranes in mammalian cells. Cell. Death Differ. 7,
666–674 (2000).
Costantini, P. et al. Oxidation of a critical thiol residue of
the adenine nucleotide translocator enforces Bcl-2independent permeability transition pore opening and
apoptosis. Oncogene 19, 307–314 (2000).
Brenner, C. et al. Bcl-2 and Bax regulate the channel
activity of the mitochondrial adenine nucleotide
translocator. Oncogene 19, 329–336 (2000).
Shimizu, S., Ide, T., Yanagida, T. & Tsujimoti, Y.
Electrophysiological study of a novel large pore formed
by Bax and the voltage-dependent anion channel that is
permeable to cytochrome c. J. Biol. Chem. 275,
12321–12325 (2000).
Shimizu, S., Konishi, A., Kodama, T. & Tsujimoto, Y. BH4
domain of antiapoptotic Bcl-2 family members closes
voltage-dependent anion channel and inhibits apoptotic
mitochondrial changes and cell death. Proc. Natl Acad.
Sci. USA 97, 3100–3105 (2000).
Jürgensmeier, J. M. et al. Bax directly induces release of
cytochrome c from isolated mitochondria. Proc. Natl
Acad. Sci. USA 95, 4997–5002 (1998).
Narita, M. et al. Bax interacts with the permeability
transition pore to induce permeability transition and
cytochrome c release in isolated mitochondria. Proc. Natl
Acad. Sci. USA 95, 14681–14686 (1998).
Pastorino, J. G. et al. Functional consequences of
sustained or transient activation by Bax of the
mitochondrial permeability transition pore. J. Biol. Chem.
274, 31734–31739 (1999).
Vande Velde, C. et al. BIP3 and genetic control of
necrosis-like cell death through the mitochondrial
permeability transition pore. Mol. Cell. Biol. 20,
5454–5468 (2000).
Harris, M. H., Vander Heiden, M. G., Kron, S. J. &
Thompson, C. B. Role of oxidative phosphorylation in
Bax toxicity. Mol. Cell. Biol. 20, 3590–3596 (2000).
Matsuyama, S., Xu, Q., Velours, J. & Reed, J. C.
Mitochondrial F0F1-ATPase proton pump is required for
function of proapoptotic protein Bax in yeast and
mammalian cells. Mol. Cell 1, 327–336 (1998).
Basañez, G. et al. Bax, but not Bcl-xL, decreases the
lifetime of planar phospholipid bilayer membranes at
subnanomolar concentrations. Proc. Natl Acad. Sci. USA
96, 5492–5497 (1999).
Kudla, G. et al. The destabilization of lipid membrane
induced by the C-terminal fragment of caspase 8cleaved Bid is inhibited by the N-terminal fragment. J.
Biol. Chem. 275, 22713–22718 (2000).
Schendel, S. L. et al. Ion channel activity of the BH3 only
bcl-2 family member, BID. J. Biol. Chem. 274,
21932–21936 (1999).
Saito, M., Korsmeyer, S. J. & Schlesinger, P. H. Baxdependent transport of cytochrome c reconstituted in
pure liposomes. Nature Cell Biol. 2, 553–555 (2000).
Shimizu, S. & Tsujimoto, Y. Proapoptotic BH3-only Bcl-2
family members induce cytochrome c release, but not
mitochondrial membrane potential loss, and do not
directly modulate voltage-dependent anion channel
activity. Proc. Natl Acad. Sci. USA 97, 577–582 (2000).
Acknowledgements
The authors’ work is supported by a special grant from the
Ligue Nationale contre le Cancer, as well as grants from ANRS,
FRM and EC (to G.K.).
VOLUME 2 | JANUARY 2001 | 7 1
© 2001 Macmillan Magazines Ltd