JOURNAL OF CELLULAR PHYSIOLOGY 195:158167 (2003)

REVIEW ARTICLES
Cell Death Regulation by the Bcl-2 Protein
Family in the Mitochondria
YOSHIHIDE TSUJIMOTO*
Osaka University Graduate School of Medicine, Department of Post-Genomics and Diseases,
Laboratory of Molecular Genetics, CREST of Japanese Science and Technology (JST),
Suita, Osaka, Japan
An increase in the permeability of the outer mitochondrial membrane is central to
apoptoticcell death, sinceit leads totherelease of several apoptogenicfactors, such
as cytochrome c and Smac/Diablo, into the cytoplasm that activate downstream
death programs. During apoptosis, the mitochondria also release AIF and endo-
nuclease G, both of which are translocated to the nucleus and are implicated in
apoptotic nuclear changes that occur in a caspase-independent manner. Mito-
chondrial membrane permeability is directly controlled by the major apoptosis
regulator, i.e., the Bcl-2 family of proteins, mainly through regulation of the for-
mation of apoptotic protein-conducting pores in the outer mitochondrial mem-
brane, although the precise molecular mechanisms are still not completely
understood. Here, I focus on the mechanisms by which Bcl-2 family members
control the permeability of mitochondrial membrane during apoptosis. J. Cell.
Physiol. 195: 158167, 2003. 2003 Wiley-Liss, Inc.
Programmedcell deathor apoptosis plays anintegral
role in a variety of biological events, including morpho-
genesis, tissue homeostasis, and removal of unwanted
or harmful cells. Dysregulation of programmed cell
death leads to various diseases in humans, including
cancer and certain neurodegenerative diseases. Exten-
sive studies performed over the last 10 years have
revealed a large part of the molecular basis of cell
death, including apoptosis and some forms of necrosis.
The mitochondriaplay anessential role inthe apoptotic
death of mammalian cells by releasing various apop-
togenic proteins, including cytochrome c, into the
cytoplasm (Martinou and Green, 2001; Zamzami and
Kroemer, 2001) (Fig. 1). Similar mitochondrial changes
also take place during necrotic cell death either as a
consequence or as an initiating event. In the process of
apoptosis, release of cytochrome c into the cytoplasm
activates death-promoting proteolytic enzymes called
caspases, which in turn cleave a set of cellular proteins
and promote the death program (Thornberry and
Lazebnik, 1998; Wang, 2001). The Bcl-2 family of
proteins regulates these mitochondrial changes during
both apoptosis and necrosis (Adams and Cory, 1998;
Tsujimoto and Shimizu, 2000a). Although the mito-
chondrial contribution to apoptotic cell death is
well established in mammals, no compelling evi-
dence of mitochondrial involvement has been obtained
in C. elegans, despite its well-conserved apoptotic
machinery.
This review is primarily focused on the mechanisms
underlying the regulation of outer mitochondrial mem-
brane permeability during apoptosis by the Bcl-2 family
of proteins.
Bcl-2 PROTEIN FAMILY IN
THE MITOCHONDRIA
Bcl-2 (Tsujimoto et al., 1985; Tsujimoto and Croce,
1986) was rst suggested to play a role incell survival by
a study on the IL-3-deprivation-induced death of a
lymphoid cell line (Vaux et al., 1998). It was subse-
quently reported that Bcl-2 also inhibits cell death
induced by various stimuli such as a chemotherapeutic
agent, ethanol, and heat shock (Tsujimoto, 1989),
establishing Bcl-2 as a negative regulator of cell death.
The anti-death role was then demonstrated in vivo by
generation of mice lacking the bcl-2 gene, which showed
a variety of abnormalities, most of which could be ex-
plained by excessive cell death (Veis et al., 1993;
Nakayama et al., 1994; Kamada et al., 1995). On the
basis of various structural and functional characteris-
tics, the Bcl-2 family of proteins is divided into three
2003 WILEY-LISS, INC.
Contract grant sponsor: Ministry of Education, Science, Sports,
and Culture of Japan (Scientic Research); Contract grant
sponsor: Grant for Scientic Research on Priority Areas; Contract
grant sponsor: Center of Excellence Research.
