JOURNAL OF CELLULAR PHYSIOLOGY 195:158167 (2003)

REVIEW ARTICLES

Cell Death Regulation by the Bcl-2 ProteinFamily 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 toapoptotic cell death, since it leads to the release of several apoptogenic factors, suchas cytochrome c and Smac/Diablo, into the cytoplasm that activate downstreamdeath programs. During apoptosis, the mitochondria also release AIF and endo-nuclease G, both of which are translocated to the nucleus and are implicated inapoptotic nuclear changes that occur in a caspase-independent manner. Mito-chondrial membrane permeability is directly controlled by the major apoptosisregulator, 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 completelyunderstood. Here, I focus on the mechanisms by which Bcl-2 family memberscontrol the permeability of mitochondrial membrane during apoptosis. J. Cell.Physiol. 195: 158167, 2003. 2003 Wiley-Liss, Inc.

Programmed cell death or apoptosis plays an integralrole in a variety of biological events, including morpho-genesis, tissue homeostasis, and removal of unwantedor harmful cells. Dysregulation of programmed celldeath leads to various diseases in humans, includingcancer and certain neurodegenerative diseases. Exten-sive studies performed over the last 10 years haverevealed a large part of the molecular basis of celldeath, including apoptosis and some forms of necrosis.The mitochondria play an essential role in the apoptoticdeath of mammalian cells by releasing various apop-togenic proteins, including cytochrome c, into thecytoplasm (Martinou and Green, 2001; Zamzami andKroemer, 2001) (Fig. 1). Similar mitochondrial changesalso take place during necrotic cell death either as aconsequence or as an initiating event. In the process ofapoptosis, release of cytochrome c into the cytoplasmactivates death-promoting proteolytic enzymes calledcaspases, which in turn cleave a set of cellular proteinsand promote the death program (Thornberry andLazebnik, 1998; Wang, 2001). The Bcl-2 family ofproteins regulates these mitochondrial changes duringboth apoptosis and necrosis (Adams and Cory, 1998;Tsujimoto and Shimizu, 2000a). Although the mito-chondrial contribution to apoptotic cell death iswell established in mammals, no compelling evi-dence of mitochondrial involvement has been obtainedin C. elegans, despite its well-conserved apoptoticmachinery.

This review is primarily focused on the mechanismsunderlying the regulation of outer mitochondrial mem-brane permeability during apoptosis by the Bcl-2 familyof proteins.

Bcl-2 PROTEIN FAMILY INTHE MITOCHONDRIA

Bcl-2 (Tsujimoto et al., 1985; Tsujimoto and Croce,1986) was first suggested to play a role in cell survival bya study on the IL-3-deprivation-induced death of alymphoid cell line (Vaux et al., 1998). It was subse-quently reported that Bcl-2 also inhibits cell deathinduced by various stimuli such as a chemotherapeuticagent, 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 bygeneration of mice lacking the bcl-2 gene, which showeda 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 thebasis 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 (Scientific Research); Contract grantsponsor: Grant for Scientific Research on Priority Areas; Contractgrant sponsor: Center of Excellence Research.

*Correspondence to: Yoshihide Tsujimoto, Osaka UniversityGraduate School of Medicine, Department of Post-Genomics andDiseases, 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 andShimizu, 2000a) (Fig. 2). The anti-apoptotic subfamilycomprises Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1 (Bfl-1).Some viruses possess Bcl-2 homologues (Cuconati andWhite, 2002), including BHRF1 (Epstein-Barr virus),ks-Bcl-2 (human herpes virus 8), LHW5-HL (Africanswine fever virus), and ORF16 (herpesvirus saimiri).These proteins share sequence homology within some ofthe four Bcl-2 homology (BH) regions, BH1 throughBH4. The multi-domain pro-apoptotic subfamily con-sists of Bax, Bak, Mtd (Bok), and Bcl-rambo. Theseproteins 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. Theseproteins only share sequence homology at the BH3region.