*Correspondence to: Yoshihide Tsujimoto, Osaka University
Graduate School of Medicine, Department of Post-Genomics and
Diseases, Laboratory of Molecular Genetics, 2-2 Yamadaoka,
Suita, Osaka 565-0871, Japan.
E-mail: tsujimot@gene.med.osaka-u.ac.jp
Received 15 November 2002; Accepted 10 December 2002
DOI: 10.1002/jcp.10254
subfamilies (Adams and Cory, 1998; Tsujimoto and
Shimizu, 2000a) (Fig. 2). The anti-apoptotic subfamily
comprises Bcl-2, Bcl-x
L
, Bcl-w, Mcl-1, and A1 (B-1).
Some viruses possess Bcl-2 homologues (Cuconati and
White, 2002), including BHRF1 (Epstein-Barr virus),
ks-Bcl-2 (human herpes virus 8), LHW5-HL (African
swine fever virus), and ORF16 (herpesvirus saimiri).
These proteins share sequence homology within some of
the four Bcl-2 homology (BH) regions, BH1 through
BH4. The multi-domain pro-apoptotic subfamily con-
sists of Bax, Bak, Mtd (Bok), and Bcl-rambo. These
proteins also share sequence homology at BH1, BH2,
and BH3. The still growing BH3-only protein sub-
family consists of numerous pro-apoptotic members: Bik
(Nbk), Bad, Bid, Bim(Bod), Hrk(DP5), Noxa, Blk, Bnip3
(Nix), Bnip3L, Puma, p193, Bmf, and Bcl-G. These
proteins only share sequence homology at the BH3
region.
The major function of the Bcl-2 family is to directly
modulate mitochondrial membrane permeability and
thereby regulate the release of apoptogenic factors from
the intermembrane space into the cytoplasm (Green,
2000; Tsujimoto and Shimizu, 2000a,b; Martinou and
Green, 2001; Zamzami andKroemer, 2001). Factors that
are known to be released fromthe mitochondria and are
implicated in cell death include cytochrome c (Liu et al.,
1996), Smac/Diablo (Du et al., 2000; Verhagen et al.,
2000), AIF (Susin et al., 1999), heat shock protein 60
(Samali et al., 1999; Xanthoudakis et al., 1999), HtrA2/
Omi (Suzuki et al., 2001; Hegde et al., 2002; Martins
et al., 2002; Verhagen et al., 2002; Van Loo et al., 2002)
and endonuclease G(Li et al., 2001) (Fig. 1). Cytochrome
c is directly involved in the activation of caspases, while
Smac/Diablo and HtrA2/Omi facilitate caspase activa-
tion by inhibiting proteins from the Iap family, such as
Xiap, which are caspase inhibitors. AIF and endonu-
clease G are thought to a play role in the induction of
caspase-independent apoptotic changes in nuclei. Anti-
apoptotic members of the Bcl-2family inhibit the release
of these apoptogenic factors, whereas pro-apoptotic
members promote it.
HOW ARE MULTI-DOMAIN PRO-APOPTOTIC
MEMBERS ACTIVATED?
As described above, the pro-apoptotic members of the
Bcl-2 family are categorized into two groups based on
their structure, i.e., multi-domain members and BH3-
only members. Proteins in these two categories are also
functionally distinct. Multi-domain members such as
Bax and Bak act as a gateway for a variety of apoptotic
signals, since cells from Bax/Bak-double knockout mice
are totally resistant to numerous apoptotic stimuli,
including transfection of DNA expressing various BH3-
only proteins such as tBid and Bim (Lindsten et al.,
2000; Wei et al., 2001b). Bax and Bak are functionally
redundant in many types of cells, although functional
differences between them have been suggested in cer-
tain types of cells (Putcha et al., 2002). Bax is generally
Fig. 1. Apoptotic signal transduction pathways. The intrinsic path-
way involving the mitochondria and the extrinsic pathway triggered
by death receptors are shown. In the intrinsic pathway, various
apoptotic signals are transmitted to the mitochondria, leading to the
release of proteins from the intermembrane space to the cytoplasm,
including several apoptogenic factors such as cytochrome c, Smac/
Diablo, HtrA2/Omi, AIF, and endonuclease G. The interaction of death
factors with death receptors induces the activation of apical caspases
such as capsase-8, followed by transmission directly to either effector
caspases or to the mitochondria via Bid, a BH3-only member of the
Bcl-2 family.
Bcl-2 PROTEIN FAMILY 159
considered to exist as monomers in living cell, although
it is still unclear whether or not it binds to other
proteins. It has recently been shown that Bax interacts
withsome isoforms of 14-3-3, whichmay be important in
sequestering Bax to the cytoplasm (Samuel et al., 2001;
Nomura et al., 2003). In response to apoptotic stimula-
tion, however, Bax and Bak form homo-multimers/
complex in the mitochondrial membrane, which have
been detected by gel ltration (Bax) (Antonsson et al.,
2001) and by protein cross-linking to detect homo-
oligomers (Bax and Bak) (Eskes et al., 2000; Antonsson
et al., 2001; Wei et al., 2001a), although it is not
known whether the multimers also interact with other
proteins. Bax is present in the cytoplasm or peri-
mitochondrial location of living cells, but it undergoes
translocation and integration into the mitochondrial
membrane after stimulation (Wolter et al., 1997),
whereas Bakis already present as anintegral mitochon-
drial membrane protein in living cells. It is considered
that Bax and Bak undergo a conformational change
upon apoptotic stimulation, based on the following ob-
servations: (1) some antibodies that recognize the
N-terminal portion of Bax and Bak only react with these
proteins after induction of apoptosis (Desagher et al.,
1999; Grifths et al., 1999), (2) the N-terminal portion of
Bax becomes accessible to limited proteolysis by trypsin
or proteinase K after induction of apoptosis (Goping
et al., 1998), (3) Bax and Bak form multimers after in-
duction of apoptosis (Eskes et al., 2000; Antonsson et al.,
2001; Wei et al., 2001a), and (4) Bax multimers are
formed from monomeric Bax in the presence of deter-
gents such as Non-idet-40 (Antonsson et al., 2000).
Currently, the conformational changes and multimer
formation by Bax and Bak have not been separated
experimentally. Bax multimer but not Bax monomer
induces cytochrome c release from isolated mitochon-
dria (Antonsson et al., 2000), suggesting that Bax
multimer represents an active form. Critical molecules
which trigger Bax/Bak activation are the BH3-only
members of the Bcl-2 family.
HOW DO BH3-ONLY PROTEINS ACT?
BH3-only proteins seem to function as death signal
sensors (Puthalakath and Strasser, 2002). Most BH3-
only proteins (including Bid, Bim, Bmf, and Bad) are
localized outside the mitochondria in living cells. After
apoptotic stimulations, these proteins are modied by
several different mechanisms and translocated to the
mitochondria, leading to increased permeability of the
outer mitochondrial membrane (Fig. 3). Bid is activated
bycleavagewithproteases, includingcaspase-8(Li et al.,
1998; Luo et al., 1998) and granzyme B (Heibein et al.,
Fig. 2. Schematic drawing of Bcl-2 and its related proteins. Bcl-2 family members are classied into
three categories. BH1 to BH4 are conserved sequence motifs. Several functional domains of Bcl-2 are
shown. a1 to a7 indicate helices identied in Bcl-x
L
, in which a core of two hydrophobic helices (a5 and a6)
is surrounded by ve amphipathic helices. The region between a1 and a2 is called a loop, which appears to
have a regulatory role. The membrane-anchoring domain is not carried by all members of the family.