The major function of the Bcl-2 family is to directlymodulate mitochondrial membrane permeability andthereby regulate the release of apoptogenic factors fromthe intermembrane space into the cytoplasm (Green,2000; Tsujimoto and Shimizu, 2000a,b; Martinou andGreen, 2001; Zamzami and Kroemer, 2001). Factors thatare known to be released from the mitochondria and areimplicated 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; Martinset al., 2002; Verhagen et al., 2002; Van Loo et al., 2002)and endonuclease G (Li et al., 2001) (Fig. 1). Cytochromec is directly involved in the activation of caspases, whileSmac/Diablo and HtrA2/Omi facilitate caspase activa-tion by inhibiting proteins from the Iap family, such asXiap, which are caspase inhibitors. AIF and endonu-clease G are thought to a play role in the induction ofcaspase-independent apoptotic changes in nuclei. Anti-apoptotic members of the Bcl-2 family inhibit the releaseof these apoptogenic factors, whereas pro-apoptoticmembers promote it.

HOW ARE MULTI-DOMAIN PRO-APOPTOTICMEMBERS ACTIVATED?

As described above, the pro-apoptotic members of theBcl-2 family are categorized into two groups based ontheir structure, i.e., multi-domain members and BH3-only members. Proteins in these two categories are alsofunctionally distinct. Multi-domain members such asBax and Bak act as a gateway for a variety of apoptoticsignals, since cells from Bax/Bak-double knockout miceare 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 functionallyredundant in many types of cells, although functionaldifferences 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 triggeredby death receptors are shown. In the intrinsic pathway, variousapoptotic signals are transmitted to the mitochondria, leading to therelease 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 deathfactors with death receptors induces the activation of apical caspasessuch as capsase-8, followed by transmission directly to either effectorcaspases or to the mitochondria via Bid, a BH3-only member of theBcl-2 family.

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considered to exist as monomers in living cell, althoughit is still unclear whether or not it binds to otherproteins. It has recently been shown that Bax interactswith some isoforms of 14-3-3, which may be important insequestering 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 havebeen detected by gel filtration (Bax) (Antonsson et al.,2001) and by protein cross-linking to detect homo-oligomers (Bax and Bak) (Eskes et al., 2000; Antonssonet al., 2001; Wei et al., 2001a), although it is notknown whether the multimers also interact with otherproteins. Bax is present in the cytoplasm or peri-mitochondrial location of living cells, but it undergoestranslocation and integration into the mitochondrialmembrane after stimulation (Wolter et al., 1997),whereas Bak is already present as an integral mitochon-drial membrane protein in living cells. It is consideredthat Bax and Bak undergo a conformational changeupon apoptotic stimulation, based on the following ob-servations: (1) some antibodies that recognize theN-terminal portion of Bax and Bak only react with theseproteins after induction of apoptosis (Desagher et al.,1999; Griffiths et al., 1999), (2) the N-terminal portion ofBax becomes accessible to limited proteolysis by trypsin

or proteinase K after induction of apoptosis (Gopinget 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 areformed from monomeric Bax in the presence of deter-gents such as Non-idet-40 (Antonsson et al., 2000).Currently, the conformational changes and multimerformation by Bax and Bak have not been separatedexperimentally. Bax multimer but not Bax monomerinduces cytochrome c release from isolated mitochon-dria (Antonsson et al., 2000), suggesting that Baxmultimer represents an active form. Critical moleculeswhich trigger Bax/Bak activation are the BH3-onlymembers of the Bcl-2 family.

HOW DO BH3-ONLY PROTEINS ACT?

BH3-only proteins seem to function as death signalsensors (Puthalakath and Strasser, 2002). Most BH3-only proteins (including Bid, Bim, Bmf, and Bad) arelocalized outside the mitochondria in living cells. Afterapoptotic stimulations, these proteins are modified byseveral different mechanisms and translocated to themitochondria, leading to increased permeability of theouter mitochondrial membrane (Fig. 3). Bid is activatedby cleavagewithproteases, includingcaspase-8 (Lietal.,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 classified intothree categories. BH1 to BH4 are conserved sequence motifs. Several functional domains of Bcl-2 areshown. a1 to a7 indicate helices identified in Bcl-xL, in which a core of two hydrophobic helices (a5 and a6)is surrounded by five amphipathic helices. The region between a1 and a2 is called a loop, which appears tohave a regulatory role. The membrane-anchoring domain is not carried by all members of the family.