160 TSUJIMOTO
2000). Bad is normally phosporylated and sequestered
to 14-3-3t in living cells, while apoptotic stimulation
causes Bad to undergo dephosphorylation and frees it
from14-3-3 (Zha et al., 1996). Bimand Bmf are bound to
elements of the cytoskeleton (microtubules and actin
laments, respectively) via dyneinmotor complexes and
myosin V motor complexes, respectively (Puthalakath
et al., 1999, 2001). Some of the BH3-only proteins such
as Noxa and Puma, are newly synthesized during DNA
damage-induced cell death after transcriptional activa-
tionbyp53(Odaet al., 2000; Nakano andVousden, 2001;
Yu et al., 2001). It is still not fully understood how the
BH3-only proteins transmit signals to the multi-domain
members Bax and Bak. It has been suggested that tBid
transiently binds to Bax or Bak and induces a conforma-
tional change (Desagher et al., 1999; Wei et al., 2001a)
(Fig. 4). tBid is able to fully activate Bax to form Bax
multimers inthe mitochondria, but not insynthetic lipid
membranes (Roucou et al., 2002b), suggesting that a
mitochondrial factor(s) is required for this process.
Interestingly, it was found that Bax can formmultimers
in the absence of tBid in yeast mitochondria (Roucou
et al., 2002b), suggesting the possibility that Bax mono-
mer itself has an intrinsic multimer-forming activity in
the mitochondria, and that mammalian mitochondria
may contain an inhibitor of Bax multimerization. Ac-
cordingly, a possible role of tBid is to inactivate this
inhibitor. Alternatively, tBid may activate Bax by
affecting the lipid membrane rather than via a direct
interaction with Bax. In this context, it is of great in-
terest to note that tBid binds to cardiolipin(Lutter et al.,
2000), which is abundant in the mitochondrial inner
membrane and contact sites, and that tBid has lipid
transfer activity (Esposti et al., 2001) as well as causing
membrane disruption (Kudla et al., 2000). In damaged
mitochondria, for example, after being subjected to
repeated freezing and thawing, the conformational
change of Bax is induced in the absence of tBid
(Yamaguchi and Wang, 2002), so a change in mitochon-
drial membrane lipids may be crucial for multimeriza-
tion of Bax, although it is also possible that an inhibitor
of Bax multimerization in the mitochondrial membrane
might be inactivated or a mitochondrial BH3-only
proteinmight be activated during freezing and thawing.
Based on the observation that some BH3-only pro-
teins such as tBid bind to both anti-apoptotic members
and pro-apoptotic multi-domain members of the Bcl-2
family, whereas other BH3-only members such as Bad
bind preferentially to the anti-apoptotic members, it has
been suggested that BH3-only proteins may be categor-
ized into two groups, one being activators of Bax/Bak
and the other being inactivator of anti-apoptotic mem-
bers of the Bcl-2 family (Letal et al., 2002). Consistent
with this idea, Bim alone reportedly does not induce
cytochrome c release from isolated mitochondria, but
cancels the inhibitory activity of anti-apoptotic proteins
(Terradillos et al., 2002), suggesting that Bim may only
inactivate anti-apoptotic members in the mitochondria
to facilitate the activation of Bax/Bak. However, BimEL
has also beenshownto induce cytochrome c release from
Fig. 3. BH3-only proteins as death signal sensors. Many BH3-only proteins are localized outside the
mitochondria in living cells. Apoptotic stimulation causes translocation of BH3-only proteins to the mito-
chondria by different mechanisms. Some Bcl-2 family members, such as Noxa and Puma, are trans-
criptionally activated during DNA damage-induced apoptosis.
Bcl-2 PROTEIN FAMILY 161
isolated mitochondria in a BH3 domain-dependent
manner (Yamaguchi and Wang, 2002; Sugiyama et al.,
2002). Recently, the classication of BH3-only proteins
into two categories was challengedby a study using BH3
domainpeptides fromseveral BH3-only proteins includ-
ing tBid, Bim, Bik, Bak, and Noxa (Letai et al., 2002). It
was shown that both tBid- and BimBH3-peptides
activated Bax and Bak in isolated mitochondria, where-
as Bad- and BikBH3 peptides did not activate Bax/
Bak, but instead caused inactivation of Bcl-2. Since the
BH3-peptide may not have exactly the same activity as
the full-size proteins, a similar study needs to be per-
formed using intact proteins. Bim and tBid have also
beenshownto induce cytochrome c release fromisolated
mitochondria in a Bax (Bak)-independent manner
(Grinberg et al., 2002; Sugiyama et al., 2002), although
the details of the mechanisms involved are unknown.
MOLECULAR BASIS OF INCREASED
MITOCHONDRIAL OUTER
MEMBRANE PERMEABILITY
All of the mitochondrial apoptogenic factors, except
for endonuclease G(Li et al., 2001) which is found in the
mitochondrial matrix, are mainly localized to the inter-
membrane space betweenthe outer and inner mitochon-
drial membranes. Therefore, release of these factors
requires an increase in the permeability of the outer
membrane. Exogenous cytochrome c canreachthe inner
mitochondrial membrane of apoptotic mitochondria
(Kluck et al., 1999; Mootha et al., 2001), suggesting that
the discontinuity in the outer membrane caused by
apoptosis is bi-directional. Release of cytochrome c is
quite rapid and is independent of any enzymatic reac-
tions, since it still occurs at an equivalent rate at lower
temperatures (Goldstein et al., 2000). Therefore, this
discontinuity could represent the formation of protein-
conducting pores in the outer membrane or could be due
to physical rupture or tear of the membrane. Since Bax
tetramer is able to transmit cytochrome c from inside
articial liposomes (Saito et al., 2000), Bax (or Bak)
multimer could represent such a protein-conducting
pore. Apoptosis-induced Bax (Bak) multimer was visua-
lized by immunoelectron microscopy, revealing that
most of the multimer (huge aggregates) was localized
near (but outside) the mitochondria and did not seem to
form pores in the outer membrane (Nechushtan et al.,
2001). Thus only a small fraction of Bax multimer may
form protein-conducting pores. Bax multimer (gener-
ated by using detergents such as octylglucoside) but not
Bax monomer induces cytochrome c release from isolat-
ed mitochondria as well as from synthetic lipid mem-
branes (liposomes) containing phosphatidylcholin (PC)
and phosphatidylglycerol (PG) (Roucou et al., 2002a).
Although tBid induces multimer formation of Bax in
isolated mitochondria and cytochrome c release, tBid
and Bax monomers are unable to induce cytochrome c
Fig. 4. Activation of Bax/Bak by BH3-only proteins. Once activated, BH3-only proteins are translocated
to the mitochondria to inactivate anti-apoptotic members of the Bcl-2 family and activate multi-domain
members, resulting in an increase of outer mitochondrial membrane permeability. An outer mito-
chondrial membrane channel, the VDAC, also contributes to this change of membrane permeability.
162 TSUJIMOTO
release from liposomes (PE/PG) (Roucou et al., 2002a),
suggesting that other factors are required for full
activation of Bax (probably an event involving Bax
multimerization). Recently, Kuwana et al. (2002) has
succeeded in reconstituting in liposomes containing
cardiolipin using only tBid and Bax, Bcl-x
L
-inhibitable
supramolecular openings, which are permeable to very
large (2 MDa) dextran molecules. It was hypothesized
that activated Bax promotes localized alterations in the
structure of the lipid bilayer, for example, forming large
transient lipid pores or inverted micelles. However,
Roucou et al. (2002a) reported that tBid was able to
activate Bax in mitochondria but not in cardiolipin-
containing liposomes, liposomes reconstituted from
mitochondrial lipid or proteinase K-treated mitochon-
dria, suggesting an involvement of a mitochondrial
factor(s) that is not phospholipid, but rather a protein,
for tBid-induced Bax multimerization.