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2000). Bad is normally phosporylated and sequesteredto 14-3-3t in living cells, while apoptotic stimulationcauses Bad to undergo dephosphorylation and frees itfrom 14-3-3 (Zha et al., 1996). Bim and Bmf are bound toelements of the cytoskeleton (microtubules and actinfilaments, respectively) via dynein motor complexes andmyosin V motor complexes, respectively (Puthalakathet al., 1999, 2001). Some of the BH3-only proteins suchas Noxa and Puma, are newly synthesized during DNAdamage-induced cell death after transcriptional activa-tion by p53 (Oda et al., 2000; Nakano and Vousden, 2001;Yu et al., 2001). It is still not fully understood how theBH3-only proteins transmit signals to the multi-domainmembers Bax and Bak. It has been suggested that tBidtransiently 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 Baxmultimers in the mitochondria, but not in synthetic lipidmembranes (Roucou et al., 2002b), suggesting that amitochondrial factor(s) is required for this process.Interestingly, it was found that Bax can form multimersin the absence of tBid in yeast mitochondria (Roucouet al., 2002b), suggesting the possibility that Bax mono-mer itself has an intrinsic multimer-forming activity inthe mitochondria, and that mammalian mitochondriamay contain an inhibitor of Bax multimerization. Ac-cordingly, a possible role of tBid is to inactivate thisinhibitor. Alternatively, tBid may activate Bax byaffecting the lipid membrane rather than via a directinteraction 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 innermembrane and contact sites, and that tBid has lipidtransfer activity (Esposti et al., 2001) as well as causingmembrane disruption (Kudla et al., 2000). In damagedmitochondria, for example, after being subjected torepeated freezing and thawing, the conformationalchange 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 inhibitorof Bax multimerization in the mitochondrial membranemight be inactivated or a mitochondrial BH3-onlyprotein might be activated during freezing and thawing.

Based on the observation that some BH3-only pro-teins such as tBid bind to both anti-apoptotic membersand pro-apoptotic multi-domain members of the Bcl-2family, whereas other BH3-only members such as Badbind preferentially to the anti-apoptotic members, it hasbeen suggested that BH3-only proteins may be categor-ized into two groups, one being activators of Bax/Bakand the other being inactivator of anti-apoptotic mem-bers of the Bcl-2 family (Letal et al., 2002). Consistentwith this idea, Bim alone reportedly does not inducecytochrome c release from isolated mitochondria, butcancels the inhibitory activity of anti-apoptotic proteins(Terradillos et al., 2002), suggesting that Bim may onlyinactivate anti-apoptotic members in the mitochondriato facilitate the activation of Bax/Bak. However, BimELhas also been shown to induce cytochrome c release from

Fig. 3. BH3-only proteins as death signal sensors. Many BH3-only proteins are localized outside themitochondria 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.

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isolated mitochondria in a BH3 domain-dependentmanner (Yamaguchi and Wang, 2002; Sugiyama et al.,2002). Recently, the classification of BH3-only proteinsinto two categories was challenged by a study using BH3domain peptides from several BH3-only proteins includ-ing tBid, Bim, Bik, Bak, and Noxa (Letai et al., 2002). Itwas shown that both tBid- and BimBH3-peptidesactivated 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 theBH3-peptide may not have exactly the same activity asthe full-size proteins, a similar study needs to be per-formed using intact proteins. Bim and tBid have alsobeen shown to induce cytochrome c release from isolatedmitochondria in a Bax (Bak)-independent manner(Grinberg et al., 2002; Sugiyama et al., 2002), althoughthe details of the mechanisms involved are unknown.

MOLECULAR BASIS OF INCREASEDMITOCHONDRIAL OUTER

MEMBRANE PERMEABILITY

All of the mitochondrial apoptogenic factors, exceptfor endonuclease G (Li et al., 2001) which is found in themitochondrial matrix, are mainly localized to the inter-membrane space between the outer and inner mitochon-drial membranes. Therefore, release of these factorsrequires an increase in the permeability of the outermembrane. Exogenous cytochrome c can reach the inner

mitochondrial membrane of apoptotic mitochondria(Kluck et al., 1999; Mootha et al., 2001), suggesting thatthe discontinuity in the outer membrane caused byapoptosis is bi-directional. Release of cytochrome c isquite rapid and is independent of any enzymatic reac-tions, since it still occurs at an equivalent rate at lowertemperatures (Goldstein et al., 2000). Therefore, thisdiscontinuity could represent the formation of protein-conducting pores in the outer membrane or could be dueto physical rupture or tear of the membrane. Since Baxtetramer is able to transmit cytochrome c from insideartificial liposomes (Saito et al., 2000), Bax (or Bak)multimer could represent such a protein-conductingpore. Apoptosis-induced Bax (Bak) multimer was visua-lized by immunoelectron microscopy, revealing thatmost of the multimer (huge aggregates) was localizednear (but outside) the mitochondria and did not seem toform pores in the outer membrane (Nechushtan et al.,2001). Thus only a small fraction of Bax multimer mayform protein-conducting pores. Bax multimer (gener-ated by using detergents such as octylglucoside) but notBax 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 inisolated mitochondria and cytochrome c release, tBidand Bax monomers are unable to induce cytochrome c

Fig. 4. Activation of Bax/Bak by BH3-only proteins. Once activated, BH3-only proteins are translocatedto the mitochondria to inactivate anti-apoptotic members of the Bcl-2 family and activate multi-domainmembers, 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.

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release from liposomes (PE/PG) (Roucou et al., 2002a),suggesting that other factors are required for fullactivation of Bax (probably an event involving Baxmultimerization). Recently, Kuwana et al. (2002) hassucceeded in reconstituting in liposomes containingcardiolipin using only tBid and Bax, Bcl-xL-inhibitablesupramolecular openings, which are permeable to verylarge (2 MDa) dextran molecules. It was hypothesizedthat activated Bax promotes localized alterations in thestructure of the lipid bilayer, for example, forming largetransient lipid pores or inverted micelles. However,Roucou et al. (2002a) reported that tBid was able toactivate Bax in mitochondria but not in cardiolipin-containing liposomes, liposomes reconstituted frommitochondrial lipid or proteinase K-treated mitochon-dria, suggesting an involvement of a mitochondrialfactor(s) that is not phospholipid, but rather a protein,for tBid-induced Bax multimerization.

In the presence of pro-apoptotic Bax or Bak, theVDAC, a channel in the mitochondrial outer membrane,becomes permeable to cytochrome c in liposomes(Shimizu et al., 1999) (Fig. 4). Conversion of VDAC to acytochrome c-conducting pore was also induced byoxygen radicals in the absence of Bax (Madesh andHajnoczky, 2001), suggesting that the VDAC itself hasthe intrinsic ability to become to a protein-conductingpore, probably through a conformational change. There-fore, the role of Bax may be simply to trigger a VDACconformational change rather than to be a stoichio-metric component of the cytochrome c-conductingchannel. 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 aprotein-conducting pore in lipid membranes impliesthat the VDAC contributes to the release of apoptogenicfactors in a permeability transition-independent man-ner. In contrast to Bax (Bak), Bcl-xL inhibits VDACactivity in liposomes (Shimizu et al., 1999, 2000c), al-though a controversial finding was also reported, sug-gesting that Bcl-xL maintains the VDAC in an openstate (Vander Heiden et al., 2001). The VDAC is alsoinhibited by other anti-apoptotic proteins such asgelsolin (Kusano et al., 2000) and c-Raf-1 (Le Mellayet al., 2002). Consistent with VDAC inhibition by theanti-apoptotic proteins, microinjection into cells ofantibodies which are specific to the VDAC and inhibitVDAC activity also significantly reduce apoptotic celldeath induced by the microinjection of Bax as well asother pro-apoptotic reagents such as etoposide (Shimizuet al., 2001), suggesting an important role of the VDACin apoptosis of mammalian cells. This is supported bythe observation that an anti-VDAC antibody also inhi-bits ischemia-induced neuronal damage (Velazquezet al., 2000). In addition to formation of protein-conducting pores, the VDAC may also be involved inmitochondrial reorganization that may occur duringapoptosis. A large fraction of cytochrome c is localized inthe cristae rather than in the intermembrane space.Since most cytochrome c is released from the mitochon-dria during apoptosis, mitochondrial reorganizationthat promotes the migration of cytochrome c into theintermembrane space may need to occur (Scorrano et al.,2002). This process appears to be cyclosporin A-sensitive