In the presence of pro-apoptotic Bax or Bak, the
VDAC, a channel inthe mitochondrial outer membrane,
becomes permeable to cytochrome c in liposomes
(Shimizu et al., 1999) (Fig. 4). Conversion of VDAC to a
cytochrome c-conducting pore was also induced by
oxygen radicals in the absence of Bax (Madesh and
Hajnoczky, 2001), suggesting that the VDAC itself has
the intrinsic ability to become to a protein-conducting
pore, probably through a conformational change. There-
fore, the role of Bax may be simply to trigger a VDAC
conformational change rather than to be a stoichio-
metric component of the cytochrome c-conducting
channel. Although the VDAC modulates the mitochon-
drial permeability transition (MPT) (Shimizu et al.,
2001) resulting in physical rupture of the outer mito-
chondrial membrane (see below), its ability to form a
protein-conducting pore in lipid membranes implies
that the VDACcontributes to the release of apoptogenic
factors in a permeability transition-independent man-
ner. In contrast to Bax (Bak), Bcl-x
L
inhibits VDAC
activity in liposomes (Shimizu et al., 1999, 2000c), al-
though a controversial nding was also reported, sug-
gesting that Bcl-x
L
maintains the VDAC in an open
state (Vander Heiden et al., 2001). The VDAC is also
inhibited by other anti-apoptotic proteins such as
gelsolin (Kusano et al., 2000) and c-Raf-1 (Le Mellay
et al., 2002). Consistent with VDAC inhibition by the
anti-apoptotic proteins, microinjection into cells of
antibodies which are specic to the VDAC and inhibit
VDAC activity also signicantly reduce apoptotic cell
death induced by the microinjection of Bax as well as
other pro-apoptotic reagents such as etoposide (Shimizu
et al., 2001), suggesting an important role of the VDAC
in apoptosis of mammalian cells. This is supported by
the observation that an anti-VDAC antibody also inhi-
bits ischemia-induced neuronal damage (Velazquez
et al., 2000). In addition to formation of protein-
conducting pores, the VDAC may also be involved in
mitochondrial reorganization that may occur during
apoptosis. Alarge fraction of cytochrome c is localized in
the cristae rather than in the intermembrane space.
Since most cytochrome c is released from the mitochon-
dria during apoptosis, mitochondrial reorganization
that promotes the migration of cytochrome c into the
intermembrane space mayneedto occur (Scorrano et al.,
2002). This process appears to be cyclosporinA-sensitive
(Scorrano et al., 2002), indicating the involvement of the
MPT, which has been shown to require VDAC activity
(Shimizu et al., 2001) (see below).
By patch-clamping the mitochondria and proteoli-
posmes prepared fromouter mitochondrial membranes,
a novel channel called the mitochondrial apoptosis-
induced channel (MAC) has been detected, the forma-
tion of which is correlated with the onset of apoptosis
(Pavlov et al., 2001). The diameter of the pores with the
largest conductance is approximately 4 nm. Although
the electrophysiological characteristics of this channel
are similar to those of the apoptotic VDAC channel, the
biochemical nature of MAC is unknown.
Rupture of the outer membrane is another possible
mechanismfor the release of mitochondrial apoptogenic
factors (Zamzami and Kroemer, 2001) (Fig. 5). This
could be caused by MPT-dependent mitochondrial swel-
ling. The MPT, which is triggered by various agents,
including Ca
2
and reactive oxygen species, and is
denedby the loss of DC(the potential formedacross the
inner membrane) andthe release of solutes smaller than
1,500 Da from the mitochondria (Zoratti and Szabo,
1995), leading to mitochondrial swelling followed by
physical rupture of the outer membrane. These changes
are inhibited by cyclosporin A, which targets cyclophilin
D, and by bonkrekic acid, which targets adenine
nucleotide translocator (ANT) (Zamzani et al., 1996a;
Kroemer and Reed, 2000). ANT, VDAC, and cyclophilin
D, possibly together with other protein(s), constitute a
protein complex called the MPT pore complex at the site
of contact between the outer and inner mitochondrial
membranes, although the exact molecular architecture
of this complex is still unknown. It has been proposed
that the MPTpore complex maybe anon-specic protein
aggregate (He and Lemasters, 2002). The MPT itself is
generally considered to be involved in necrotic cell
death, although some forms of apoptotic cell death also
involve it because apoptosis is sometimes efciently
blocked by MPT inhibitors such as cyclosporin A and
bonkrekic acid (Marchetti et al., 1996; Zamzami et al.,
1996b). Bax and Bak are able to induce the MPT, which
may be directly mediated by interaction of Bax (Bak)
with the VDAC (Shimizu et al., 1999, 2000c) or ANT
(Marzo et al., 1998). Rupture of the outer membrane
canalso be achievedby a MPT-independent mechanism.
For example, some pro-apoptotic Bcl-2 family members
have been shown to directly induce instability of lipid
membranes (Kudla et al., 2000). Given that various
mitochondrial proteins up to 100 kDa (such as a dimmer
of sulde oxidase) are rapidly released during apoptosis
(Kluck et al., 1999), a tear of the outer membrane
may have advantages over the formation of protein-
conducting pores. It is certainly conceivable that more
than one mechanismmay collaborate simultaneously or
sequentially in destroying the barrier of the outer
membrane.
ANTI-APOPTOTIC MEMBERS ACT VIA
BOTH HETERODIMERIZATION-DEPENDENT
AND -INDEPENDENT MECHANISMS
Bcl-2 binds to multi-domain members as well as the
BH3-only proteins via interaction of the BH3 domain of
pro-apoptotic members with a hydrophobic pocket form-
ed in anti-apoptotic members. Since Bcl-2 is mainly
Bcl-2 PROTEIN FAMILY 163
localized on the mitochondrial membrane and most
BH3-only proteins or Bax are localized in the non-
mitochondrial compartment of living cells, Bcl-2 prob-
ably interacts with activated pro-apoptotic proteins.
This concept is consistent with the observation that the
interactionof Bcl-2withBax occurs or is enhancedinthe
presence of non-ionic detergents such as NP40 and
octylglucoside, which induce a conformational change of
Bax that is probably equivalent to that of the activated
form(Antonssonet al., 2000). However, since Bcl-2Bax
interaction is not readily detected in the absence of
detergents, Bcl-2 activity to interact with Bax might not
represent a main role of Bcl-2. Alternatively, Bcl-2
might bind efciently to activated Bax and Bak in the
mitochondrial membrane upon apoptotic stimulation,
although this is not experimentally supported. The
major role of Bcl-2 thus seems to be trapping of activated
BH3-only proteins (Cheng et al., 2001). On the other
hand, many studies have been done using various
mutants, and have suggested that the interaction of
Bcl-2 with pro-apoptotic members of the Bcl-2 family is
not essential for its anti-apoptotic activity (Cheng et al.,
1996, 1997; Minn et al., 1999). The heterodimerization-
independent anti-apoptotic activity of Bcl-2 is well
documented, for example, by the nding that a mutant
of Bcl-2 lacking the BH4 domainalmost completely loses
its anti-apoptotic activity, but still binds as strongly to
various pro-apoptotic members, including Bax, Bak,
Bik, Bid, and Bim, as the wild type Bcl-2 (Huang et al.,
1998). This heterodimerization-independent activity
was also demonstrated by the observation that Bcl-2 is
able to protect the Ca
2
-induced permeability transition
(Zamzami et al., 1996b; Shimizu et al., 1998) indepen-
dently of multi-domain pro-apoptotic members of the
Bcl-2 family (Wei et al., 2001a) and that Bcl-x
L
inhibits
the PT-like event in yeast mitochondria which do not
have any homologue of the Bcl-2 family (Shimizu et al.,
2000b). This heterodimerization-independent activity
of Bcl-2 may be ascribable to its ability to inhibit
mitochondrial channels such as the VDAC, since VDAC
inhibition by antibodies blocks the MPT (Shimizu
et al., 2001).