(Scorrano et al., 2002), indicating the involvement of theMPT, which has been shown to require VDAC activity(Shimizu et al., 2001) (see below).

By patch-clamping the mitochondria and proteoli-posmes prepared from outer 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 thelargest conductance is approximately 4 nm. Althoughthe electrophysiological characteristics of this channelare similar to those of the apoptotic VDAC channel, thebiochemical nature of MAC is unknown.

Rupture of the outer membrane is another possiblemechanism for the release of mitochondrial apoptogenicfactors (Zamzami and Kroemer, 2001) (Fig. 5). Thiscould be caused by MPT-dependent mitochondrial swel-ling. The MPT, which is triggered by various agents,including Ca2 and reactive oxygen species, and isdefined by the loss ofDC (the potential formed across theinner membrane) and the release of solutes smaller than1,500 Da from the mitochondria (Zoratti and Szabo,1995), leading to mitochondrial swelling followed byphysical rupture of the outer membrane. These changesare inhibited by cyclosporin A, which targets cyclophilinD, and by bonkrekic acid, which targets adeninenucleotide translocator (ANT) (Zamzani et al., 1996a;Kroemer and Reed, 2000). ANT, VDAC, and cyclophilinD, possibly together with other protein(s), constitute aprotein complex called the MPT pore complex at the siteof contact between the outer and inner mitochondrialmembranes, although the exact molecular architectureof this complex is still unknown. It has been proposedthat the MPT pore complex may be a non-specific proteinaggregate (He and Lemasters, 2002). The MPT itself isgenerally considered to be involved in necrotic celldeath, although some forms of apoptotic cell death alsoinvolve it because apoptosis is sometimes efficientlyblocked by MPT inhibitors such as cyclosporin A andbonkrekic acid (Marchetti et al., 1996; Zamzami et al.,1996b). Bax and Bak are able to induce the MPT, whichmay 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 membranecan also be achieved by a MPT-independent mechanism.For example, some pro-apoptotic Bcl-2 family membershave been shown to directly induce instability of lipidmembranes (Kudla et al., 2000). Given that variousmitochondrial proteins up to 100 kDa (such as a dimmerof sulfide oxidase) are rapidly released during apoptosis(Kluck et al., 1999), a tear of the outer membranemay have advantages over the formation of protein-conducting pores. It is certainly conceivable that morethan one mechanism may collaborate simultaneously orsequentially in destroying the barrier of the outermembrane.

ANTI-APOPTOTIC MEMBERS ACT VIABOTH HETERODIMERIZATION-DEPENDENT

AND -INDEPENDENT MECHANISMS

Bcl-2 binds to multi-domain members as well as theBH3-only proteins via interaction of the BH3 domain ofpro-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 mostBH3-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 theinteraction of Bcl-2 with Bax occurs or is enhanced in thepresence of non-ionic detergents such as NP40 andoctylglucoside, which induce a conformational change ofBax that is probably equivalent to that of the activatedform (Antonsson et al., 2000). However, since Bcl-2Baxinteraction is not readily detected in the absence ofdetergents, Bcl-2 activity to interact with Bax might notrepresent a main role of Bcl-2. Alternatively, Bcl-2might bind efficiently to activated Bax and Bak in themitochondrial membrane upon apoptotic stimulation,although this is not experimentally supported. Themajor role of Bcl-2 thus seems to be trapping of activatedBH3-only proteins (Cheng et al., 2001). On the otherhand, many studies have been done using variousmutants, and have suggested that the interaction ofBcl-2 with pro-apoptotic members of the Bcl-2 family isnot 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 welldocumented, for example, by the finding that a mutantof Bcl-2 lacking the BH4 domain almost completely losesits anti-apoptotic activity, but still binds as strongly tovarious pro-apoptotic members, including Bax, Bak,Bik, Bid, and Bim, as the wild type Bcl-2 (Huang et al.,1998). This heterodimerization-independent activitywas also demonstrated by the observation that Bcl-2 isable to protect the Ca2-induced permeability transition(Zamzami et al., 1996b; Shimizu et al., 1998) indepen-