POSSIBLE ROLE OF CED-9
(C. elegans Bcl-2 HOMOLOGUE)
IN THE MITOCHONDRIA
Since yeast mitochondria undergo an event similar to
the MPT, it is conceivable that the mitochondria of
C. elegans also do so under certain circumstances.
Occurrence of the MPT may be one reason why Ced-9
mainly localizes to the mitochondria (Chen et al., 2000),
like Bcl-2. In addition to sequestering Ced-4 (Apaf-1
homologue) to inhibit the main pathway of programmed
cell death (Liu and Hengartner, 1999), Ced-9 may
associate with the mitochondria to prevent MPT-like
events and thereby preserve mitochondrial function.
Because the BH4 domain is bothessential and sufcient
to close the VDAC (Shimizu et al., 2000a), and because
Ced-9 possesses a BH4 domain, it is reasonable to expect
that Ced-9 can close the VDACor similar channels. This
possible action of Ced-9 might be related to inhibition of
the release of endonuclease G (Parrish et al., 2001),
which probably cleaves nuclear DNA during program-
med cell death in C. elegans (Parrish et al., 2001).
Ced-9sequesters Ced-4(acaspase activator) viadirect
interaction to prevent PCD, while loss-of-function-
Fig. 5. Release of mitochondrial proteins by formation of apoptotic protein-conducting pores or physical
rupture. Pore formation that leads to release of proteins from the mitochondrial intermembrane space or
physical rupture due to the MPT are both suppressed by anti-apoptotic members of the Bcl-2 family.
164 TSUJIMOTO
mutants of Ced-9 lose this activity (Chinnaiyan et al.,
1997; Spector et al., 1997; Wu et al., 1997; Chen et al.,
2000). Human Bcl-2 is able to inhibit programmed cell
death in C. elegans (Vaux et al., 1992) which may be
ascribed at least partly to its activity to bind to Ced-4
(Chinnaiyan et al., 1997; Wu et al., 1997; Huang et al.,
1998). However, there may be an additional crucial step,
possibly occurring in the mitochondria, that prevents
PCDandiscontrolledbyCed-9. Thisadditional activityof
Ced-9 might be related to possible regulationof the MPT.
PERSPECTIVES
In mammals, a variety of death signals converge on
the mitochondria, which subsequently showanincrease
of outer membrane permeability, leading to release of
proteins from the intermembrane space, including
several apoptogenic factors. One major function of the
Bcl-2 protein family is to directly control membrane
permeability, although the precise mechanisms by
which Bcl-2 family members do so are still to be deter-
mined. Some Bcl-2 family members are also localized on
the endoplasmic reticulum, and it would be of interest to
determine their functions in relation to those located on
the mitochondria. Despite an essential role of the
mitochondria in the death of mammalian cells, it is still
unclear whether or not the mitochondria play a role in
cell death in other organisms, including the fruit y and
nematode, although proteins belonging to the Bcl-2
family are well conserved in these organisms and are
localized inor target the mitochondria as in mammalian
cells. Although the overall structure of the apoptotic
machinery has been unveiled, there are still many
issues that need to be claried to obtain a complete
understanding of the regulation of cell death.
ACKNOWLEDGMENTS
I thank all members of my laboratory at Osaka
University Graduate School of Medicine for helpful
discussion. I am particularly grateful to Dr. Shigeomi
Shimizufor his major contributionto our recent work on
the Bcl-2 family of proteins and Dr. Yutaka Eguchi for
continuous discussion. The work performed in my
laboratory was supported in part by a Grant for
Scientic Research on Priority Areas, by a Center of
Excellence Research grant, and by grants for Scientic
Research from the Ministry of Education, Science,
Sports, and Culture of Japan.
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