dently of multi-domain pro-apoptotic members of theBcl-2 family (Wei et al., 2001a) and that Bcl-xL inhibitsthe PT-like event in yeast mitochondria which do nothave any homologue of the Bcl-2 family (Shimizu et al.,2000b). This heterodimerization-independent activityof Bcl-2 may be ascribable to its ability to inhibitmitochondrial channels such as the VDAC, since VDACinhibition by antibodies blocks the MPT (Shimizuet al., 2001).

POSSIBLE ROLE OF CED-9(C. elegans Bcl-2 HOMOLOGUE)

IN THE MITOCHONDRIA

Since yeast mitochondria undergo an event similar tothe MPT, it is conceivable that the mitochondria ofC. elegans also do so under certain circumstances.Occurrence of the MPT may be one reason why Ced-9mainly localizes to the mitochondria (Chen et al., 2000),like Bcl-2. In addition to sequestering Ced-4 (Apaf-1homologue) to inhibit the main pathway of programmedcell death (Liu and Hengartner, 1999), Ced-9 mayassociate with the mitochondria to prevent MPT-likeevents and thereby preserve mitochondrial function.Because the BH4 domain is both essential and sufficientto close the VDAC (Shimizu et al., 2000a), and becauseCed-9 possesses a BH4 domain, it is reasonable to expectthat Ced-9 can close the VDAC or similar channels. Thispossible action of Ced-9 might be related to inhibition ofthe 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-9 sequesters Ced-4 (a caspase activator) via directinteraction to prevent PCD, while loss-of-function-

Fig. 5. Release of mitochondrial proteins by formation of apoptotic protein-conducting pores or physicalrupture. Pore formation that leads to release of proteins from the mitochondrial intermembrane space orphysical rupture due to the MPT are both suppressed by anti-apoptotic members of the Bcl-2 family.

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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 celldeath in C. elegans (Vaux et al., 1992) which may beascribed 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 preventsPCDandiscontrolled byCed-9.Thisadditionalactivity ofCed-9 might be related to possible regulation of the MPT.

PERSPECTIVES

In mammals, a variety of death signals converge onthe mitochondria, which subsequently show an increaseof outer membrane permeability, leading to release ofproteins from the intermembrane space, includingseveral apoptogenic factors. One major function of theBcl-2 protein family is to directly control membranepermeability, although the precise mechanisms bywhich Bcl-2 family members do so are still to be deter-mined. Some Bcl-2 family members are also localized onthe endoplasmic reticulum, and it would be of interest todetermine their functions in relation to those located onthe mitochondria. Despite an essential role of themitochondria in the death of mammalian cells, it is stillunclear whether or not the mitochondria play a role incell death in other organisms, including the fruit fly andnematode, although proteins belonging to the Bcl-2family are well conserved in these organisms and arelocalized in or target the mitochondria as in mammaliancells. Although the overall structure of the apoptoticmachinery has been unveiled, there are still manyissues that need to be clarified to obtain a completeunderstanding of the regulation of cell death.

ACKNOWLEDGMENTS

I thank all members of my laboratory at OsakaUniversity Graduate School of Medicine for helpfuldiscussion. I am particularly grateful to Dr. ShigeomiShimizu for his major contribution to our recent work onthe Bcl-2 family of proteins and Dr. Yutaka Eguchi forcontinuous discussion. The work performed in mylaboratory was supported in part by a Grant forScientific Research on Priority Areas, by a Center ofExcellence Research grant, and by grants for ScientificResearch from the Ministry of Education, Science,Sports, and Culture of Japan.

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